The fibroblast growth factor receptor inhibitor, derazantinib, has strong efficacy in human gastric tumor models and synergizes with paclitaxel in vivo.

Derazantinib (DZB) is an inhibitor of fibroblast growth factor receptors 1-3 (FGFR1-3), with additional activity against colony-stimulating-factor-1 receptor (CSF1R). We have profiled the activity of DZB in gastric cancer (GC) as monotherapy and combined with paclitaxel, and explored means of stratifying patients for treatment. The antiproliferative potency of DZB in vitro was quantified in 90 tumor cell lines and shown to correlate significantly with FGFR expression (<0.01) but not with FGFR DNA copy-number (CN) or FGFR mutations. In four GC cell lines in vitro , little or no synergy was observed with paclitaxel. In athymic nude mice, bearing cell-line derived xenografts (CDX) or patient-derived xenograft (PDX) GC models, DZB efficacy correlated highly significantly with FGFR gene expression ( r2 = 0.58; P = 0.0003; n = 18), but not FGFR mutations or DNA-CN. In FGFR-driven GC models, DZB had comparable efficacy to three other FGFR inhibitors and was more efficacious than paclitaxel. DZB had dose-dependent plasma pharmacokinetics but showed low brain penetration at all doses. GC models (one CDX and six PDX) were tested for sensitivity to the combination of DZB and paclitaxel and characterized by immunohistochemistry. The combination showed synergy (5) or additivity (2), and no antagonism, with synergy significantly associated ( P < 0.05) with higher levels of M2-type macrophages. The association of strong efficacy of the combination in vivo with M2 macrophages, which are known to express CSF1R, and the absence of synergy in vitro is consistent with the tumor microenvironment also being a factor in DZB efficacy and suggests additional means by which DZB could be stratified for cancer treatment in the clinic.

Derazantinib, a fibroblast growth factor receptor inhibitor, inhibits colony-stimulating factor receptor-1 in macrophages and tumor cells and in combination with a murine programmed cell death ligand-1-antibody activates the immune environment of murine syngeneic tumor models.

Derazantinib (DZB) is an inhibitor of the fibroblast growth factor receptors 1-3 (FGFRi) with similar potency against colony-stimulating factor receptor-1 (CSF1R), a protein important in the recruitment and function of tumor-associated macrophages. DZB inhibited pCSF1R in the macrophage cell line RAW264.7, and tumor cells GDM-1 and DEL, and had the same potency in HeLa cells transiently over-expressing FGFR2. DZB exhibited similar potency against pCSF1R expressed by isolated murine macrophages, but as in the cell lines, specific FGFRi were without significant CSF1R activity. DZB inhibited growth of three tumor xenograft models with reported expression or amplification of CSF1R, whereas the specific FGFRi, pemigatinib, had no efficacy. In the FGFR-driven syngeneic breast tumor-model, 4T1, DZB was highly efficacious causing tumor stasis. A murine PD-L1 antibody was without efficacy in this model, but combined with DZB, increased efficacy against the primary tumor and further reduced liver, spine and lung metastases. Immunohistochemistry of primary 4T1 tumors showed that the combination favored an antitumor immune infiltrate by strongly increasing cytotoxic T, natural killer and T-helper cells. Similar modulation of the tumor microenvironment was observed in an FGFR-insensitive syngeneic bladder model, MBT-2. These data confirm CSF1R as an important oncology target for DZB and provide mechanistic insight for the ongoing clinical trials, in which DZB is combined with the PD-L1 antibody, atezolizumab.

Preclinical modeling in glioblastoma patient-derived xenograft (GBM PDX) xenografts to guide clinical development of lisavanbulin-a novel tumor checkpoint controller targeting microtubules.

BACKGROUND: Glioblastoma (GBM) is an incurable disease with few approved therapeutic interventions. Radiation therapy (RT) and temozolomide (TMZ) remain the standards of care. The efficacy and optimal deployment schedule of the orally bioavailable small-molecule tumor checkpoint controller lisavanbulin alone, and in combination with, standards of care were assessed using a panel of IDH-wildtype GBM patient-derived xenografts. METHODS: Mice bearing intracranial tumors received lisavanbulin +/-RT +/-TMZ and followed for survival. Lisavanbulin concentrations in plasma and brain were determined by liquid chromatography with tandem mass spectrometry, while flow cytometry was used for cell cycle analysis. RESULTS: Lisavanbulin monotherapy showed significant benefit (P < .01) in 9 of 14 PDXs tested (median survival extension 9%-84%) and brain-to-plasma ratios of 1.3 and 1.6 at 2- and 6-hours postdose, respectively, validating previous data suggesting significant exposure in the brain. Prolonged lisavanbulin dosing from RT start until moribund was required for maximal benefit (GBM6: median survival lisavanbulin/RT 90 vs. RT alone 69 days, P = .0001; GBM150: lisavanbulin/RT 143 days vs. RT alone 73 days, P = .06). Similar observations were seen with RT/TMZ combinations (GBM39: RT/TMZ/lisavanbulin 502 days vs. RT/TMZ 249 days, P = .0001; GBM26: RT/TMZ/lisavanbulin 172 days vs. RT/TMZ 121 days, P = .04). Immunohistochemical analyses showed a significant increase in phospho-histone H3 with lisavanbulin treatment (P = .01). CONCLUSIONS: Lisavanbulin demonstrated excellent brain penetration, significant extension of survival alone or in RT or RT/TMZ combinations, and was associated with mitotic arrest. These data provide a strong clinical rationale for testing lisavanbulin in combination with RT or RT/TMZ in GBM patients.

Treating ICB-resistant glioma with anti-CD40 and mitotic spindle checkpoint controller BAL101553 (lisavanbulin).

Glioblastoma is a highly malignant brain tumor with no curative treatment options, and immune checkpoint blockade has not yet shown major impact. We hypothesized that drugs targeting mitosis might affect the tumor microenvironment and sensitize cancer cells to immunotherapy. We used 2 glioblastoma mouse models with different immunogenicity profiles, GL261 and SB28, to test the efficacy of antineoplastic and immunotherapy combinations. The spindle assembly checkpoint activator BAL101553 (lisavanbulin), agonistic anti-CD40 antibody, and double immune checkpoint blockade (anti-programmed cell death 1 and anti-cytotoxic T lymphocyte-associated protein 4; anti-PD-1 and anti-CTLA-4) were evaluated individually or in combination for treating orthotopic GL261 and SB28 tumors. Genomic and immunological analyses were used to predict and interpret therapy responsiveness. BAL101553 monotherapy increased survival in immune checkpoint blockade-resistant SB28 glioblastoma tumors and synergized with anti-CD40 antibody, in a T cell-independent manner. In contrast, the more immunogenic and highly mutated GL261 model responded best to anti-PD-1 and anti-CTLA-4 therapy and more modestly to BAL101553 and anti-CD40 combination. Our results show that BAL101553 is a promising therapeutic agent for glioblastoma and could synergize with innate immune stimulation. Overall, these data strongly support immune profiling of glioblastoma patients and preclinical testing of combination therapies with appropriate models for particular patient groups.

Phase 1/2a trial of intravenous BAL101553, a novel controller of the spindle assembly checkpoint, in advanced solid tumours.

BACKGROUND: BAL101553 (lisavanbulin), the lysine prodrug of BAL27862 (avanbulin), exhibits broad anti-proliferative activity in human cancer models refractory to clinically relevant microtubule-targeting agents. METHODS: This two-part, open-label, phase 1/2a study aimed to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of 2-h infusion of BAL101553 in adults with advanced or recurrent solid tumours. The MTD was determined using a modified accelerated titration design in phase I. Patients received BAL101553 at the MTD and at lower doses in the phase 2a expansion to characterise safety and efficacy and to determine the recommended phase 2 dose (RP2D). RESULTS: Seventy-three patients received BAL101553 at doses of 15-80 mg/m2 (phase 1, n = 24; phase 2a, n = 49). The MTD was 60 mg/m2; DLTs observed at doses ≥60 mg/m2 were reversible Grade 2-3 gait disturbance with Grade 2 peripheral sensory neuropathy. In phase 2a, asymptomatic myocardial injury was observed at doses ≥45 mg/m2. The RP2D for 2-h intravenous infusion was 30 mg/m2. The overall disease control rate was 26.3% in the efficacy population. CONCLUSIONS: The RP2D for 2-h infusion of BAL101553 was well tolerated. Dose-limiting neurological and myocardial side effects were consistent with the agent's vascular-disrupting properties. CLINICAL TRIAL REGISTRATION: EudraCT: 2010-024237-23.

EB1-dependent long survival of glioblastoma-grafted mice with the oral tubulin-binder BAL101553 is associated with inhibition of tumor angiogenesis.

Glioblastoma (GBM) are aggressive brain tumors with limited treatment options. Cancer stem-like cells (CSLCs) contribute to GBM invasiveness, representing promising targets. BAL101553, a prodrug of BAL27862, is a novel small molecule tubulin-binding agent, promoting tumor cell death through spindle assembly checkpoint activation, which is currently in Phase 1/2a in advanced solid tumor patients including GBM. This study aimed to evaluate long-term daily oral BAL101553 treatment of mice orthotopically grafted with GBM CSLCs (GBM6) according to EB1 expression-level, and to decipher its mechanism of action on GBM stem cells. Oral treatment with BAL101553 for 100 days provoked a large EB1 expression level-dependent survival benefit, together with a decrease in tumor growth and brain invasion. Formation of vascular structures by the fluorescent GBM6-GFP-sh0 cells, mimicking endothelial vascular networks, was observed in the brains of control grafted mice. Following BAL101553 treatment, vessels were no longer detectable, suggesting inhibition of the endothelial trans-differentiation of GBM stem cells. In vitro, BAL27862 treatment resulted in a switch to the endothelial-like phenotype of GBM6 towards an astrocytic phenotype. Moreover, the drug inhibited secretion of VEGF, thus preventing normal endothelial cell migration activated by CSLCs. The decrease in VEGF secretion was confirmed in a human GBM explant following drug treatment. Altogether, our data first confirm the potential of EB1 expression as a response-predictive biomarker of BAL101553 in GBM we previously published and add new insights in BAL101553 long-term action by counteracting CSLCs mediated tumor angiogenesis. Our results strongly support BAL101553 clinical studies in GBM patients.

Dual Inhibition of the Lactate Transporters MCT1 and MCT4 Is Synthetic Lethal with Metformin due to NAD+ Depletion in Cancer Cells.

Highly glycolytic cancer cells prevent intracellular acidification by excreting the glycolytic end-products lactate and H+ via the monocarboxylate transporters 1 (MCT1) and 4 (MCT4). We report that syrosingopine, an anti-hypertensive drug, is a dual MCT1 and MCT4 inhibitor (with 60-fold higher potency on MCT4) that prevents lactate and H+ efflux. Syrosingopine elicits synthetic lethality with metformin, an inhibitor of mitochondrial NADH dehydrogenase. NAD+, required for the ATP-generating steps of glycolysis, is regenerated from NADH by mitochondrial NADH dehydrogenase or lactate dehydrogenase. Syrosingopine treatment leads to high intracellular lactate levels and thereby end-product inhibition of lactate dehydrogenase. The loss of NAD+ regeneration capacity due to combined metformin and syrosingopine treatment results in glycolytic blockade, leading to ATP depletion and cell death. Accordingly, ATP levels can be partly restored by exogenously provided NAD+, the NAD precursor nicotinamide mononucleotide (NMN), or vitamin K2. Thus, pharmacological inhibition of MCT1 and MCT4 combined with metformin treatment is a potential cancer therapy.

The novel microtubule targeting agent BAL101553 in combination with radiotherapy in treatment-refractory tumor models.

BACKGROUND AND PURPOSE: Resistance to microtubule targeting agents (MTA) represents a major drawback in successful cancer therapy with MTAs. Here we investigated the combined treatment modality of the novel MTA BAL101553 in combination with radiotherapy in paclitaxel and epothilone-resistant tumor models. MATERIAL AND METHODS: Multiple regimens of BAL101553, or its active moiety BAL27862 for in vitro experiments, were probed in combination with radiotherapy in P-glycoprotein-overexpressing, human colon adenocarcinoma cells (SW480) and in tubulin-mutated human NSCLC cells (A549EpoB40) and tumors thereof. RESULTS: BAL27862 reduced the proliferative activity of SW480 and A549EpoB40 tumor cells with similar potency as in A549 wildtype cells. Combined treatment of BAL27862 with ionizing radiation in vitro resulted in an additive reduction of clonogenicity. Moreover, treatment of paclitaxel- and epothilone-resistant tumors with fractionated irradiation and different regimens of BAL101553 (a single i.v. bolus vs. oral daily) suppressed tumor growth and resulted in an extended additive tumor growth delay with strong reduction of tumor proliferation and mean tumor vessel density. CONCLUSIONS: BAL101553 is a promising alternative in taxane- and epothilone-refractory tumors as part of a combined treatment modality with ionizing radiation. Its potent antitumor effect is not only tumor cell-directed but also targets the tumor microenvironment.

Syrosingopine sensitizes cancer cells to killing by metformin.

We report that the anticancer activity of the widely used diabetic drug metformin is strongly potentiated by syrosingopine. Synthetic lethality elicited by combining the two drugs is synergistic and specific to transformed cells. This effect is unrelated to syrosingopine's known role as an inhibitor of the vesicular monoamine transporters. Syrosingopine binds to the glycolytic enzyme α-enolase in vitro, and the expression of the γ-enolase isoform correlates with nonresponsiveness to the drug combination. Syrosingopine sensitized cancer cells to metformin and its more potent derivative phenformin far below the individual toxic threshold of each compound. Thus, combining syrosingopine and codrugs is a promising therapeutic strategy for clinical application for the treatment of cancer.

The Novel Tubulin-Binding Checkpoint Activator BAL101553 Inhibits EB1-Dependent Migration and Invasion and Promotes Differentiation of Glioblastoma Stem-like Cells.

Glioblastoma patients have limited treatment options. Cancer stem-like cells (CSLC) contribute to glioblastoma invasiveness and repopulation; hence, they represent promising targets for novel therapies. BAL101553 is a prodrug of BAL27862, a novel microtubule-destabilizing agent inhibiting tumor cell proliferation through activation of the spindle assembly checkpoint, which is currently in phase I/II clinical development. Broad anticancer activity has been demonstrated against human cancer models, including tumors refractory to conventional treatments. We have shown that overexpression of microtubule + end-binding 1-protein (EB1) correlates with glioblastoma progression and poor survival. Here, we show that BAL27862 inhibits the growth of two glioblastoma CSLCs. As EB1 is overexpressed in the CSLC line GBM6, which displays a high tumorigenicity and infiltrative pattern of migration in vivo, we investigated drug activity on GBM6 according to EB1 expression. BAL27862 inhibited migration and colony formation at subcytotoxic concentrations in EB1-expressing control cells (GBM6-sh0) but only at cytotoxic concentrations in EB1-downregulated (GBM-shE1) cells. Three administrations of BAL101553 were sufficient to provoke an EB1-dependent survival benefit in tumor-bearing mice. Patterns of invasion and quantification of tumor cells in brain demonstrated that GBM6-sh0 cells were more invasive than GBM6-shEB1 cells, and that the antiproliferative and anti-invasive effects of BAL101553 were more potent in mice bearing control tumors than in EB1-downregulated tumors. This was associated with inhibition of stem cell properties in the GBM6-sh0 model. Finally, BAL27862 triggered astrocytic differentiation of GBM6 in an EB1-dependent manner. These results support the potential of BAL101553 for glioblastoma treatment, with EB1 expression as a predictive biomarker of response. Mol Cancer Ther; 15(11); 2740-9. ©2016 AACR.

Effectiveness and molecular interactions of the clinically active mTORC1 inhibitor everolimus in combination with tamoxifen or letrozole in vitro and in vivo.

INTRODUCTION: Strategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1 (mTORC1), which regulate cell-cycle progression and are supported by recent clinical results. METHODS: BC cell lines expressing aromatase (AROM) and modeling endocrine-sensitive (MCF7-AROM1) and human epidermal growth factor receptor 2 (HER2)-dependent de novo resistant disease (BT474-AROM3) and long-term estrogen-deprived (LTED) MCF7 cells that had acquired resistance associated with HER2 overexpression were treated in vitro and as subcutaneous xenografts with everolimus (RAD001-mTORC1 inhibitor), in combination with tamoxifen or letrozole. End points included proliferation, cell-cycle arrest, cell signaling, and effects on ER-mediated transactivation. RESULTS: Everolimus caused a concentration-dependent decrease in proliferation in all cell lines, which was associated with reductions in S6 phosphorylation. Everolimus plus letrozole or tamoxifen enhanced the antiproliferative effect and G1-accumulation compared with monotherapy, as well as increased phosphorylation (Ser10) and nuclear accumulation of p27 and pronounced dephosphorylation of Rb. Sensitivity was greatest to everolimus in the LTED cells but was reduced by added estrogen. Increased pAKT occurred in all circumstances with everolimus and, in the BT474 and LTED cells, was associated with increased pHER3. Decreased ER transactivation suggested that the effectiveness of everolimus might be partly related to interrupting cross-talk between growth-factor signaling and ER. In MCF7-AROM1 xenografts, letrozole plus everolimus showed a trend toward enhanced tumor regression, versus the single agents. In BT474-AROM3 xenografts, everolimus alone was equally effective at reducing tumor volume as were the combination therapies. CONCLUSIONS: The results provide mechanistic support for recent positive clinical data on the combination of everolimus and endocrine therapy, as well as data on potential routes of escape via enhanced HER2/3 signaling. This merits investigation for further improvements in treatment efficacy.

Everolimus and PTK/ZK show synergistic growth inhibition in the orthotopic BL16/BL6 murine melanoma model.

PURPOSE: Everolimus (RAD001, Afinitor) is an mTORC1 pathway inhibitor, and vatalanib (PTK/ZK) is a pan VEGF-R tyrosine kinase inhibitor (TKI). These two drugs have been shown to have overlapping but also distinct anti-angiogenic effects. Consequently, we investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of their combination in vivo. METHODS: Murine melanoma B16/BL6 cells were grown orthotopically in BL6/C57 mice by injection into the derma of both ears to create a primary tumour which metastasized rapidly to the cervical lymph nodes. Mice were treated daily p.o. with PTK/ZK (100 mg/kg) or everolimus (1 mg/kg) or their combination, and anti-tumour efficacy (PD) assessed. In the same model, plasma PK of everolimus was measured following single doses of the monotherapy or combination schedules. RESULTS: Two independent experiments showed that combination of everolimus and PTK/ZK caused at least additive increases in anti-tumour activity compared to either monotherapy, without increases in toxicity. Pooling the data to improve the statistical power demonstrated the interactions to be synergistic. PK modelling showed that although PTK/ZK increased everolimus plasma concentrations by about twofold, this PK drug-drug interaction could not account for the increased anti-tumour effect of the combination. Modelling of the PTK/ZK dose-response curve in this model suggested that any effect of everolimus on the PK of PTK/ZK was unlikely to affect efficacy. Measurement of changes in tumour and plasma VEGF levels at the endpoint of therapy confirmed earlier observations of differential effects of these two agents. CONCLUSIONS: The combination of everolimus and PTK/ZK hold promise for the treatment of human cancers.

Evaluation of the mTOR inhibitor, everolimus, in combination with cytotoxic antitumor agents using human tumor models in vitro and in vivo.

The aim was to determine the potential of the allosteric mammalian target of rapamycin inhibitor, everolimus, to act in combination with cytotoxic anticancer compounds in vitro and in vivo. A concomitant combination in vitro showed no evidence of antagonism, but enhanced the antiproliferative effects (additive to synergistic) with cisplatin, doxorubicin, 5-fluorouracil, gemcitabine, paclitaxel, and patupilone. Everolimus (1-5 mg/kg/d orally) was evaluated for antitumor activity in vivo alone or in combination with suboptimal cytotoxic doses using athymic nude mice bearing subcutaneous human H-596 lung, KB-31 cervical, or HCT-116 colon tumor xenografts. Everolimus monotherapy was very well tolerated and caused inhibition of tumor growth, rather than regression, and this was associated with a dose-dependent decline in tumor pS6 levels, a key downstream protein of mammalian target of rapamycin. At the doses used, the cytotoxics inhibited tumor growth and caused tolerable body-weight loss. Concomitant combinations of cisplatin, doxorubicin, paclitaxel, or patupilone with everolimus produced cooperative antitumor effects, in some cases producing regressions without clinically significant increases in toxicity. In contrast, combinations with gemcitabine and 5-fluorouracil were less well tolerated. Alternative administration schedules were tested for cisplatin, gemcitabine, or paclitaxel combined with everolimus: these did not dramatically affect cisplatin or gemcitabine activity or tolerability but were antagonistic for paclitaxel. Everolimus showed promising maintenance activity after treatment with doxorubicin or paclitaxel ceased. Overall, the results confirm that everolimus is an effective, well-tolerated suppressor of experimental human tumor growth, and although it did not show strong potentiation of efficacy, antitumor activity in vivo was increased without marked increases in toxicity, supporting clinical use of everolimus as a partner for conventional cytotoxics.

Research and innovation in the development of everolimus for oncology.

INTRODUCTION: The critical role of increased activity of mammalian target of rapamycin (mTOR) in the pathophysiology of multiple diseases is well established. Inhibition of the mTOR pathway may block disease progression and improve patient outcomes. Everolimus, an mTOR inhibitor, began in clinical development as part of a regimen (Certican, Zortress) for prevention of organ transplant rejection and is now an approved oncology agent. AREAS COVERED: The objective of this review is to discuss the history of key findings and innovative cancer research undertaken to successfully develop everolimus as an oncology therapy (Afinitor) now approved for patients with advanced renal cell carcinoma (RCC) and for subependymal giant cell astrocytomas (SEGAs) associated with tuberous sclerosis. In addition, data for the use of everolimus in the treatment of other cancers and rare diseases are also discussed. A PubMed search of English articles without time restrictions was conducted using the search terms 'everolimus or rapamycin' and 'cancer'. Bibliographies of retrieved articles were manually searched for additional relevant articles. Major cancer congresses were also searched. EXPERT OPINION: The clinical efficacy of everolimus alone and in combination with other agents has been observed in recently completed Phase II-III studies in a wide spectrum of tumors, including RCC, neuroendocrine tumors, tuberous sclerosis complex, SEGAs and angiomyolipomas, lymphoma and gastric, breast and hepatocellular cancers. These findings emphasize the importance of mTOR in diverse cancers and rare diseases and underscore the potential role for everolimus as an effective agent in multiple indications.

Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET.

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[(18)F]fluoro-glucose (FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC(50) values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 microM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.

Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition.

Mammalian target of rapamycin (mTOR) regulates cellular processes important for progression of human cancer. RAD001 (everolimus), an mTORC1 (mTOR/raptor) inhibitor, has broad antitumor activity in preclinical models and cancer patients. Although most tumor lines are RAD001 sensitive, some are not. Selective mTORC1 inhibition can elicit increased AKT S473 phosphorylation, involving insulin receptor substrate 1, which is suggested to potentially attenuate effects on tumor cell proliferation and viability. Rictor may also play a role because rictor kinase complexes (including mTOR/rictor) regulate AKT S473 phosphorylation. The role of raptor and rictor in the in vitro response of human cancer cells to RAD001 was investigated. Using a large panel of cell lines representing different tumor histotypes, the basal phosphorylation of AKT S473 and some AKT substrates was found to correlate with the antiproliferative response to RAD001. In contrast, increased AKT S473 phosphorylation induced by RAD001 did not correlate. Similar increases in AKT phosphorylation occurred following raptor depletion using siRNA. Strikingly, rictor down-regulation attenuated AKT S473 phosphorylation induced by mTORC1 inhibition. Further analyses showed no relationship between modulation of AKT phosphorylation on S473 and T308 and AKT substrate phosphorylation patterns. Using a dual pan-class I phosphatidylinositol 3-kinase/mTOR catalytic inhibitor (NVP-BEZ235), currently in phase I trials, concomitant targeting of these kinases inhibited AKT S473 phosphorylation, eliciting more profound cellular responses than mTORC1 inhibition alone. However, reduced cell viability could not be predicted from biochemical or cellular responses to mTORC1 inhibitors. These data could have implications for the clinical application of phosphatidylinositol 3-kinase/mTOR inhibitors.

Phase II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer.

PURPOSE: Cross-talk between the estrogen receptor (ER) and the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways is a mechanism of resistance to endocrine therapy, and blockade of both pathways enhances antitumor activity in preclinical models. This study explored whether sensitivity to letrozole was enhanced with the oral mTOR inhibitor, everolimus (RAD001). PATIENTS AND METHODS: Two hundred seventy postmenopausal women with operable ER-positive breast cancer were randomly assigned to receive 4 months of neoadjuvant treatment with letrozole (2.5 mg/day) and either everolimus (10 mg/day) or placebo. The primary end point was clinical response by palpation. Mandatory biopsies were obtained at baseline and after 2 weeks of treatment (ie, day 15). Samples were assessed for PI3K mutation status (PIK3CA) and for pharmacodynamic changes of Ki67, phospho-S6, cyclin D1, and progesterone receptor (PgR) by immunohistochemistry. RESULTS: Response rate by clinical palpation in the everolimus arm was higher than that with letrozole alone (ie, placebo; 68.1% v 59.1%), which was statistically significant at the preplanned, one-sided, alpha = 0.1 level (P = .062). Marked reductions in progesterone receptor and cyclin D1 expression occurred in both treatment arms, and dramatic downregulation of phospho-S6 occurred only in the everolimus arm. An antiproliferative response, as defined by a reduction in Ki67 expression to natural logarithm of percentage positive Ki67 of less than 1 at day 15, occurred in 52 (57%) of 91 patients in the everolimus arm and in 25 (30%) of 82 patients in the placebo arm (P < .01). The safety profile was consistent with historical results of everolimus monotherapy; grades 3 to 4 adverse events occurred in 22.6% of patients who received everolimus and in 3.8% of patients who received placebo. CONCLUSION: Everolimus significantly increased letrozole efficacy in neoadjuvant therapy of patients with ER-positive breast cancer.

mTOR inhibitor RAD001 (everolimus) has antiangiogenic/vascular properties distinct from a VEGFR tyrosine kinase inhibitor.

PURPOSE: Comparison of the antiangiogenic/vascular properties of the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and the vascular endothelial growth factor receptor (VEGFR) inhibitor vatalanib (PTK/ZK). EXPERIMENTAL DESIGN: Antiproliferative activity against various tumor histotypes and downstream effects on the mTOR pathway were measured in vitro. In vivo, antitumor activity, plasma, and tumor RAD001 levels were measured. Activity in several different angiogenic/vascular assays in vitro and in vivo was assessed and compared with PTK/ZK. RESULTS: RAD001 inhibited proliferation in vitro (IC50 values<1 nmol/L to >1 micromol/L), and in sensitive and insensitive tumor cells, pS6 kinase and 4E-BP1 were inhibited. Activity in vitro did not correlate with activity in vivo and significant responses were seen in tumors with IC50 values>10-fold higher than tumor RAD001 concentrations. In vitro, RAD001 inhibited the proliferation of VEGF-stimulated and fibroblast growth factor-stimulated human endothelial cells but not dermal fibroblasts and impaired VEGF release from both sensitive and insensitive tumor cells but did not inhibit migration of human endothelial cells. In vivo, in tumor models derived from either sensitive or insensitive cells, RAD001 reduced Tie-2 levels, the amount of mature and immature vessels, total plasma, and tumor VEGF. RAD001 did not affect blood vessel leakiness in normal vasculature acutely exposed to VEGF nor did it affect tumor vascular permeability (Ktrans) as measured by dynamic contrast-enhanced magnetic resonance imaging. However, the pan-VEGFR inhibitor PTK/ZK inhibited endothelial cell migration and vascular permeability but had less effect on mature vessels compared with RAD001. CONCLUSIONS: VEGFR and mTOR inhibitors show similar but also distinct effects on tumor vascular biology, which has implications for their clinical activity alone or in combination.

AKT/mTOR pathway activation and BCL-2 family proteins modulate the sensitivity of human small cell lung cancer cells to RAD001.

PURPOSE: The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology. We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. EXPERIMENTAL DESIGN: The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. RESULTS: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001 treatment impaired basal and growth factor-stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001 showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2-specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. CONCLUSION: Together, our data show that inhibiting mTOR signaling with RAD001 potently disrupts growth and survival signaling in human SCLC cells.

Optimal targeting of the mTORC1 kinase in human cancer.

A central sensor of the availability of growth factors, nutrients and energy sources, the mammalian target of rapamycin complex 1 (mTORC1) kinase plays a key role in tumor biology. Consequently, mTORC1 inhibitors have been shown to have broad antitumor activity pre-clinically in experimental tumor models as well as clinically in cancer patients. Strikingly, certain tumor types appear to be predisposed to respond to mTORC1 inhibition, a phenomenon related to deregulation of critical elements of the PI3K/mTORC1 pathway. In this review we address optimization of clinical development in the context of mTORC1 inhibitor-induced activation of survival pathways, crosstalk between different signaling modules involved in malignant transformation, definition of rational target combination scenarios and biologically based dosing and patient stratification strategies. Emphasis is given where possible to mTORC1 drug development decisions based on full clinical publications.