Combination Of mTOR Inhibitors Research Articles

Cancer Battlefield: Six Characters in Search of an Author

ImmunotherapyVol. 4, No. 8 EditorialCancer battlefield: six characters in search of an authorLeslie Huye & Barbara SavoldoLeslie HuyeCenter for Cell & Gene Therapy, Departments of Pediatrics, Baylor College of Medicine, The Methodist Hospital & Texas Children’s Hospital, Houston, TX 77030, USASearch for more papers by this author & Barbara SavoldoCenter for Cell & Gene Therapy, Departments of Pediatrics, Baylor College of Medicine, The Methodist Hospital & Texas Children’s Hospital, Houston, TX 77030, USASearch for more papers by this authorPublished Online:4 Sep 2012https://doi.org/10.2217/imt.12.77AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: chimeric T cell receptorimmune evasionimmunotherapytumor microenvironmentReferences1 Dudley M, Wunderlich J, Yang JC et al. 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Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood119(18),4133–4141 (2012).Crossref, Medline, CAS, Google Scholar16 Chmielewski M, Kopecky C, Hombach AA, Abken H. IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression. Cancer Res.71(17),5697–5706 (2011).Crossref, Medline, CAS, Google Scholar17 Bollard CM, Rossig C, Calonge MJ et al. Adapting a transforming growth factor β-related tumor protection strategy to enhance antitumor immunity. Blood99(9),3179–3187 (2002).Crossref, Medline, CAS, Google Scholar18 Vanneman M, Dranoff G. Combining immunotherapy and targeted therapies in cancer treatment. Nat. Rev. Cancer12(4),237–251 (2012).Crossref, Medline, CAS, Google Scholar19 De Angelis B, Dotti G, Quintarelli C et al. Generation of Epstein–Barr-virus-specific cytotoxic T lymphocytes resistant to the immunosuppressive drug tacrolimus (FK506). Blood114(23),4784–4791 (2009).Crossref, Medline, CAS, Google Scholar20 Huye LE, Nakazawa Y, Patel MP et al. Combining mTor inhibitors with rapamycin-resistant T cells: a two-pronged approach to tumor elimination. Mol. Ther.19(12),2239–2248 (2011).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByStrategies to Enhance Migration and Persistence of Chimeric Antigen Receptor (CAR)-T Cells into Tumors31 August 2016 Vol. 4, No. 8 Follow us on social media for the latest updates Metrics Downloaded 150 times History Published online 4 September 2012 Published in print August 2012 Information© Future Medicine LtdKeywordschimeric T cell receptorimmune evasionimmunotherapytumor microenvironmentFinancial & competing interests disclosureThis work was supported by the William Lawrence and Blanche Hughes Foundation, by NIH-NCI Lymphoma SPORE P50 CA126752, NIH R01CA131027 and by a Leukemia and Lymphoma Society Translational Research grant. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

Clinical activity of mTOR inhibition in combination with cyclophosphamide in the treatment of recurrent nonresectable chondrosarcomas.

10043 Background: Chondrosarcomas (CS) represent a heterogeneous group of rare sarcomas, poorly responsive to chemotherapy or radiotherapy. When local therapies in recurrent or metastatic disease are exhausted, chemotherapy plays a marginal role. Different molecular pathways have been shown to be activated in CS. In this retrospective study we summarize our experience in treating a cohort of patients with recurrent nonresectable CS with a combination of sirolimus and cyclophosphamide. Methods: Nine consecutive patients with nonresectable CS were offered off-label treatment with sirolimus and cyclophosphamide between 2007-2011. Tumor response, progression-free survival (PFS), adverse events and other relevant clinical data were analyzed. Results: The median patients’ age was 52 (range, 35-68). Median disease-free interval (DFI) since the primary diagnosis was 24 months. Median time from the disease recurrence to initiation of sirolimus and cyclophosphamide treatment was 20.9 months due to additional local surgical treatments/excision of metastases/slow and asymptomatic progression. One (11%) objective response was observed and five (56%) patients had stabilization of disease for at least 6 months. One patient has been treated for only 3.5 months and reported symptomatic improvement at this stage, two patients had progressive disease. Median time to treatment failure (TTF) was 15 months (range, 2.8-30.3). No significant adverse events were observed. Conclusions: Although advanced CS remains an incurable disease, our experience suggests that a combination of sirolimus and cyclophosphamide is well-tolerated and has meaningful clinical activity with clinical benefit rate of 67%. Further prospective studies are warranted.

NCCTG N057K phase II trial of everolimus, temozolomide, and radiotherapy in patients with newly diagnosed glioblastoma: A North Central Cancer Treatment Group trial.

2031 Background: The mammalian target of rapamycin (mTOR) functions within the PI3K/Akt pathway as a critical modulator of cell survival. Preclinical studies in GBM indicate that the combination of mTOR inhibitors, such as everolimus (RAD001), with either radiation therapy (RT) or temozolomide (TMZ) provide increased tumor cell killing. Methods: Newly diagnosed GBM pts were eligible for the study. RAD001 was dosed orally at 70 mg/week weekly, starting 1 week prior to RT/TMZ, and continued throughout RT/TMZ, adjuvant TMZ and then until progression. This was a single arm phase II design powered to detect a true overall survival at 12 months (OS12) of 73% (vs 58% in historical controls). Secondary endpoints were toxicity, response rate, and time to progression (TTP). A subgroup of patients with measurable residual disease were eligible for the PET imaging component of the study, consisting of an 18FLT-PET/CT scan performed before and after the initial two doses of RAD001 but before the first dose of RT or TMZ. Results: 103 patients were accrued to phase II of which 100 were evaluable. The median age was 60.5 years (23-81), median ECOG PS was 1, 46 patients had GTR, 33 STR, and the remainder had biopsy at diagnosis. Treatment tolerance was acceptable: 17% patient had at least one grade 3 hematologic toxicity; 14% had at least one grade 4 hematologic toxicity, 42% had at least one grade 3 non-heme toxicity, while 12% had at least one grade 4 non-heme toxicity. No increased incidence of infectious complications was observed and there were no treatment related deaths. Median PFS was 5.3 months (1.3-21.4), with 22 patients progression free. Mature OS data will be available at the meeting. MGMT methylation status analysis is ongoing. Of the pts who had evaluable FLT-PET data, three of eight (37.5%) had a drop in SUVmax >25% after two treatments of RAD001. Conclusions: RAD001 with standard of care chemoradiation had moderate toxicity. Serial 18FLT-PET was feasible for evaluating drug-induced changes in tumor proliferation following RAD001. Final outcome data and association of MGMT status with outcome will be reported at the meeting.

Abstract 1868: Response biomarkers to IGF1R and mTOR inhibitor combination therapy in ovarian carcinoma

Abstract Ovarian cancer, the sixth most frequent cause of cancer death among women in the developed world, is a heterogeneous disease characterized by a diverse set of genetic alterations. These factors underscore the need for more effective treatment options accompanied by biomarker strategies to identify patients who will have a greater likelihood to respond to novel therapy. A combination strategy to target the PI3K pathway with the mTOR inhibitor ridaforolimus and anti-IGF1R antibody dalotuzumab is currently undergoing clinical development. Previous translational work has suggested that low RAS activity, as determined by a RAS gene expression signature score, and high levels of IGF1R pathway activation may enrich for response to this combination therapy. Ranking of tumors in the Moffitt tumor database with a low RAS and high IGF profile suggested that ER+ breast and ovarian cancers are enriched for these putative response biomarkers. Consistent with these observations, clinical responses were noted for several ER+ breast or ovarian cancer patients in a Phase I trial for ridaforolimus and dalotuzumab combination therapy. To provide further support for low RAS and high IGF as response biomarkers, the anti-tumor activity of ridaforolimus and dalotuzumab was assessed in 12 patient derived primary ovarian cancer xenograft models developed at START. These models have been extensively characterized by the South Texas Accelerated Research and Therapeutics (START) group for response to standard of care drugs, and the status of many commonly mutated genes in ovarian cancer. Molecular analyses of these tumors suggest that they represent a diverse cross section of ovarian cancer. Similarly, responses to ridaforolimus and dalotuzumab combination therapy ranged from minimal to significant regression. Importantly, the responsive tumor models were associated with a low RAS gene signature and a moderate to high IGF expression level. Tumors with KRAS mutations or a high RAS gene score were generally resistant to therapy. These results support the further development of low RAS and high IGF as enrichment biomarkers for ridaforolimus and dalotuzumab combination therapy in ovarian carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1868. doi:1538-7445.AM2012-1868

Combined mTOR Inhibition and OX40 Agonism Enhances CD8+ T Cell Memory and Protective Immunity Produced by Recombinant Adenovirus Vaccines

The memory CD8(+) T cell population elicited by immunization with recombinant human adenovirus serotype 5 (rHuAd5) vaccines is composed primarily of effector and effector memory cells (T(EM)) with limited polyfunctionality. In this study, we investigated whether treatment with immunomodulators could enhance and/or redistribute the CD8(+) memory population elicited by rHuAd5. Vaccination in combination with both rapamycin (to modulate differentiation) and an OX40 agonist (to enhance costimulation) increased both the quantity and polyfunctionality of the CD8(+) memory T cell population, with expansion of the T(EM) and memory precursor populations. Furthermore, this intervention enhanced protection against multiple virus challenges. Attenuation of adenovirus transgene expression was required to enable the combination rapamycin + OX40 agonist immunomodulatory treatment to further enhance skewing towards central memory formation, indicating that persistence of antigen expression ultimately limits development of this memory population following rHuAd5 immunization. These results demonstrate that during the expansion phase following adenovirus immunization, the level of mammalian target of rapamycin (mTOR) activity, the amount of costimulation and the duration of antigen availability act together to define the magnitude, phenotype, and functionality of memory CD8(+) T cells. Modulation of these factors can be used to selectively manipulate memory formation.

Combining mTor Inhibitors With Rapamycin-resistant T Cells: A Two-pronged Approach to Tumor Elimination

Despite activity as single agent cancer therapies, Rapamycin (rapa) and its rapalogs may have their greatest effects when combined with other therapeutic modalities. In addition to direct antitumor activity, rapalogs reverse multiple tumor-intrinsic immune evasion mechanisms. These should facilitate tumor-specific T cell activity, but since rapa directly inhibits effector T cells, this potential immune enhancement is lost. We hypothesized that if T cells were rendered resistant to rapa they could capitalize on its downregulation of tumor immune evasion. We therefore modified T cells with a rapa-resistant mutant of mTor, mTorRR, and directed them to B lymphomas by coexpressing a chimeric antigen receptor (CAR) for CD19 (CAR.CD19-28ζ). T cells expressing transgenic mTorRR from a piggyBac transposon maintain mTor signaling, proliferate in the presence of rapa and retain their cytotoxic function and ability to secrete interferon-γ (IFNγ) after stimulation, effector functions that were inhibited by rapa in control T cells. In combination, rapa and rapa-resistant-CAR.CD19-28ζ-expressing T cells produced greater antitumor activity against Burkitt's lymphoma and pre-B ALL cell lines in vitro than CAR.CD19-28ζ T cells or rapa alone. In conclusion, the combination of rapa and rapa-resistant, CAR.CD19-28ζ-expressing T cells may provide a novel therapy for the treatment of B cell malignancies and other cancers.

Inhibitors of Mtor and PI3K Synergistically Block Plasmacytoma Cell Growth

Abstract 2903 Introduction:Both, the phosphoinositide-3-kinase (PI3K)-AKT pathway as well as its nutrient-dependent downstream target, the mToR (mammalian target of rapamycin) kinase, are essential for the growth and survival of malignant plasma cells. PI3K-AKT can be activated by the loss of the tumor suppressor phosphatase and tensin homolog (PTEN) or by stimulation with cytokines such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1). Inhibitors of the mToR pathway like sirolimus, everolimus and temsirolimus are approved for immunosuppression and treatment of renal cell cancer. In fact, they show activity in multiple myeloma patients. However, the clinical activity of mToR inhibitors may be limited by feedback loops in tumor cells that lead to activation of AKT upon inhibition of the rapamycin-sensitive Raptor complex. Selective PI3K inhibitors (Ly294002, NVP-BKM120) as well as dual PI3K-mToR inhibitors (NVP-BEZ235) are in clinical development. Here, the inhibitory effect of a panel of mToR and PI3K inhibitors, alone and in combination, was evaluated in malignant plasma cell lines and primary samples from myeloma patients. Results:In five human plasma cell lines, rapamycin, everolimus, Ly294002, NVP-BKM120 and NVP-BEZ235 (kindly provided by Novartis) induced a dose-dependent growth inhibition as measured by MTS assay. Despite the observed strong anti-myeloma activity of the mToR inhibitors rapamycin and everolimus with IC50 values in the nm range, the AKT pathway was activated as indicated by increased phosphorylation at Ser473. This observation was also made in explanted plasma cell tumors of INA-6 xenografted SCID mice that were treated with rapamycin, indicating that this feedback loop is also active in vivo. Therefore, combining mToR and PI3K inhibitors could be an effective strategy to overcome rapamycin-induced AKT activation. In fact, rapamycin in combination with the selective PI3K inhibitor Ly294002 or NVP-BKM120 led to synergistic growth inhibition in plasma cell lines, as calculated by the Calcusyn™ software (Biosoft). This abrogation of AKT activation was also seen in Western blot analysis. Combined treatment also enhanced the induction of apoptosis in cell lines as well as in plasmacytoma cells. Interestingly, the activity of the dual inhibitor NVP-BEZ235 could be even further enhanced by the addition of rapamycin. Conclusion:First clinical trials with mToR inhibitors showed anti-tumor activity and an acceptable safety profile in patients with multiple myeloma. The data presented here suggest that a combination of mToR inhibitors with PI3K targeting compounds or the use of dual inhibitors may have a favorable therapeutic potential. Disclosures:Guenther:Novartis, Celgene: Consultancy, Research Funding. Gramatzki:Novartis, Celgene: Consultancy, Research Funding.

Single-Cell Pharmacodynamic Monitoring of S6 Ribosomal Protein in AML Blasts During Trials Combining Sirolimus and Intensive Chemotherapy: Target Inhibition Enhances Response

Abstract 230 Background:Mammalian target of rapamycin (mTOR) inhibitors enhance cytotoxic chemotherapy effects in primary acute leukemia samples in preclinical assays, prompting multi-center evaluation of regimens combining mTOR inhibitors plus induction chemotherapy in AML. As mTOR is frequently but not uniformly activated in primary AML samples, it is unclear which patients benefit from this targeted approach. Thus, we sought to monitor mTOR kinase activity during therapy to determine whether target activation and/or inhibition predicted clinical response. We previously reported the feasibility of real-time, pharmacodynamic monitoring of ribosomal S6 phosphorylation (pS6) in leukemic blasts using flow cytometry of fixed whole blood (Perl et al, ASH 2009, #413). mTOR directly regulates the p70S6 kinase and its phosphorylation of S6 at serines 235/6 is inhibited by rapamycin. Thus pS6 provides a surrogate marker of mTOR kinase activity. Fixing whole blood and/or marrow preserves phosphorylation states in the presence of administered signal transduction inhibitors, thus avoiding cell-processing effects. Here we update our experience monitoring pS6 in AML blasts by flow during clinical trials combining sirolimus and AML induction chemotherapy. Methods:Subjects had relapsed/refractory AML or untreated AML with unfavorable risk factors (e.g. therapy-related, prior MDS or MPN, or age >60 with non-favorable karyotype) and received oral sirolimus (12 mg on day 1, then 4 mg daily on days 2–9) plus MEC (mitoxantrone 8 mg/m2/day, etoposide 100 mg/m2/day, cytarabine 1 gm/m2/d on days 4–8) on one of two successive clinical trials. Clinical response was assessed at hematologic recovery or day 42 using IWG criteria (CR, CRp, PR vs. non-response). Pharmacodynamic samples were collected from blood or marrow at baseline, 2 hours post-sirolimus dose on days 1 and 4, and at trough on day 4 (prior to chemotherapy administration). Concurrent blood rapamycin concentration was measured by immunoassay or HPLC. Whole blood/marrow fixation was performed using published methods (Chow & Hedley, Cytometry A, 2005). Positive gates for pS6 were created by comparing blasts in ex vivo stimulated (phorbol ester/PMA) and inhibited (rapamycin) conditions and/or autofluorescence (FMO) controls. Results:27 subjects provided paired day 1 and day 4 flow samples and were evaluable for clinical response at the time of submission. Mean peak and trough rapamycin levels were 22.8 and 9.2 ng/ml, respectively, and did not differ among clinically responding and non-responding subjects. 17/27 (63%) subjects’ blasts had constitutive S6 phosphorylation at baseline. Consistent with prior reports, pS6 was heterogeneous and typically present in a subset of blasts. In these 17 subjects, we observed a median of 13% pS6+ blasts (mean 14, range 2–33). 14/17 showed a therapy-induced reduction in pS6+ blasts to a mean of 4.5% (median 2.5, range 0.4–22) on day 4. The remainder had either no change or increased pS6+ blasts. Comparing the percentage of pS6+ cells on day 4 to baseline, the median reduction in pS6 differed among clinically responding and non-responding subjects (72% and 43%, respectively). The clinical response rate was 9/17 (53%, 6 CR 3 PR) among subjects with baseline S6 phosphorylation and 4/10 (40%, 3 CR, 1 PR) in patients with no baseline S6 phosphorylation. Subjects with >50% reduction in pS6 blasts on day 4 were considered to be biochemically sensitive to rapamycin, while subjects with <50% reduction or increased pS6 were considered rapamycin-resistant. The response rate in rapamycin-sensitive patients was 8/12 (67%), while in resistant subjects was 1/5 (20%). 5/15 (33%) subjects who either lacked baseline pS6 or were rapamycin resistant responded to the regimen. Conclusions:Data from these ongoing trials suggest that sirolimus plus MEC preferentially benefits the subset of patients with demonstrable baseline mTOR activation. The greatest response is seen when mTOR is activated in leukemic cells at baseline and its function is potently inhibited during therapy. Future trials may benefit from enrichment for subjects with mTOR activation and/or rapamycin sensitivity assessment. These data provide in vivo evidence that mTOR inhibitors augment cytotoxic chemotherapy effect in AML and demonstrate the utility of fixed whole blood flow cytometry for real-time pharmacodynamic evaluation of novel signal transduction inhibitors. Disclosures:Off Label Use: Rapamycin. FDA approved for solid organ transplant. Investigational use for treatment of leukemia. Carroll:Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

Phase I and pharmacokinetic study of capecitabine and the oral mTOR inhibitor everolimus in patients with advanced solid malignancies

Summary Background Everolimus is an oral mTOR-inhibitor. Preclinical data show synergistic effects of mTOR inhibition in combination with 5-fluorouracil-based anticancer therapy. The combination of everolimus with capecitabine seems therefore an attractive new, orally available, treatment regimen. Patients and methods Safety, preliminary efficacy and pharmacokinetics of everolimus in combination with capecitabine were investigated in patients with advanced solid malignancies. Patients were treated with fixed dose everolimus 10 mg/day continuously, plus capecitabine bid for 14 days in three-weekly cycles. Dose escalation of capecitabine proceeded according to the standard 3 × 3 phase I design in four predefined dose levels (500–1,000 mg/m2 bid). Results In total, 18 patients were enrolled. Median (range) treatment duration with everolimus was 70 days (21–414). Capecitabine 1,000 mg/m2 bid combined with 10 mg/day everolimus was declared the maximum tolerated dose, at which level one patient developed dose-limiting toxicity (stomatitis grade 3). Drug-related adverse events were mostly grade ≤2 and included mainly fatigue (56%), stomatitis (50%), and hand-foot syndrome (33%). Partial response was documented in three patients, and four had stable disease. There was no pharmacokinetic interaction between everolimus and capecitabine. Conclusion Everolimus 10 mg/day continuously combined with capecitabine 1,000 mg/m2 bid for 14 days every 3 weeks is a patient-convenient, safe and tolerable oral treatment regimen. This is the first study to demonstrate feasibility of this combination at doses with proven single agent efficacy in a number of tumors. Prolonged clinical benefit was observed in an encouraging 39% of patients with advanced solid malignancies.

Highlights of This Issue

The PI3K/AKT/mTOR pathway is frequently activated in human cancers and mTOR is a central regulator of this pathway. To inhibit both mTORC1 and mTORC2 activity, Bhagwat and colleagues have discovered OSI-027, a selective, small molecule ATP competitive inhibitor that is mechanistically and functionally distinct from rapamycin.OSI-027 showed significant mechanistic inhibition of mTORC1 and mTORC2 and broad-spectrum tumor growth inhibition in several xenograft models including those harboring PTEN deficiencies/mutations and PIK3CA mutations. The breadth of these results indicate a global effect of mTOR signaling pathways and a potential to treat a variety of human cancers that may not be as responsive to rapalogs.Ovarian cancers commonly exhibit upregulation of the PI3K/mTOR signaling. Using a genetically engineered ovarian cancer mouse model (Pten deletion;KrasG12D),Kinross and colleagues show that PF-04691502, a PI3K and mTOR inhibitor in clinical development, potently inhibits PI3K/mTOR signaling, FDG-PET activity, and inhibits tumor growth in vivo. However, PF-04691502 exhibited only short-term benefit as a single agent.Long-term therapeutic benefit could be achieved through combined therapy of PF-04691502 with MEK inhibitor PD-0325901, which, via coordinated activation of apoptosis, led to tumor regression. This study identifies combination PF-04691502 and PD-0325901 therapy as a promising new approach for the treatment of ovarian cancers.Triple-negative breast cancers have the worst prognosis of any subset of breast cancer, at least in part because of a lack of viable therapeutic targets.Epidermal growth factor receptor (EGFR) is overexpressed in approximately 50% of triple-negative breast cancers, but attempts at targeting this receptor in clinical trials have, to date, produced disappointing results. Liu and colleagues show that mTOR inhibition in combination with EGFR inhibition leads to synergistic effects in triple-negative breast cancers in vitro and suppressed tumor growth in vivo. They are currently evaluating the combination of the EGFR inhibitor, lapatinib, and the mTOR inhibitor, everolimus, in patients with metastatic triplenegative breast cancer.Misexpression of microRNAs (miRNA) is widespread in human cancers, with aberrations including overexpression of oncogenic miRs and downregulation of tumor suppressor miRs (TSG-miR). Pramanik and colleagues developed a method of restituting expression of downregulated TSG-miRs in vivo using a lipid-based nanoparticle for systemic delivery of miRNA expression vectors (nanovector).Selecting two candidate TSG-miRs (miR-34a and the miR-143/145 cluster), the authors show the usefulness of the nanovector platform—one with potential broad applicability in systemic miRNA delivery to cancer cells—in both subcutaneous and orthotopic xenograft models of pancreatic cancer.

Combination of PI3K/mTOR Inhibitors: Antitumor Activity and Molecular Correlates

The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway is a major target for cancer therapy. As a strategy to induce the maximal inhibition of this pathway in cancer cells, we combined allosteric mTOR inhibitors (rapamycin and RAD001) with a dual PI3K/mTOR kinase inhibitor (PI-103). Both in vitro and in vivo, the combination exhibited more activity than single agents in human ovarian and prostate cancer cells that harbor alterations in the pathway. At the molecular level, combined inhibition of mTOR prevented the rebound activation of Akt that is seen after treatment with rapamycin and its analogues and caused more sustained inhibition of Akt phosphorylation. Furthermore, the combination strongly inhibited the expression of PI3K/Akt/mTOR downstream proteins. In particular, it showed greater activity than the single agents in inhibiting the phosphorylation of 4EBP1, both in vitro and in vivo, resulting in selective inhibition of CAP-dependent translation. A proteomic approach was used to confirm the identification of c-Myc as the key regulator for the reduction in downstream proteins affected by the combined inhibition of mTOR. In conclusion, the combination of a catalytic and an allosteric inhibitor of mTOR shows greater activity, without a concomitant increase in toxicity, than either drug alone, and this may have therapeutic implications for inhibiting this pathway in the clinical setting.

Evaluating rational non-cross-resistant combination therapy in advanced clear cell renal cell carcinoma: combined mTOR and AKT inhibitor therapy

Inhibition of the mammalian target of rapamycin (mTOR), a regulator of hypoxia inducible factor (HIF), is an established therapy for advanced renal cell cancer (RCC). Inhibition of mTOR results in compensatory AKT activation, a likely resistance mechanism. We evaluated whether addition of the Akt inhibitor perifosine to the mTOR inhibitor rapamycin would synergistically inhibit RCC. Select RCC cell lines were studied [786-O, A498 (VHL mutant), CAKI-1 (VHL wild type), and 769-P (VHL methylated)] with single agent and combination therapy. Growth inhibition was assessed by MTT and cell cycling by flow cytometry. Phospho-AKT (S473) and HIF-2α were assessed by Western blot. Total RNA was isolated from 786-O cells subjected to single agent and combination treatments. In these cells, genome-wide expression profiles were assessed, and real-time PCR was used to confirm a limited set of expression results. Three out of four cell lines (CAKI-1, 769-P, and 786-O) were sensitive to single-agent perifosine with 50% inhibitory concentrations ranging from 5 to 10μM. Perifosine blocked phosphorylation of AKT induced by rapamycin and inhibited HIF-2α expression in 786-O and CAKI-1. Combined treatment resulted in sub-additive growth inhibition. GeneChip analysis and pathway modeling revealed inhibition of the IL-8 pathway by these agents, concomitant with up-regulation of the KLF2 gene, a known suppressor of HIF1α. Perifosine is active in select RCC lines, abrogating the induction of AKT phosphorylation mediated by mTOR inhibition. Combined mTOR and AKT inhibition resulted in the modulation of pro-angiogenesis pathways, providing a basis for future investigations.

Utility of mTOR Inhibition in Hematologic Malignancies

The mammalian target of rapamycin (mTOR) is an intracellular serine/threonine kinase that exists as a downstream component of numerous signaling pathways. The activation of mTOR results in the production of proteins involved in cell metabolism, growth, proliferation, and angiogenesis. Aberrant activation of mTOR signaling has been identified in a number of cancers, and targeted inhibition of mTOR has been successful in achieving tumor responses, prolonging progression-free survival, and increasing overall survival in various oncologic patient populations. In particular, persistent activation of mTOR signaling has been identified in cell lines and patient samples with leukemias, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), and Waldenström's macroglobulinemia (WM). In vitro and preclinical studies using agents that inhibit mTOR signaling have demonstrated cytostatic and cytotoxic effects in these hematologic malignancies, suggesting that mTOR is a rational target for therapy in these disease states. In addition, the combination of mTOR inhibitors with traditional therapies may help to overcome the development of resistance and may improve response rates over those seen with established regimens through synergistic or additive effects. Inhibitors of mTOR signaling currently are being investigated in clinical trials of hematologic malignancies as single agents and as components of combination regimens. Thus far, promising results have been seen with the application of mTOR inhibitors as single agents in patients with relapsed or refractory leukemia, HL, NHL, MM, and WM.

Phase I study of temsirolimus (Tem) and pazopanib (Paz) in solid tumors with emphasis on renal cell carcinoma (RCC).

e15113 Background: Phase III trials have shown that either inhibition of VEGFR- (Paz) or mTOR-signaling (Tem) can improve outcomes in patients (pts) with advanced RCC. To investigate the hypothesis that simultaneous “vertical inhibition” of VEGFR and mTOR pathways is feasible and results in enhanced efficacy, we conducted a phase I trial of Tem plus Paz to determine the maximum tolerated dose (MTD), safety, and recommended phase II dose. Methods: Pts with advanced solid tumors (emphasis on RCC), PS 0-2, no prior mTOR inhibitor or Paz, and acceptable end-organ function were eligible. A typical 3 + 3 design was used starting at dose level (DL) 1 with de-escalation to DL -2 if DL 1 was not well tolerated. Dose limiting toxicity (DLT) was defined as attributable grade 3 (Gr3) or higher non-hematologic adverse events (AEs) in cycle 1. MTD was defined as the maximum dose level at which less than 2 pts experienced DLT. Cycle length was 28 days. Results: Eight pts were enrolled. Median age was 57 years; 38% had RCC. One pt at DL1 was not evaluable due to treatment deviation in cycle 1 (see Table). At DL1, 2 pts had 4 DLTs (Gr3 anorexia, fatigue, hyponatremia, hypophosphatemia). After reduction to DL-2, 1 of 3 pts had DLT (Gr3 fatigue). The 1st pt in DL-2 expansion had Gr3 hypophosphatemia. Attributable Gr3+ AEs: hypophosphatemia (2 pts); neutropenia (2 pts); anorexia, hyperglycemia, hypokalemia, hyponatremia, mucositis and proteinuria (1 pt each). Gr2 nausea, elevated alk phos and rash were seen in at least 2 pts. Four of 6 pts evaluable for response had tumor reduction (not meeting RECIST response) including 2 with RCC and 1 with adrenal cancer. Conclusions: Tem plus Paz was not feasible in this population due to Gr3 fatigue and electrolyte disturbances. This and other trials of combined mTOR inhibitor and VEGFR-TKI therapy confirm that although active, this strategy is associated with unacceptable toxicity. Patient Dose Level Tem (mg IV weekly) Paz (mg/day) DLTs 1 1 15 400 None 2 1 15 400 Anorexia 3 1 15 400 Not evaluable 4 1 15 400 Fatigue, hyponatremia,hypophosphatemia 5 -2 10 200 Fatigue 6 -2 10 200 None 7 -2 10 200 None 8 -2 10 200 Hypophosphatemia

Cixutumumab combined with temsirolimus in patients with refractory Ewing’s sarcoma.

10031 Background: In this multicenter NCI CTEP-sponsored trial [funded by R21CA13763301A1 (AN), U01CA62461 (RK), and U01CA62487 (PL)], we combined the mTOR inhibitor temsirolimus with cixutumumab, a fully humanized IgG1 monoclonal antibody directed at insulin growth factor-1 receptor (IGF-1R), on the basis of preclinical data suggesting that the combination could putatively overcome the resistance observed when the individual agents were used as monotherapy in Ewing’s sarcoma. Methods: Patients received cixutumumab 6 mg/kg IV weekly and temsirolimus 25 mg IV weekly (four week cycles) with restaging after eight weeks. Results: Seventeen Ewing’s sarcoma patients were enrolled. Ten patients (59%) were men and the median age was 27.5 years (range, 14 to 41 years). The median number of prior therapies was 6. Six patients had previously received an IGFR inhibitor and two had previously received temsirolimus. The most frequent drug-related toxicities were thrombocytopenia (82%), mucositis (76%), hypertriglyceridemia (71%), hypercholesterolemia (65%), and hyperglycemia (65%). Only 3/17 patients experienced a dose-limiting toxicity, which was grade 3 mucositis. Patients with well-controlled diabetes were not excluded. With early referral to an endocrinologist, hyperglycemia was controlled in one patient with metformin and in another patient with metformin and insulin. One of 17 (6%) patients, who had previously developed resistance to a different IGFR inhibitor, achieved a complete response (CR) of 14+ months in this study.Tumor regression was seen in 4/17 (24%) patients, who had 50%, 23%, 23%, and 27% regressions lasting 6+, 10+, 8 and 15 months, respectively. Conclusions: The combination ofcixutumumab and temsirolimus was well tolerated. Prolonged objective tumor reduction (>6 months) and one CR were seen in 29% of heavily pretreated Ewing’s sarcoma patients, suggesting that anti-IGF1R inhibitor and mTOR inhibitor combinations merit further study.

Abstract 5368: Combination therapy with mTOR and PI3 kinase inhibitors is broadly synergistic in a wide variety of endometrial cancer cells

Abstract Background- Dysregulation of mammalian target of rapamycin (mTOR) signaling has been found in many human tumors, including endometrial cancer, and mTOR inhibitors have been utilized in clinical trials as targeted therapies. Although the mTORC1 inhibitor temsirolimus initially exhibited broad antitumor activity in preclinical models and Phase II/III clinical trials, many patients do not respond to therapy and relapse is common. Several resistance mechanisms have been hypothesized, including an increase in AKT phosphorylation after treatment with temsirolimus and subsequent activation of AKT-dependent pro-survival pathways. Hypothesis and Approach – In order to identify a viable treatment alternative that overcomes temsirolimus-induced AKT phosphorylation in endometrial cancer, we assessed the therapeutic benefit of dual mTOR/PI3K inhibition using a panel of endometrial cancer cell lines. Results – Of the six drugs tested, NVP-BEZ235 (dual PI3K/mTOR inhibitor) and ZSTK474 (PI3K inhibitor) successfully blocked temsirolimus-induced AKT phosphorylation. Proliferation assays revealed that in a majority of cell lines tested, BEZ235 or ZSTK474 combined with temsirolimus (1 nM) synergistically decreased cell growth compared to cells treated with any of the agents alone. Our data suggest temsirolimus and BEZ235 inhibit different components of the AKT/mTOR signaling pathway to accomplish synergistic pathway inhibition. Specifically, temsirolimus partially blocked phosphorylation of mTOR and completely abolished phosphorylation of p70S6K and its downstream substrate, rS6, at low (1 nM) concentrations. In contrast, BEZ235 inhibited 4E-BP1 phosphorylation, another substrate for mTOR, whereas temsirolimus had no effect on 4E-BP1 phosphorylation. Furthermore, G0/G1 cell cycle arrest and up-regulation of p27 was observed after combination treatment of temsirolimus and BEZ235. Strikingly, cell death occurred through autophagy and not apoptosis. Finally, molecular profiling revealed that elevated basal levels of AKT phosphorylation and a loss of the tumor suppressor PTEN correlated with sensitivity to combinatorial treatment. Conclusions – While previous studies have established that endometrial cancer cells are responsive to drugs that inhibit PI3K or mTOR, we observed a novel synergistic effect when the specific inhibitors were combined. In particular, the mechanism of synergy is through blocking two arms of the PI3K/mTOR signaling pathways: p70S6K and rS6 by temsirolimus with high efficacy and AKT and 4E-BP1 by BEZ235. Combined inhibition of mTOR and PI3K is necessary for therapeutic efficacy to abrogate the increased signaling through AKT that occurs with mTOR inhibition alone. Loss of PTEN and baseline AKT phosphorylation appeared to be the most sensitive predictors for response to the PI3K/mTOR inhibitor combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5368. doi:10.1158/1538-7445.AM2011-5368

Abstract 4500: Combined mTOR inhibition and autophagy inhibition: Phase I trial of temsirolimus and hydroxchloroquine in patients with advanced solid tumors

Abstract Background: Therapy-induced autophagy may be a key resistance mechanism that explains the low rates of clinical benefit observed in trials of mTOR inhibitors in multiple cancers. For example, the mTOR inhibitor temsirolimus produced a 0% stable disease rate in a prior phase II trial in patients with metastatic melanoma. Hydroxychloroquine (HCQ) is an autophagy inhibitor that augments the antitumor efficacy of a number of anticancer therapies including mTOR inhibitors in preclinical models. To determine the safety and pharmacodynamic (PD) effects of combining an mTOR inhibitor with an autophagy inhibitor, we report the results of a phase I clinical trial of temsirolimus and HCQ in patients with advanced solid tumors. Methods: We conducted a traditional 3+3 phase I dose escalation clinical trial in which patients were treated with 1 week single agent temsirolimus 25 mg IV weekly followed by combined weekly temsirolimus with increasing doses of daily continuous HCQ. Patients with advanced solid tumors with any number of prior therapies were eligible. The primary objective was to determine the maximal tolerated dose (MTD) of HCQ in this combination. Secondary objectives included toxicity rate, response rate, measurement of therapy-induced accumulation of autophagic vesicles (AV) in serial peripheral blood mononuclear cells (PBMC) and tumor tissue, and pharmacokinetic (PK) analysis of temsirolimus, sirolimus and HCQ to explore drug interactions and establish a PK-PD relationship. A 12 patient expansion at the MTD or HCQ 600 mg bid (final planned dose level) in patients with metastatic melanoma with serial FDG-PET scans is planned. Results: 23 patients were enrolled with 14 evaluable for response and toxicity. The median number of prior treatments was 4. HCQ was successfully dose escalated from 200 mg daily to 600 mg bid in 4 cohorts. There was one dose limiting toxicity (DLT) at 200 mg HCQ, grade 4 thrombocytopenia with bleeding, that lead to dose expansion. The 800 mg dose cohort was also expanded due to a death from streptococcal pneumonia in month 3 of treatment. No additional bleeding, infections, or other DLTs were observed. Anorexia, nausea, and fatigue were common grade 2 toxicities. Stable disease was achieved in 10/14 evaluable patients, including 4/5 patients with metastatic melanoma. After 6 weeks of treatment, a significant accumulation of AV was observed in PBMC of patients treated with this combination compared to pretreatment samples providing PD evidence of consistent autophagy inhibition. Conclusions: The combination of temsirolimus and HCQ demonstrated significant clinical activity, and a manageable safety profile in a highly treatment-refractory patient population. PD evidence of autophagy inhibition was observed. The recommended phase II dose, PK-PD analysis, response rate, and FDG-PET results in the melanoma expansion will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4500. doi:10.1158/1538-7445.AM2011-4500

Temsirolimus and rituximab in patients with relapsed or refractory mantle cell lymphoma: a phase 2 study

Temsirolimus is a mammalian target of rapamycin (mTOR) inhibitor with single-agent antitumour activity in patients with mantle cell lymphoma. We therefore tested its efficacy and toxicity in combination with rituximab (an antiCD20 antibody) in patients with relapsed or refractory mantle cell lymphoma. In a phase 2 study, patients (aged ≥18 years) at 35 centres in the USA were given temsirolimus 25 mg/week, and rituximab 375 mg/m(2) per week for 4 weeks during the first cycle and thereafter a single dose of rituximab every other 28-day cycle. Both drugs were administered intravenously. Responding patients after six cycles could continue treatment for a total of 12 cycles, and were then observed without additional maintenance treatment. The primary endpoint was the proportion of patients with either rituximab-sensitive or rituximab-refractory disease who had at least a partial response. The analyses were done on all patients who were treated. The study was registered with ClinicalTrials.gov, number NCT00109967. 71 patients with mantle cell lymphoma were enrolled and 69 were assessable and were included in the final analysis. The overall response rate (ORR) was 59% (41 of 69 patients)-13 (19%) patients had complete responses and 28 (41%) had partial responses. The ORR was 63% (30 of 48; 95% CI 47-76) for rituximab-sensitive patients, and 52% (11 of 21; 30-74) for rituximab-refractory patients. The most common treatment-related grade 3 or 4 adverse events in rituximab-sensitive and rituximab-refractory patients were thrombocytopenia (eight [17%] and eight [38%], respectively), neutropenia (ten [21%] and five [24%], respectively), fatigue (eight [17%] and two [10%], respectively), leucopenia (six [13%] and three [14%], respectively), pneumonia (five [10%] and two [10%], respectively), lymphopenia (five [10%] and two [10%], respectively), pneumonitis (four [8%] and none, respectively), oedema (four [8%] and none, respectively), dyspnoea (three [6%] and two [10%], respectively), and hypertriglyceridaemia (three [6%] and two [10%], respectively). mTOR inhibitors in combination with rituximab could have a role in the treatment of patients with relapsed and refractory mantle cell lymphoma. National Institutes of Health and the Predolin Foundation.

The inhibition of MAPK potentiates the anti-angiogenic efficacy of mTOR inhibitors

The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.

Future directions of mammalian target of rapamycin (mTOR) inhibitor therapy in renal cell carcinoma.

With an explosion of available treatments for metastatic renal cell carcinoma (mRCC) in recent years, it is important to recognize that approved targeted therapies fall broadly into only two mechanistic categories. The first category, vascular endothelial growth factor (VEGF)-directed therapies, includes sunitinib, pazopanib, sorafenib and bevacizumab. The second category includes inhibitors of the mammalian target of rapamycin (mTOR), namely everolimus and temsirolimus. A pivotal trial of everolimus supports use of the agent in patients with mRCC refractory to VEGF- tyrosine kinase inhibitors (TKI) therapy, while pivotal data for temsirolimus supports use in poor-prognosis patients as first-line therapy. Multiple reviews exist to delineate the laboratory and clinical development of mTOR inhibitors. This paper will outline the future applications of these therapies. It will explore ongoing trials evaluating combinations of mTOR inhibitors with other targeted therapies, along with sequencing strategies and biomarker discovery efforts. The application of mTOR inhibitors in unique populations is also described.