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PDF file - 210K, RTK antibody array analysis in SYO-1 cells treated with vehicle or 1 nM rapamycin for 6 hours; RTK array performed upon SYO-1 cells after treatment with vehicle or rapamycin

PDF file - 220K, Rapamycin decreases IRS-1 phosphorylation and Grb10 levels in HS-SY-II cells, but not SYO-1 cells; western blots evaluating changes in IRS-1 phosphorylation and Grb10 levels with rapamycin treatment

PDF file - 73K, Viability assays quantify the percent decrease in viability achieved with rapamycin and R1507 alone or in combination in sarcoma cell lines; viability assays performed in the 12 cell line sarcoma panel

PDF file - 64K

Peptide nucleic acid (PNA) oligomers are DNA mimics, which are capable of binding gene sequences 1000-fold more avidly than complementary native DNA by strand invasion and effectively obstruct transcription. Irreversibly obstructing the transcription or replication of a gene sequence, such as BRAFV600E, offers a potential route to specifically target the cancer cell itself. We have employed PNA oligomers to target BRAFV600E in a sequence-specific complementary manner. These PNAs have been modified by appending configurationally stabilizing cationic peptides in order to improve their cellular delivery and target avidity. Our results indicate that exposure of the melanoma cell lines to a modified PNA-peptide conjugate complementary to BRAFV600E mutation sequence results in a concentration-dependent and time-dependent inhibition of cell growth that is specific for the BRAFV600E-mutant melanoma cell lines with inhibition of mRNA and protein expression. Xenograft mouse trials show increased tumor growth delay and necrosis with the BRAFV600E-complementary PNA-peptide conjugates as compared with the saline and scrambled PNA sequence controls. Similarly, quantitative measurement shows a 2.5-fold decrease in Ki67 and a 3-fold increase in terminal deoxynucleotidyl transferase dUTP nick end labeling expression with this approach. PNA-delivery peptide conjugates represent a novel way to target BRAFV600E and represent a new approach in targeting selective oncogenes that induce tumor growth.

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that exists in two complexes (mTORC1 and mTORC2) and integrates extracellular and intracellular signals to act as a master regulator of cell growth, survival, and metabolism. The PI3K/AKT/mTOR prosurvival pathway is often dysregulated in multiple sarcoma subtypes. First-generation allosteric inhibitors of mTORC1 (rapalogues) have been extensively tested with great preclinical promise, but have had limited clinical utility. Here, we report that MLN0128, a second-generation, ATP-competitive, pan-mTOR kinase inhibitor, acts on both mTORC1 and mTORC2 and has potent in vitro and in vivo antitumor activity in multiple sarcoma subtypes. In vitro, MLN0128 inhibits mTORC1/2 targets in a concentration-dependent fashion and shows striking antiproliferative effect in rhabdomyosarcoma (RMS), Ewing sarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, osteosarcoma, and liposarcoma. Unlike rapamycin, MLN0128 inhibits phosphorylation of 4EBP1 and NDRG1 as well as prevents the reactivation of pAKT that occurs via negative feedback release with mTORC1 inhibition alone. In xenograft models, MLN0128 treatment results in suppression of tumor growth with two dosing schedules (1 mg/kg daily and 3 mg/kg b.i.d. t.i.w.). At the 3 mg/kg dosing schedule, MLN0128 treatment results in significantly better tumor growth suppression than rapamycin in RMS and Ewing sarcoma models. In addition, MLN0128 induces apoptosis in models of RMS both in vitro and in vivo. Results from our study strongly suggest that MLN0128 treatment should be explored further as potential therapy for sarcoma. Mol Cancer Ther; 14(2); 395–406. ©2014 AACR.

Summary Background Preclinical studies have shown synergistic antitumour activity by inhibition of insulin-like growth factor-1 receptor (IGF-1R) and mTOR. The expression of IGF-1R seems to be crucial for this effect. We investigated the safety and efficacy of the combination of the IGF-1R antibody cixutumumab and the mTOR inhibitor temsirolimus in patients with chemotherapy-refractory bone and soft-tissue sarcomas according to IGF-1R expression by immunohistochemistry. Methods We undertook a multicentre, open-label, phase 2 study in 19 cancer centres in the USA. Patients aged at least 16 years with a histologically confirmed diagnosis of bone or soft-tissue sarcoma were allocated on the basis of IGF-1R expression by immunohistochemistry to one of three treatment groups: IGF-1R-positive soft-tissue sarcoma (group A), IGF-1R-positive bone sarcomas (group B), or IGF-1R-negative bone and soft-tissue sarcoma (group C). Patients received weekly treatment with cixutumumab (6 mg/kg, intravenous) and temsirolimus (25 mg, intravenous flat dose) in 6-week cycles. A Simon optimal two-stage design was used for every arm. The primary endpoint was progression-free survival (PFS) at 12 weeks by intention-to-treat analysis in the first 54 patients assigned to every treatment arm. Although patients still remain on treatment, this trial has completed enrolment and this represents the final analysis. This study is registered with ClinicalTrials.gov, number NCT01016015. Findings Between Nov 18, 2009, and April 11, 2012, 388 patients were screened for IGF-1R expression and 54 were assigned to each arm. 17 of 54 patients in the IGF-1R-positive soft-tissue sarcoma group (31%; one-sided 95% CI lower bound 21%; two-sided 90% CI 21–43), 19 of 54 in IGF-1R-positive bone sarcoma group (35%; one-sided 95% CI lower bound 24%; two-sided 90% CI 24–47), and 21 of 54 in the IGF-1R-negative group (39%, one-sided 95% CI lower bound 28%; two-sided 90% CI 28–51) were progression free at 12 weeks. On April 6, 2011, the protocol was amended to include three additional patients in the IGF-1R-positive soft-tissue sarcoma group (total of 57 patients) and nine more in the IGF-1R-negative group (total of 63 patients). There were 2546 adverse events reported during the study, 214 (8%) of which were grade 3–4. The most common grade 3–4 toxicities in the 174 treated patients were anaemia in 16 (9%) patients, hyperglycaemia in 18 (10%), hypophosphataemia in 16 (9%), lymphopenia in 25 (14%), oral mucositis in 19 (11%), and thrombocytopenia in 19 (11%). Interpretation The combination of cixutumumab and temsirolimus shows clinical activity in patients with sarcoma and forms a basis for future trials. However, IGF-1R expression by immunohistochemistry is not predictive of clinical outcome after treatment with this combination. Funding National Cancer Institute and CycleforSurvival Fund, Memorial Sloan-Kettering Cancer Center.

Akt activation by the IGF-1 receptor (IGF-1R) has been posited to be a mechanism of intrinsic resistance to mTORC1 inhibitors (rapalogues) for sarcomas. Here we show that rapamycin-induced phosphorylation of Akt can occur in an IGF-1R–independent manner. Analysis of synovial sarcoma cell lines showed that either IGF-1R or the PDGF receptor alpha (PDGFRA) can mediate intrinsic resistance to rapamycin. Repressing expression of PDGFRA or inhibiting its kinase activity in synovial sarcoma cells blocked rapamycin-induced phosphorylation of Akt and decreased tumor cell viability. Expression profiling of clinical tumor samples revealed that PDGFRA was the most highly expressed kinase gene among several sarcoma disease subtypes, suggesting that PDGFRA may be uniquely significant for synovial sarcomas. Tumor biopsy analyses from a synovial sarcoma patient treated with the mTORC1 inhibitor everolimus and PDGFRA inhibitor imatinib mesylate confirmed that this drug combination can impact both mTORC1 and Akt signals in vivo. Together, our findings define mechanistic variations in the intrinsic resistance of synovial sarcomas to rapamycin and suggest therapeutic strategies to address them. Cancer Res; 72(17); 4515–25. ©2012 AACR.

Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.

Our aim is to target tumor cells specifically by directly suppressing their oncogenes with peptide nucleic acid (PNA) oligonucleotide analogues. PNA oligonucleotides bind to DNA over 1000-fold more avidly than its native complement, are completely resistant to intra and extra cellular enzymatic degradation, show no nonspecific toxicity at therapeutic levels, and when conjugated to delivery peptides can be made nuclear and cell membrane permeable. We have employed these PNA oligomers to target BRAFV600E which is prevalent in cutaneous melanoma in a sequence-specific complementary manner so as to disrupt transcription by strand invasion. For these studies, we have developed a novel delivery peptide conjugated to PNA modified to increase both cellular delivery and PNA stability towards its target. Our results indicate that exposure of the melanoma cell lines to a modified PNA-peptide conjugate selective for BRAFV600E results in a concentration-dependent inhibition of cell growth that is specific for the BRAFV600E mutant melanoma cell lines with an IC50 range of 250 to 500 nM. Moreover, there is no inhibition of BRAFWT cell growth at these concentrations. This is associated with suppression of BRAFV600E protein over time with no effect on BRAFWT protein levels. Furthermore, BRAFV600E protein expression was suppressed for up to 6 days following initial exposure proving the durability of this type of inhibition. Exposure to this modified PNA-peptide down-regulates BRAFV600E mRNA transcription exclusively in the mutant cell lines. Live cell imaging of BRAFV600E mutant cells confirms localization of fluorescein-labeled PNA-delivery peptide specific to BRAFV600E to the nucleus within 3 hours of treatment. Our results indicate that these PNA-peptide derivatives could represent a novel and promising new therapy for patients with BRAFV600E mutant melanoma, and this technology could be applied to a multitude of other cancers either with specific translocations or mutations differing from wild-type cells even by only a single base pair. Citation Format: Jeffrey H. Rothman, Oliver Surriga, Shyamprasad D. Vasudeva, Grazia Ambrosini, Ouathek Ouerfelli, Gary K. Schwartz. PNA as novel targeted cancer therapy for BRAFV600E mutant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2926. doi:10.1158/1538-7445.AM2014-2926

KPT-330 is a small molecule Selective Inhibitor of Nuclear Export (SINE) that irreversibly block the major nuclear export protein CRM1 (exportin 1, XPO1). The Chromosome Region Maintenance 1 (CRM1) protein is responsible for the nuclear export of snRNAs and of proteins bearing a leucine-rich nuclear export signal (NES). This class of drugs is expected to induce forced nuclear accumulation of tumor suppressor proteins (TSPs) and is believed to initiate a ‘genome survey’ leading to the death of cancer cells with normal cells undergoing transient, reversible cell cycle arrest. Our effort was to investigate the effects of KPT-330 in sarcomas and the mechanism by which it exerts the effect of tumor suppression. Our data clearly shows KPT-330 as active against a broad panel of human bone and soft tissue sarcoma cell lines at low nanomolar concentrations. The effect was mainly due to the induction of apoptosis. Target specificity of the drug was confirmed by the decrease in CRM1 protein level. An induction of p53 and p21 protein levels was observed. However, the effect of apoptosis or inhibition in proliferation seems to be independent of p53 status of the cell line. The effect of KPT-330 was further tested in xenograft models using malignant peripheral nerve sheath (MPNST) and de-differentiated liposarcoma (LS141) cell lines. Tumor suppression was observed in both the sarcoma models and its target specificity was confirmed in the xenografts. KPT-330 may represent a new drug for the treatment of bone and soft tissue sarcoma. Citation Format: Jayasree S. Nair, William Tap, Shyamprasad Deraje Vasudeva, Jason K. Sicklick, Michael Kauffman, Sharon Shacham, Gary K. Schwartz. KPT-330, a selective small molecule inhibitor of nuclear export, is active in bone and soft tissue sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5210. doi:10.1158/1538-7445.AM2013-5210

Adenoid cystic carcinoma (ACC) is a rare variant of adenocarcinoma that most commonly arises in the salivary glands. Rarely, ACC can originate at other sites, including the lung and breast. c-myb overexpression has been identified in over 70% of all ACCs, suggesting that it is a critical driver of oncogenesis for this disease. Recently, a translocation between chromosomes 6 and 9 has been described in ACC which creates a fusion gene between the transcription factors c-myb and NFIB. The C-terminal truncation of the c-myb gene in the t(6;9) translocation leads to loss of negative regulatory microRNA binding sites and hence elevated c-myb protein levels. Full length c-myb is also reportedly overexpessed in fusion negative tumors presumably via alternate mechanisms. Based upon previous reports that the phosphatidylinositol-3 phosphate kinase (PI3K)/Akt pathway is activated in ACC tumors as well as data from other tumor models that this pathway is critical for maintaining c-myb protein stability, we hypothesized that inhibiting the PI3K/Akt pathway would be a novel therapeutic strategy for targeting c-myb in ACC tumors. Given the lack of validated ACC tumor models, we tested this hypothesis by transiently transfecting hemagglutinin (HA) tagged full length c-myb or the MYB-NFIB fusion gene in the breast cancer cell line MDA-468. The impact of three PI3K/Akt pathway inhibitors was tested: the PI3K/mTOR inhibitor LY294002, the mTOR inhibitor rapamycin, and the Akt inhibitor MK-2206 (Merck)(all drugs were obtained commercially). While all three drugs downregulated full-length c-myb, Akt inhibition with MK-2206 led to the greatest degree of c-myb protein reduction. For the c-myb/NFIB fusion, again MK-2206 was the most effective for downregulating c-myb/NFIB levels. The impact of MK-2206 upon c-myb/NFIB levels was dose dependent and correlated to loss of Akt phosphorylation at serine 473. Akt inhibition with MK-2206 also resulted in increased c-myb/NFIB protein ubiquitination, supporting the hypothesis that Akt inhibition may lead to ubiquitin mediated degradation of the fusion protein. To confirm the importance of PI3K/Akt signaling for maintaining c-myb/NFIB stability, we utilized an RNA interference approach to downregulate expression of p110α, the catalytic subunit of PI3K, in cells expressing c-myb/NFIB. Transfection of the p110α siRNA downregulated the target gene, decreased Akt phosphorylation, and lowered c-myb/NFIB levels relative to cells treated with an siRNA control. Taken together, these results argue that Akt is critical for maintaining stability of both overexpressed full length c-myb and the MYB-NFIB fusion gene product. This study proposes Akt as a rational therapeutic target for ACC, and suggests that c-myb overexpression may be a potential biomarker of response to Akt inhibition. 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 638. doi:10.1158/1538-7445.AM2011-638