Sabita Sankar

REDD1, an inhibitor of mTOR signalling, is regulated by the CUL4A–DDB1 ubiquitin ligase

The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxia‐inducible factor‐1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post‐translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin‐mediated degradation mediated by the CUL4A–DDB1–ROC1–β‐TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3β. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.

Discovery and SAR exploration of a novel series of imidazo[4,5-b]pyrazin-2-ones as potent and selective mTOR kinase inhibitors.

We report here the discovery of a novel series of selective mTOR kinase inhibitors. A series of imidazo[4,5-b]pyrazin-2-ones, represented by screening hit 1, was developed into lead compounds with excellent mTOR potency and exquisite kinase selectivity. Potent compounds from this series show >1000-fold selectivity over the related PI3Kα lipid kinase. Further, compounds such as 2 achieve mTOR pathway inhibition, blocking both mTORC1 and mTORC2 signaling, in PC3 cancer cells as measured by inhibition of pS6 and pAkt (S473).

CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization.

mTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K–AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogues, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematologic and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematologic cancer cell lines. Correlative analysis revealed that IRF4 expression level associates with resistance, whereas mTOR pathway activation seems to associate with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223–treated tumors suggests that the observed antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clinical trials. Mol Cancer Ther; 14(6); 1295–305. ©2015 AACR.

Optimization of a Series of Triazole Containing Mammalian Target of Rapamycin (mTOR) Kinase Inhibitors and the Discovery of CC-115

We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity, and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC-115 for clinical development.

Use of core modification in the discovery of CC214-2, an orally available, selective inhibitor of mTOR kinase

We report here the discovery of a novel series of selective mTOR kinase inhibitors and the identification of CC214-2, a compound with demonstrated anti-tumor activity upon oral dosing in a PC3 prostate cancer xenograft model. A series of 4,6-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were discovered through a core modification of our original compound series. Analogs from this series have excellent mTOR potency and maintain selectivity over the related PI3Kα lipid kinase. Compounds such as CC214-2 were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC3 cancer cells, in vitro and in vivo.

CC-4047 promotes Th1 cell differentiation and reprograms polarized human Th2 cells by enhancing transcription factor T-bet

The IMiDs immunomodulatory drugs are an expanding family of compounds under investigation in a broad range of diseases because they exhibit immunomodulatory and anti-tumorigenic properties. Although the molecular targets remain unidentified, the broad activity of select IMiDs immunomodulatory drugs on cell signaling pathways and transcription regulation has been partly described. One characteristic of these compounds is their ability to act as a co-stimulus of TCR ligation leading to increased IL-2, TNF-alpha and IFN-gamma expression indicative of a Th1 phenotype. Because clinical evidence for this response has been observed in thalidomide and lenalidomide treated patients, we investigated the effect of CC-4047 on T cell activation and differentiation at the molecular level. We used primary human CD4(+) T cells as a model and found that CC-4047 enhances the expression of transcription factor T-bet in both naive and pre-polarized Th2 cells. This modulation leads to upregulation of Th1 markers and cytokine production. By increasing the expression of T-bet, CC-4047 promotes the differentiation of naive T-cells to Th1 as well as effectively reverting Th2 cells into Th1-like effector cells in vitro. These findings elucidate a novel mechanism of action of CC-4047 on T cell differentiation, suggesting that certain IMiDs immunomodulatory drugs may have expanded clinical application in treating both allergic diseases and certain T cell lymphomas where a predominant Th2 phenotype is displayed.

Discovery of Mammalian Target of Rapamycin (mTOR) Kinase Inhibitor CC-223

We report here the synthesis and structure-activity relationship (SAR) of a novel series of mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were optimized for in vivo efficacy. These efforts resulted in the identification of compounds with excellent mTOR kinase inhibitory potency, with exquisite kinase selectivity over the related lipid kinase PI3K. The improved PK properties of this series allowed for exploration of in vivo efficacy and ultimately the selection of CC-223 for clinical development.

CC-115, a dual inhibitor of mTOR Kinase and DNA-PK, blocks DNA damage repair pathways and selectively inhibits ATM-deficient cell growth in vitro

CC-115, a selective dual inhibitor of the mammalian target of rapamycin (mTOR) kinase and DNA-dependent protein kinase (DNA-PK), is undergoing Phase 1 clinical studies. Here we report the characterization of DNA-PK inhibitory activity of CC-115 in cancer cell lines. CC-115 inhibits auto-phosphorylation of the catalytic subunit of DNA-PK (DNA-PKcs) at the S2056 site (pDNA-PK S2056), leading to blockade of DNA-PK-mediated non-homologous end joining (NHEJ). CC-115 also indirectly reduces the phosphorylation of ataxia-telangiectasia mutated kinase (ATM) at S1981 and its substrates as well as homologous recombination (HR). The mTOR kinase and DNA-PK inhibitory activity of CC-115 leads to not only potent anti-tumor activity against a large panel of hematopoietic and solid cancer cell lines but also strong induction of apoptosis in a subset of cancer lines. Mechanistically, CC-115 prevents NHEJ by inhibiting the dissociation of DNA-PKcs, X-ray repair cross-complementing protein 4 (XRCC4), and DNA ligase IV from DNA ends. CC-115 inhibits colony formation of ATM-deficient cells more potently than ATM-proficient cells, indicating that inhibition of DNA-PK is synthetically lethal with the loss of functional ATM. In conclusion, CC-115 inhibits both mTOR signaling and NHEJ and HR by direct inhibition of DNA-PK. The mechanistic data not only provide selection of potential pharmacodynamic (PD) markers but also support CC-115 clinical development in patients with ATM-deficient tumors.

Abstract A165: Antitumor activity of mTOR kinase inhibitor CC-223 in a mouse model of prostate cancer.

CC-223 is an orally active selective mammalian target of rapamycin (mTOR) kinase inhibitor which is currently in clinical development. In biochemical assays CC-223 was highly selective to mTOR over 249 kinases. In cellular assays CC-223 inhibited both mTORC1 and mTORC2 as evidenced by inhibition of pS6RP(Ser 235/236) and pAkt(Ser 473). The current study was aimed to determine the pharmacokinetic/pharmacodynamic (PK/PD) relationship, antitumor activity, and mechanism of action of CC-223 in mice with PC3 human prostate cancer tumors. In PK/PD studies with PC3 tumor-bearing mice, CC-223 exhibited a dose-dependent inhibition mTOR pathway biomarkers, pS6RP, and pAkt, which correlated with plasma exposure. Antitumor activity of CC-223 was tested in mice with PC3 tumors of approximately 100-150 mm3 in size. CC-223 consistently exhibited dose-dependent tumor growth inhibition. The minimum dose required to obtain >65% tumor volume reduction compared with vehicle control was 5 mg/kg BID. In addition to showing antitumor activity in smaller tumors, CC-223 also caused the regression or growth delay of larger PC3 tumors. At 10 mg/kg BID, CC-223 caused the regression of tumors, whereas 5 and 1 mg/kg BID slowed tumor growth compared to the vehicle control. The mechanism of action of CC-223 on PC3 tumors was characterized by analyzing tumors for functional markers (proliferation, apoptosis, and blood vessel markers). The number of proliferating cells in CC-223-treated tumors was significantly reduced compared to vehicle control. A significant increase in the number of apoptotic cells and a significant decrease in blood vessel density was observed in CC-223-treated tumors. In conclusion, treatment with the mTOR kinase inhibitor CC-223 significantly inhibited PC3 prostate tumor growth. Significant inhibition of mTOR pathway markers pS6RP and pAkt in CC-223-treated tumors suggests that the observed antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. Maintenance of >80% and >60% biomarker inhibition for pS6 and pAkt, respectively, and plasma levels greater than 0.2 μM through 8 hours twice daily confers good antitumor efficacy in PC3 tumors. Further immunohistochemical data demonstrated that the antitumor activity of CC-223 was not only due to the inhibition of tumor cell proliferation but also due to the increase in apoptosis and antiangiogenic activity of CC-223. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A165. Citation Format: Rama Krishna Narla, Sophie Peng, Jim Gamez, Jason Katz, Julius Apuy, Mehran Moghaddam, Kimberly E. Fultz, Sabita Sankar, Deborah S. Mortensen, Heather K. Raymon. Antitumor activity of mTOR kinase inhibitor CC-223 in a mouse model of prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A165.

Abstract A208: Mutation detection in FFPE and plasma circulating DNA with a focused ALK-EGFR-KRAS Next-Generation Sequencing panel.

Background: Mutations in ALK, EGFR and KRAS have well-established or anticipated clinical utility in predicting response to targeted therapies either approved or in development for patients with non-small cell lung cancer (NSCLC). Here we report results from a proof-of-concept study using a custom Ion AmpliSeq Next-Generation Sequencing (NGS) panel designed to sequence a range of clinically relevant exons in ALK, EGFR and KRAS genes in formalin fixed paraffin-embedded (FFPE) DNA and plasma circulating DNA. Methods: The cumulative 2.1kb regions of interest (ROI) include exons 20-25 of ALK, exons 18-22 of EGFR, and exons 2-4 of KRAS, as well as the first 3 bp of the intron at the intron-exon boundaries. The custom panel was designed by Ion AmpliSeq Designer. Sequencing data were analyzed with Torrent Suite 3.4 and MolecularMDs proprietary analysis pipeline. Results: The study demonstrated the robustness of the custom NGS assay with at least 90% of reads-on-target, an average coverage of above 5000x, and no less than 81% uniformity in the reads. In addition, the minimum coverage for each ROI in all the samples tested was no less than 1000x. The limit of detection (LOD) of the NGS assay, as determined using dilutions of cell line DNA standards, was 0.7% for single base substitution (SBS) mutations, and 1.2% for indels. In addition, the assay was able to quantify mutations with frequencies as low as ∼1% in plasma circulating DNA. The assay detected 4 KRAS SBS mutations (G12C, G12D, G12V and Q61H), 2 EGFR SBS mutations (G719A, V769M) and 1 EGFR exon 19 deletion (G746-A750del) in 10 FFPE specimens tested from colon and lung cancer patients. These variants were each confirmed using Cancer Panels (Ion Torrent AmpliSeq and Illumina TrueSeq). The assay also detected T790M, L858R, V769M and the exon 19 deletion in EGFR in 7 plasma samples obtained from NSCLC patients. Each of these variants was confirmed by Sanger sequencing, Qiagen RGQ assay, or by MolecularMDs proprietary EGFR droplet digital PCR (ddPCR) assay. A low-frequency EGFR exon 19 deletion identified by the Qiagen RGQ assay was not seen in either the NGS assay or ddPCR assay. No other potential false negatives were identified in the custom NGS assay. Conclusions: This study demonstrates the feasibility of creating a sensitive and specific ALK-EGFR-KRAS focused NGS assay that covers broader regions of the target genes than the hotspots represented in commercial panels. With a DNA input of merely 20ng of FFPE DNA, or 1-2ng of the plasma circulating DNA, the assay was able to detect both SBS and small indels in the targeted regions. Given the low DNA input requirements and the capability of plasma-based testing, this assay and other custom NGS panels may enable routine monitoring of mutation status for relevant genes in patients with various solid tumors, and may ultimately inform clinical decision-making. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A208. Citation Format: Peng Fang, Agus Darwanto, Zhenyu Yan, Weihua Liu, Kimberly Pelak, Jessica Kristof, Philip C. Mack, Sabita Sankar, Chad Galderisi, Jin Li. Mutation detection in FFPE and plasma circulating DNA with a focused ALK-EGFR-KRAS Next-Generation Sequencing panel. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A208.