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Supplementary Figures S1-S3, Tables S1-S7. Suppl. Figure S1: Prolif. Curves, PC3 Cell Cycle & solid tumor apoptosis Suppl. Figure S2: PK/PD Model Performance and eIF4E In Vivo Biomarker Suppl. Figure S3: HCT-116, MBA MD231 & A549 Efficacy Studies Suppl. Table S1: Kinase Selectivity Suppl. Table S2: Cellular Biomarker with SEM Suppl. Table S3: Cellular Proliferation with SEM Suppl. Table S4: Hematological Panel Suppl. Table S5: IRF4 Gene and Protein Data Suppl. Table S6: PK/PD Data PK Data Suppl. Table S7: In Vivo Efficacy Across Tumor Types

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.

TTK is an essential spindle assembly checkpoint enzyme in many organisms. It plays a central role in tumor cell proliferation and is aberrantly overexpressed in a wide range of tumor types. We recently reported on a series of potent and selective TTK inhibitors with strong antiproliferative activity in triple negative breast cancer (TNBC) cell lines (8: TTK IC50 = 3.0 nM; CAL-51 IC50 = 84.0 nM). Inspired by previously described potent tricyclic TTK inhibitor 6 (TTK IC50 = 0.9 nM), we embarked on a structure-enabled design and optimization campaign to identify an improved series with excellent potency, TTK selectivity, solubility, CYP inhibition profile, and in vivo efficacy in a TNBC xenograft model. These efforts culminated in the discovery of 25 (TTK IC50 = 3.0 nM; CAL-51 IC50 = 16.0 nM), which showed significant single-agent efficacy when dosed iv in a TNBC xenograft model without body weight loss.

Over the past 50 years, our understanding of cancer has grown dramatically. Conceptual models that explain the tumor development process have been formulated. The biological characteristics that account for the behavior of tumor cells in experimental systems and in vivo and the genetic alterations that account for these changes have been catalogued in detail. Additionally, new preventative strategies and therapeutic agents using this wealth of information are now being formulated. In this chapter, we present a brief overview of our current understanding of cancer as a disease as well as give specific examples of some of the approaches being adopted to combat it. Keywords: tumorigenesis; cancer; apoptosis; angiogenesis; oncogene; tumor suppressor gene; gene therapy; cell cycle

We previously described six families with Milroy congenital lymphedema, only one of which showed possible linkage to a candidate locus on chromosome 5 [Witte et al., 1998]. We have now performed a complex segregation analysis of these families, and performed linkage analyses with the other 387 markers used in our genome-wide search. Our results confirm that Milroy lymphedema is generally inherited as a dominant condition. However, this mode of inheritance, as elucidated from the segregation analyses, did not account for all observed familial correlations. The segregation analysis also suggested that shared environmental or additional genetic factors are important in explaining the observed familial aggregation. The finding of linkage to multiple locations in the largest family studied by multipoint parametric mapping (one of which was confirmed by sib-pair non-parametric mapping), suggests that Milroy congenital lymphedema may be oligogenic in this family. © 2001 Wiley-Liss, Inc.

Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene ( APC ) and show defects in APC -dependent signaling. APC influences the expression of several genes, including the c- myc oncogene and its antagonist Mad1 . Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c- myc and a modifier of APC -dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human ODC gene, which lies between two myc -binding domains. This region is known to affect ODC transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor ODC A-allele who reported using aspirin are ≈0.10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele. Mad1 selectively suppressed the activity of the ODC promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (≥10 μM) did not affect ODC allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the ODC polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the ODC polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention.

Mutation/deletion of the adenomatous polyposis coli (APC) tumor suppressor gene in germline cells of rodents and humans is associated with increased intestinal activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, and intestinal neoplasia. To study the role of APC in signaling ODC expression, we used the human colon tumor cell line HT29 (wtAPC−/−), which has been stably transfected with a zinc-inducible wild-type APC gene. The addition of ZnCl2 to HT29-APC cells increased wild-type APC protein and Mad1 RNA and protein and decreased levels of c-myc and ODC RNA and protein, relative to these parameters in HT29 cells transfected with the same plasmid containing the β-galactosidase gene in place of APC. Upon induction of APC expression, ODC promoter activity and RNA levels were suppressed. When the e-box domain in the 5′ flanking region of the ODC gene was mutated, ODC promoter activity was unaffected by wild-type APC expression. Antisense, but not missense, c-myc oligonucleotides decreased ODC activity in HT29 cells expressing mutant APC. These results demonstrated that wild-type APC suppressed c-myc and activated Mad1 expression in HT29 colon-derived cells. These proteins, in turn, regulated the transcription of target genes, including ODC. The data presented indicate that ODC is a modifier of APC-dependent signaling in intestinal cells and tissues. © 2002 Wiley-Liss, Inc.