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In addition to genetic alterations, cancer cells are characterized by myriad epigenetic changes. EZH2 is a histone methyltransferase that is over-expressed and mutated in cancer. The EZH2 gain-of-function (GOF) mutations first identified in lymphomas have recently been reported in melanoma (~2%) but remain uncharacterized. We expressed multiple EZH2 GOF mutations in the A375 metastatic skin melanoma cell line and observed both increased H3K27me3 and dramatic changes in 3D culture morphology. In these cells, prominent morphological changes were accompanied by a decrease in cell contractility and an increase in collective cell migration. At the molecular level, we observed significant alteration of the axonal guidance pathway, a pathway intricately involved in the regulation of cell shape and motility. Furthermore, the aggressive 3D morphology of EZH2 GOF-expressing melanoma cells (both endogenous and ectopic) was attenuated by EZH2 catalytic inhibition. Finally, A375 cells expressing exogenous EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. This study not only demonstrates the first functional characterization of EZH2 GOF mutants in non-hematopoietic cells, but also provides a rationale for EZH2 catalytic inhibition in melanoma.

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine ( Met ). Met is the obligate precursor of S‐adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)‐specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met ‐cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co‐regulated in models of cancer cell differentiation and co‐expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small‐molecule inhibition leads to de‐repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1‐EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA‐mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.

Phosphorylation of the retinoblastoma protein (Rb) by cyclin-dependent kinases 4 and 6 (CDK4/6) is a critical checkpoint for G1/S cell cycle progression and commitment to cellular proliferation. Human malignancies often subvert these control mechanisms through a range of genetic and biochemical adaptations. Accordingly, tumors that depend on CDK4/6 activity for proliferation and survival are particularly sensitive to inhibition of this pathway by palbociclib (Ibrance™), a highly selective inhibitor of CDK4/6 kinase activities. Treatment regimen of palbociclib with letrozole significantly improved progression-free survival in a randomized phase 2 study of women with advanced estrogen receptor-positive (ER+), HER2-negative breast cancer. Likewise, in ER+ breast cancer models palbociclib and estrogen antagonists combine for greater anti-proliferative activity, increased hallmarks of cellular senescence and prolonged durability of response following drug removal. Dual inhibition of CDK4/6 and ER signaling demonstrated robust anti-tumor activity in xenograft studies. The addition of Palbociclib to other targeted therapeutics elicits improved activity in pre-clinical models of several non-breast indications and these effects also manifest through modulation of cellular proliferation, senescence and growth arrest. Data will be presented on the molecular basis of combination benefit with Palbociclib in ER+ breast and other oncology indications. Citation Format: Stephen Dann, John Chionis, Liu Choating, Enhong Chen, Ping Wei, Koleen Eisele, David J. Shields, Paul A. Rejto, Todd VanArsdale. Mechanistic basis of Palbociclib combinatorial activity in ER+ breast cancer and non-breast indications. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A08.

Phosphorylation of the retinoblastoma protein (Rb) by cyclin-dependent kinases 4 and 6 (CDK4/6) is a critical checkpoint for G1/S cell cycle progression and commitment to cellular proliferation. Human malignancies often subvert these control mechanisms through a range of genetic and biochemical adaptations. Accordingly, tumors that depend on CDK4/6 activity for proliferation and survival are particularly sensitive to inhibition of this pathway by palbociclib (IbranceTM), a highly selective inhibitor of CDK4/6 kinase activities. Treatment regimen of palbociclib with letrozole significantly improved progression-free survival in a randomized phase 2 study of women with advanced estrogen receptor-positive (ER+), HER2-negative breast cancer. Likewise, in ER+ breast cancer models palbociclib and estrogen antagonists combine for greater anti-proliferative activity, increased hallmarks of cellular senescence and prolonged durability of response following drug removal. Dual inhibition of CDK4/6 and ER signaling demonstrated robust anti-tumor activity in xenograft studies. The addition of Palbociclib to other targeted therapeutics elicits improved activity in pre-clinical models of several non-breast indications and these effects also manifest through modulation of cellular proliferation, senescence and growth arrest. Data will be presented on the molecular basis of combination benefit with Palbociclib in ER+ breast and other oncology indications. Citation Format: Stephen Dann, Jing Yuan, John Chionis, Chaoting Liu, Tao Xie, Nathan V. Lee, Enhong Chen, Ping Wei, Paul A. Rejto, David J. Shields, Todd VanArsdale. Mechanistic basis of Palbociclib combinatorial activity in ER+ breast cancer and non-breast indications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2740.

Space is the final frontier for e-marketing. Advances in storage space, digital data transmission, and infrastructure development have created a near limitless marketspace that exists over the contemporary physical marketplaces, and as an independent market of ideas, data, experience, and content. This chapter overviews a series of key issues in the use of the new “space” for e-novation with attention given to the rise of user generated content through prosumer activity. This chapter is based on exploring how companies and individuals are currently co-creating value in the dynamic marketplace of the new collaborative platforms, and how these new concepts such as the “home shopping channel”, digital rights management, and user generated distribution channels can factor in the future success on and offline for marketing.

PKN3 is an AGC-family protein kinase implicated in growth of metastatic prostate cancer cells with phosphoinositide 3-kinase pathway deregulation. The molecular mechanism, however, by which PKN3 contributes to malignant growth and tumorigenesis is not well understood. Using orthotopic mouse tumor models, we now show that inducible knockdown of PKN3 protein not only blocks metastasis, but also impairs primary prostate and breast tumor growth. Correspondingly, overexpression of exogenous PKN3 in breast cancer cells further increases their malignant behavior and invasiveness in-vitro . Mechanistically, we demonstrate that PKN3 physically interacts with Rho-family GTPases, and preferentially with RhoC, a known mediator of tumor invasion and metastasis in epithelial cancers. Likewise, RhoC predominantly associates with PKN3 compared to its closely related PKN family members. Unlike the majority of Rho GTPases and PKN molecules, which are ubiquitously expressed, both PKN3 and RhoC show limited expression in normal tissues and become upregulated in late-stage malignancies. Since PKN3 catalytic activity is increased in the presence of Rho GTPases, the co-expression and preferential interaction of PKN3 and RhoC in tumor cells are functionally relevant. Our findings provide novel insight into the regulation and function of PKN3 and suggest that the PKN3–RhoC complex represents an attractive therapeutic target in late-stage malignancies.

In addition to numerous genetic changes underlying cellular transformation, cancer cells are also characterized by epigenetic changes that are likely to play important roles in disease progression. EZH2 is an epigenetic repressor that plays well-established roles in development. In addition to widespread overexpression in a variety of tumors, the discovery of gain of function (GOF) mutations of EZH2 in diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and melanoma strongly suggests an important function for this histone methyltransferase in cancer. To ascertain the function of elevated EZH2 catalytic activity, we expressed either wild-type EZH2 (WT) or EZH2 GOF mutants in both non-tumorigenic (immortalized epithelial cells) and tumorigenic (melanoma cells) settings. In both systems, EZH2 GOF mutants greatly increased global levels of H3K27me3 and decreased H3K27me2 levels, similar to the epigenetic pattern seen in DLBCL cell lines with endogenous EZH2 GOF mutations. In epithelial cells, expression of an EZH2 GOF mutant caused striking changes in 3D-morphology and gene changes that are indicative of cells that have undergone an epithelial to mesenchymal transition. In the disease relevant melanoma cells, several distinct EZH2 GOF mutants (but not EZH2 WT) caused prominent branching morphology in 3D-culture. Interestingly, these GOF mutants did not affect 2D-cell morphology or proliferation of melanoma cells. Furthermore, catalytic inhibition of EZH2 GOF mutants with a commercially available tool compound attenuated the 3D-phenotype. Importantly, EZH2 inhibition in melanoma cells expressing an endogenous GOF mutation also caused similar changes in 3D-morphology. RNA-seq analysis revealed genes involved in processes such as cell adhesion and axonal guidance that were down-regulated by EZH2 GOF mutants. Finally, melanoma cells expressing ectopic EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. Collectively, these results suggest that EZH2 GOF mutants may alter the interaction of tumor cells with their microenvironment and in this way provide a selective advantage to such tumors. Citation Format: Robert A. Rollins, Anthony M. Barsotti, Michael Ryskin, Wenyan Zhong, Wei-Guo Zhang, Andreas Giannakou, Christine Loreth, Veronica Diesl, Maximillian T. Follettie, Jonathon Golas, Michelle Lee, Timothy Nichols, Conglin Fan, Gary Li, Stephen Dann, Paul A. Rejto, Kim T. Arndt, Dominique Verhelle. Epigenetic reprogramming by tumor-derived EZH2 gain of function mutants leads to aggressive 3D-cell morphologies in both epithelial and melanoma cells. [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 5144. doi:10.1158/1538-7445.AM2014-5144

The phosphatidylinositol 3-kinase/mechanistic target of rapamycin (PI3K/mTOR) pathway and the mitogen-activated protein kinase (MAPK) signaling cascades are central facilitators of cellular growth, proliferation, survival and motility within a variety of tumor lineages. To date, several ongoing oncology clinical trials are currently evaluating the benefit of combining PI3K with MEK inhibition. While this dual combination is well founded pre-clinically, investigating potential mechanisms of resistance and identifying novel biological signatures of sensitivity is of great clinical importance. We sought to establish a model of acquired resistance by evaluating the co-treatment of a colorectal carcinoma model with inhibitors of PI3K/mTOR inhibitor (PF-04691502) and MEK (PD-0325901). We can associate the onset of acquired resistance in this model to re-engagement of signaling within the PI3K/mTOR pathway correlative to a loss of PTEN protein expression , relative to parental cells. In addition, next-generation sequencing (NSG) comparing parental versus resistant cells identified a unique somatic “gatekeeper” mutation located in the kinase domain of mTOR. Furthermore, a cell proliferation screen evaluating potential cross resistance was analyzed using a PI3K/mTOR inhibitor with a chemical scaffold distinct from PF-502 (PF-05212384). Treatment of PF-384 as a single agent (IC50 ∼150nM) circumvented the acquired resistance derived from the combination of PF-502+ PD-901 (IC50: ∼2uM and ∼334nM respectfully). Inhibition of growth observed with PF-384 correlated with rescuing of PI3K/mTOR signaling as measured by substrate phosphorylation readouts. Finally, co-crystal structure modeling of mTOR was used to evaluate the potential interaction of PF-502 within the ATP binding pocket and we confirm that this interaction is hindered due to the point mutation identified at mTOR. Furthermore, co-crystal modeling of mTOR with PF-384 illustrates an independence and lack of PF-384 binding to the mutated site and thus substantiates a mechanistic explanation for overcoming PF-502 acquired resistance. In summary, we have identified and characterized a novel mechanism of resistance to the combined treatment of a Pyrimidinone class PI3K/mTOR inhibitor in addition to a MEK inhibitor, and have shown that rescuing of this resistance can be achieved through the use of a Triazine class PI3K/mTOR clinical candidate. Citation Format: Scott J. Garza, Paul Lira, Stephen D. Huang, Hengmiao Cheng, Stephen G. Dann, Todd L. VanArsdale, Valeria Fantin, James Christensen, Julie LC Kan. Identifying a mechanism of acquired resistance to the combined inhibition of PI3K/mTOR and MEK in colorectal carcinoma. [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 3713. doi:10.1158/1538-7445.AM2014-3713

Within the family of protein kinase C (PKC) molecules only the novel isoform member PRKCE (PKC∈) acts as a bona fide oncogene in in-vitro and in-vivo models of tumorigenesis. Previous studies have reported cancer-specific misexpression of PKC∈ at levels well above that of normal adjacent tissue in breast, prostate and lung tumors. We find that oncogenic Ras signaling promotes PKC∈ expression and results in hyperphosphorylation of CTNND1/p120-catenin (p120ctn). In this context, loss of PKC∈ by genetic or pharmacological means results in normalization of morphology and signaling responses. In a KRasD13-dependent breast cancer model loss of PKC∈ function results in growth inhibition in 2-dimensional (2D) and 3-dimensional (3D) culture systems as well as in orthotopic xenografts concomitant with the normalization of a subset of Ras-induced signaling responses. Using phospho-proteomic profiling analysis we observe that CTNND1 (p120ctn) phosphorylation at serine 268 (S268) occurs in a strictly PKC∈ dependent manner. Treatment with a specific PKC∈ inhibitor, PF-5263555, recapitulates the genetic loss of function phenotype and interferes with breast cancer cell growth in-vitro and in-vivo. We also show that PKC∈-mediated phosphorylation at S268 further stabilizes additional p120ctn phosphorylation sites and total protein levels of β-catenin. We demonstrate that p120ctn phosphorylation at S268 represents a specific readout for PKC∈ activity and as such can serve as a suitable biomarker for PKC∈ dysregulation in human cancer and for monitoring therapeutic response. 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 1235. doi:1538-7445.AM2012-1235