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Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21(Cip1) in a p53-independent manner, identified the TGFbeta pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated beta-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient's selection and monitoring response, as well as targets for improved combination therapy.

The BULT melanoma originated at Brown University as a spontaneous, small black nodule on the tail of an adult female mouse of the LT/Ch strain. Histological examination of a portion of the tumor indicated that it was intradermal and consisted predominantly of heavily melanized, ovoid to fusiform cells with melanin-laden macrophages scattered among them. The BULT melanoma has been maintained in LT/Ch mice for approximately 5 years by periodic transplantation, at first subcutaneously on the flanks and, more recently, intramuscularly in the hind legs. The shift in transplantation site was made following a marked decline in the growth of subcutaneous grafts. The transplants have retained the uniform deep-black melanization and general histology of the primary melanoma. Numerous melanosomes at all stages of development are found within the melanoma cells. DOPA-positive cytoplasmic vesicles are abundant. Occasional autophagic vacuoles containing clusters of melanosomes are also present. A few metastases from the transplanted melanoma have been observed in lymph nodes and on one occasion in the lungs. When grown in vitro, BULT melanoma cells do not require special growth promoting agents (e.g., TPA; cAMP) in order to proliferate. The BULT melanoma differs in one or more respects from each of the other three transplantable spontaneous mouse melanomas widely used in cancer research. In addition, it arose in a strain of mice characterized by the spontaneous death of melanocytes while the latter are engaged in synthesizing eumelanin within hair follicles. Karyotypic analysis of cultured cells showed a modal chromosome number of 68 with a range of 58-72 chromosomes. [ABSTRACT FROM AUTHOR]

Basic Fibroblast Growth Factor (bFGF/FGF2) is thought to play a decisive role in malignant progression. Aberrant expression of bFGF causes constitutive autocrine activation of its cognate receptor and autonomous growth of human melanoma cells or bFGF transformed fibroblasts in culture. It remains to be determined, however, whether the endogenous bFGF confers growth advantage to tumors and what are the downstream targets of the activated FGF receptor critical for its transforming capacity. We therefore transfected metastatic melanoma cells and bFGF transformed mouse fibroblasts with a dominant-negative mutant of the murine FGF receptor 1 (fgfr1/flg), comprising the extracellular and transmembrane domains but lacking the intracellular kinase domain (dnflg). Reverse transcriptase-PCR, 125I-bFGF binding and affinity labeling analyses show that the truncated receptor is targeted to the membrane and is expressed at much higher levels than the endogenous receptor in all of the selected clones. Expression of the dnflg dramatically reduces the basal as well as bFGF induced growth of these cells in vitro and also suppresses their tumorigenic potential in nude mice. The expression of the dnflg does not significantly alter the general level of tyrosyl-phosphorylated proteins in the trunsduced melanoma cells. Rather, a major downstream affected target is a Src-family kinase, whose activity, determined by an in vitro immune kinase assay, is stimulated in normal melanocytes by exogenous bFGF, and is markedly reduced in the dnflg-expressing melanoma cells. The present study demonstrates that direct interference with the activity of FGF receptors has a deleterious effect on cell proliferation and survival in vitro and in vivo leading to the suppression of melanoma tumor progression possibly through the inactivation of a Src-family kinase. [ABSTRACT FROM AUTHOR]

Combination therapy concurrently targeting PD-1 and CTLA-4 immune checkpoints leads to remarkable antitumor effects. Although both PD-1 and CTLA-4 dampen the T cell activation, the in vivo effects of these drugs in humans remain to be clearly defined. To better understand biologic effects of therapy, we analyzed blood/tumor tissue from 45 patients undergoing single or combination immune checkpoint blockade. We show that blockade of CTLA-4, PD-1, or combination of the two leads to distinct genomic and functional signatures in vivo in purified human T cells and monocytes. Therapy-induced changes are more prominent in T cells than in monocytes and involve largely nonoverlapping changes in coding genes, including alternatively spliced transcripts and noncoding RNAs. Pathway analysis revealed that CTLA-4 blockade induces a proliferative signature predominantly in a subset of transitional memory T cells, whereas PD-1 blockade instead leads to changes in genes implicated in cytolysis and NK cell function. Combination blockade leads to nonoverlapping changes in gene expression, including proliferation-associated and chemokine genes. These therapies also have differential effects on plasma levels of CXCL10, soluble IL-2R, and IL-la. Importantly, PD-1 receptor occupancy following anti-PD-1 therapy may be incomplete in the tumor T cells even in the setting of complete receptor occupancy in circulating T cells. These data demonstrate that, despite shared property of checkpoint blockade, Abs against PD-1, CTLA-4 alone, or in combination have distinct immunologic effects in vivo. Improved understanding of pharmacodynamic effects of these agents in patients will support rational development of immune-based combinations against cancer. [ABSTRACT FROM AUTHOR]