In this paper, we report the synthesis and biological activity of a series of dihydroisocoumarin analogues conjugated with fatty acids, alcohols, or amines, of varying hydrocarbon chain length and degree of unsaturation, to the dihydroisocoumarins, kigelin and mellein, at the C-7 and C-8 positions on the core dihydroisocoumarin structure. These compounds were evaluated for their antiproliferative activity against human breast cancer (MCF-7 and MDA-MB-468) and melanoma cells (SK-MEL-28 and Malme-3M) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Two compounds conjugated with gamma-linolenyl alcohol (18:3 n-6) demonstrated potent antiproliferative activity in vitro with one of these 4-hydroxy-3-oxo-1,3-dihydro-isobenzofuran-5-carboxylic acid octadeca-6,9,12-trienyl ester, demonstrating significant antitumor activity in vivo in a number of human tumor xenograft models.

Background: Oncogenesis rewires signaling networks to confer a fitness advantage to malignant cells. For instance, the B16F0 melanoma cell model creates a cytokine sink for Interleukin-12 (IL-12) to deprive neighboring cells of this important anti-tumor immune signal. While a cytokine sink provides an indirect fitness advantage, does IL-12 provide an intrinsic advantage to B16F0 cells? Methods: Acute in vitro viability assays were used to compare the cytotoxic effect of imatinib on a melanoma cell line of spontaneous origin (B16F0) with a normal melanocyte cell line (Melan-A) in the presence of IL-12. The results were analyzed using a mathematical model coupled with a Markov Chain Monte Carlo approach to obtain a posterior distribution in the parameters that quantified the biological effect of imatinib and IL-12. Intracellular signaling responses to IL-12 were compared using flow cytometry in 2D6 cells, a cell model for canonical signaling, and B16F0 cells, where potential non-canonical signaling occurs. Bayes Factors were used to select among competing signaling mechanisms that were formulated as mathematical models. Analysis of single cell RNAseq data from human melanoma patients was used to explore generalizability. Results: Functionally, IL-12 enhanced the survival of B16F0 cells but not normal Melan-A melanocytes that were challenged with a cytotoxic agent. Interestingly, the ratio of IL-12 receptor components (IL12RB2:IL12RB1) was increased in B16F0 cells. A similar pattern was observed in human melanoma. To identify a mechanism, we assayed the phosphorylation of proteins involved in canonical IL-12 signaling, STAT4, and cell survival, Akt. In contrast to T cells that exhibited a canonical response to IL-12 by phosphorylating STAT4, IL-12 stimulation of B16F0 cells predominantly phosphorylated Akt. Mechanistically, the differential response in B16F0 cells is explained by both ligand-dependent and ligand-independent aspects to initiate PI3K-AKT signaling upon IL12RB2 homodimerization. Namely, IL-12 promotes IL12RB2 homodimerization with low affinity and IL12RB2 overexpression promotes homodimerization via molecular crowding on the plasma membrane.

16Within tissues, cells secrete protein signals that are subsequently interpreted by 17 neighboring cells via intracellular signaling networks to coordinate a cellular response. 18 However, the oncogenic process of mutation and selection can rewire these signaling 19 networks to confer a fitness advantage to malignant cells. For instance, the melanoma 20 cell model (B16F0) creates a cytokine sink for Interleukin-12 (IL-12) to deprive neigh-21 boring cells of this important extracellular signal for sustaining anti-tumor immunity. 22 Alternatively, oncogenesis may also rewire intracellular signaling networks. To test this 23 concept, we asked whether IL-12 provides an intrinsic advantage to B16F0 melanoma 24 cells. Functionally, stimulation with IL-12 promoted the survival of B16F0 cells that 25 were challenged with a cytotoxic agent but had no rescue effect on normal Melan-A 26 melanocytes. We also explored a mechanistic basis for the functional results by sur-27 veying the phosphorylation intracellular proteins involved in canonical IL-12 signaling, 28 STAT4, and cell survival, Akt. In contrast to T cells that exhibited a canonical response § Corresponding author to IL-12 by phosphorylating STAT4, IL-12 stimulation of B16F0 cells phosphorylated 30 both STAT4 and Akt. Collectively, the data suggests that B16F0 cells have shifted 31 the intracellular response to IL-12 from engaging immune surveillance to favoring cell 32 survival. In short, identifying how signaling networks are rewired can guide therapeutic 33 strategies to restore effective immunosurveillance. 34 35 36Responses of the different cell types within tissues, such as T cells and Langerhans cells 38 within the epidermal layer of skin, are coordinated by the extracellular release of protein 39 signals. One such extracellular signal is the cytokine Interleukin-12 (IL-12) [1]. IL-12 is a 40 heterodimer comprised of a p35 and p40 subunit that is produced by antigen presenting cells, 41 like Langerhans and Dendritic cells, to boost natural killer (NK) and cytotoxic T lymphocyte 42 (CTL) activity and to polarize T helper cells towards a type 1 phenotype [2]. Activating 43 NK, CTLs and type 1 T helper cells play important roles in controlling viral infections 44 and defending against malignancy. Towards this aim, local delivery of IL-12 to the tumor 45 microenvironment has been shown to promote tumor regression in both mouse models and 46 human melanoma [3][4][5][6][7][8][9]. 47Inside the cell, these extracellular signals are processed through a series of protein-protein 48 interactions that transmit this information from the cell membrane to the nucleus to orches-49 trate a cellular response. In the case of IL-12, these extracellular signals are transduced by 50 the canonical Janus-Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT) 51 signaling pathway [10]. The IL-12 receptor, composed of a β1 (IL12RB1) and β2 (IL12RB2) 52 subunit, lacks intrinsic enzymatic activity and forms a complex with two JAKs, JAK2 and 53 TYK2. Autoph...