Combining a BCL2 Inhibitor with the Retinoid Derivative Fenretinide Targets Melanoma Cells Including Melanoma Initiating Cells Nabanita Mukherjee, Steven.

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Combining a BCL2 Inhibitor with the Retinoid Derivative Fenretinide Targets Melanoma Cells Including Melanoma Initiating Cells Nabanita Mukherjee, Steven N. Reuland, Yan Lu, Yuchun Luo, Karoline Lambert, Mayumi Fujita, William A. Robinson, Steven E. Robinson, David A. Norris, Yiqun G. Shellman Journal of Investigative Dermatology Volume 135, Issue 3, Pages 842-850 (March 2015) DOI: 10.1038/jid.2014.464 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The effects of 4-HPR combined with ABT-737 in melanomas and melanocytes. (a) The MTS assay shows that 4-HPR combined with ABT-737 synergistically killed melanomas (A375, SK-MEL-28, WM852c, and 451Lu) but had little effect on melanocytes (HEMNLP2 and PIG1). Cells were treated with the indicated drugs for 48 hours before the MTS assay. (b) The annexin V assay shows massive apoptosis in melanoma cells induced by 48-hour treatment of 4-HPR and ABT-737. In all melanoma cells (either mutant BRAF or NRAS), the combination treatment significantly induced apoptosis compared with all other treatments (DMSO or single drug, P<0.01). (c) Visual appearance of cells showing cell death in combination treatments for melanoma cells but not for melanocytes. Scale bar=100 μm. Journal of Investigative Dermatology 2015 135, 842-850DOI: (10.1038/jid.2014.464) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The combination of 4-HPR with ABT-737 induced PARP cleavage, increased the NOXA/MCL-1 ratio, and caused Caspase-mediated MCL-1 degradation. (a) Immunoblot of cell lysates treated with either vehicle control (DMSO), 3.3 μM ABT-737 (ABT), 5–10 μM 4-HPR (4HPR), or the combination of the two drugs (Combo). The ratio of NOXA/MCL-1 in the control was set as “1” for each cell line. (b) Immunoblot of cell lysates treated with either vehicle control (DMSO) or the combination with or without co-treatment of 30 μM pan caspase inhibitor Z-VAD-FMK. WM852c and SK-MEL-30 are NRAS mutants, and all other cell lines shown here are BRAF mutants. The normalized MCL-1 levels in the Combo with or without the Caspase inhibitor were compared with the respective DMSO conditions. Journal of Investigative Dermatology 2015 135, 842-850DOI: (10.1038/jid.2014.464) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Knockdown of NOXA decreased drug-induced cell death. Annexin V assays of the indicated knockdown cell lines derived from A375 (a) or SK-MEL-30 (b). Immuoblots (c, d) of lysates from the cell lines used in a and b, respectively show efficient knockdown of indicated proteins. In A375, knockdown of NOXA by shRNA (shNoxa) significantly reduced combination-induced apoptosis as compared with control (shCont) (P<0.05), but knockdown of other pro-apoptotic proteins did not. In SK-MEL-30, both knockdown of NOXA and BIM significantly reduced combination-induced apoptosis as compared with control (P<0.01). Journal of Investigative Dermatology 2015 135, 842-850DOI: (10.1038/jid.2014.464) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The combination of 4-HPR with ABT-737 caused cytotoxicity in melanoma initiating cells. (a) Bright-field analysis showing complete disruption of primary spheres after 48 hours of the combination treatment. Scale bar=100 μm. (b) Quantification of the number of spheres from melanoma cell lines with either mutant BRAF or NRAS. The combination significantly reduced the number of spheres compared with the vehicle (DMSO) or ABT-737 in multiple melanoma cell lines (P<0.001). (c) Quantification of the ALDH assay. The combination treatment significantly reduced the relative percentage of ALDHhigh cells compared with the control or ABT-737 in multiple melanoma cell lines. (d) Immunoblots of lysates from the cells used in b showing that the combination induced PARP cleavage, increased NOXA, and reduced MCL-1. (e) Primary sphere assays with NOXA knockdown (shNoxa) or control (shCont.) of A375 after being treated with indicated drugs. Knockdown of NOXA significantly protected cells from the combination-induced disruption of spheres compared with the control (P<0.01). Journal of Investigative Dermatology 2015 135, 842-850DOI: (10.1038/jid.2014.464) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The Combination of 4-HPR and ABT-737 inhibited the self-renewal capacity of MICs in vitro. (a) Bright-field analysis of the secondary sphere assay showing that only the combination inhibited the formation of secondary spheres. Scale bar=100 μm. (b) Visualization of the cells with ethidium bromide/acridine orange (EtBr/AO) staining indicated that the majority of cells in the control or single drug treatment were alive (green), but the majority of cells in the combination were dead (orange). Scale bar=100 μm. (c) Quantification of the number of secondary spheres from melanoma cell lines with either mutated BRAF or NRAS. The combination significantly reduced the number of spheres compared with all other treatments in multiple melanoma cell lines (P<0.05 or less). Journal of Investigative Dermatology 2015 135, 842-850DOI: (10.1038/jid.2014.464) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The combination of 4-HPR with ABT-737 had cytotoxic effects in patient-derived xenograft (PDX) tumor samples and in a mouse xenograft model. (a) Bright-field analysis of PDX tumor samples showing complete disruption of spheres after 48 hours of combination treatment. Scale bar=100 μm. (b) Quantification of the number of spheres for the PDX samples. The combination treatment significantly reduced the number of spheres compared with vehicle (DMSO, P<0.001)), ABT-737 (P<0.01 or less), and 4-HPR (P<0.01 or less) in multiple PDX samples. (c) Relative tumor volumes in a mouse xenograft model. The tumor volume at day 0 was set as 100%. The combination significantly inhibited the tumor growth compared with control and 4-HPR. (d) Sphere assays with the tumor cells collected at the end of the xenograft experiment from c. The combination significantly reduced the number of spheres compared with vehicle or individual treatments (P<0.05). The graph represents the mean of three different sets of tumors collected. (e) A simplified model to illustrate how the combination of ABT-737 plus 4HPR triggers apoptosis by altering Bcl-2 family members. Initially, the combination eliminates the functions of multiple apoptotic inhibitors and activates apoptosis in two ways: ABT-737 alone blocks BCL-2/BCL-XL/BCL-W, and the combination induces NOXA expression, which inhibits MCL-1. Consequently, the combination treatment’s synergistic mechanism is achieved by MCL-1 degradation by a small percentage of activated caspases, which produces a positive feedback signal for further apoptosis activation. Journal of Investigative Dermatology 2015 135, 842-850DOI: (10.1038/jid.2014.464) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions