In vivo coherent Raman imaging of the melanomagenesis-associated pigment pheomelanin

Melanoma is the most deadly form of skin cancer with a yearly global incidence over 232,000 patients. Individuals with fair skin and red hair exhibit the highest risk for developing melanoma, with evidence suggesting the red/blond pigment known as pheomelanin may elevate melanoma risk through both UV radiation-dependent and -independent mechanisms. Although the ability to identify, characterize, and monitor pheomelanin within skin is vital for improving our understanding of the underlying biology of these lesions, no tools exist for real-time, in vivo detection of the pigment. Here we show that the distribution of pheomelanin in cells and tissues can be visually characterized non-destructively and noninvasively in vivo with coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. We validated our CARS imaging strategy in vitro to in vivo with synthetic pheomelanin, isolated melanocytes, and the Mc1re/e, red-haired mouse model. Nests of pheomelanotic melanocytes were observed in the red-haired animals, but not in the genetically matched Mc1re/e; Tyrc/c (“albino-red-haired”) mice. Importantly, samples from human amelanotic melanomas subjected to CARS imaging exhibited strong pheomelanotic signals. This is the first time, to our knowledge, that pheomelanin has been visualized and spatially localized in melanocytes, skin, and human amelanotic melanomas

Landscape of Targeted Anti-Cancer Drug Synergies in Melanoma Identifies a Novel BRAF-VEGFR/PDGFR Combination Treatment

<div><p>A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1) transporter inhibition. We identified drugs sensitizing cell lines that are BRAF^V600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR) and platelet derived growth factor receptor (PDGFR) family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis <i>in vitro</i> and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs), demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK) kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.</p></div

Ultra high-throughput screen to identify synergistic combinations in melanoma cells.

<p>(A) Summary of clinical development stage of 108 drugs included in the combination drug panel. (B) Example of raw UHTS data generated, demonstrating cell count information collected from DAPI channel and apoptosis data from cleaved PARP immunofluorescence; positive control of HSP90 inhibitor 17-AAG treatment is shown. (C) Summary matrix of combinatorial drug data, with each point representing the effect of one of 5,778 combinations at the standard drug concentration, as the median effect of the drug combination across all 36 melanoma cell lines on the relative cell count (left) and the calculated Bliss synergy score for that combination (right). (D) Histogram of number of cell lines a given drug combination showed synergy. Peak number of synergies were seen in one cell line, indicating many synergies are private. (E) As in (C), showing median effect of the drug combination (at standard concentration) on the relative cPARP positive proportion (left) and the calculated Bliss synergy for that cPARP level (right). (F) Graphical representation of drug combinations (drug pairs connected by an edge) that showed a significant unexpectedly high cPARP over a predicted level at the given cell count. Node size indicates the number of drug pairs that the given drug appears with other drugs on the “unexpectedly apoptotic” list. Edge color indicates the drug pair concentration (standard or low) where the elevated cPARP was found; edge pattern indicates whether the elevated cPARP was found in the setting of low cell count or normal cell count (> 80% control), with elevated cPARP in the setting of normal viability potentially representing “slow” death kinetics for that combination.</p

Cytotoxic potentiation by a MDR inhibitor.

<p>(A) Screening results showing the effects at both concentrations of vincristine and lapatinib, individually, and as a combination, showing strong synergy across most melanoma cell lines as indicated by high Bliss synergy scores. (B) Confirmation of the synergistic effect of the combination of lapatinib (5μM) in A375 (<i>n</i> = 19) but not WM451Lu (<i>n</i> = 14) cells. Error bars represent s.d. of measurement replicates. (C) Representative flow cytometry data showing lapatinib potentiates G₂/M shift of A375 cell population consistent with increased vincristine effect. (D) Log₂ relative expression of the given multi-drug resistance transporter in A375 versus WM451Lu cells, showing increased MDR1 expression in A375 cells. Error bars represent s.d. of measurement replicates (<i>n</i> = 9). (E) Calcein dye flux experiments showing increased fluorescence intensity (indicating decreased MDR flux) in the presence of lapatinib or control MDR inhibitor verapamil (both at 5μM); quantitation of cell grey values shown at left. Error bars represent s.d. of measurement replicates (<i>n</i> = 4, > 200 cells per replicate). (F) MDR1 knockdown by siRNA causes a synergistic effect on cell viability in the presence of 5nM vincristine. Error bars represent s.d. of measurement replicates (<i>n</i> = 7). (G) Overexpression of MDR1 (compared to GFP control) in WM451Lu decreases sensitivity to vincristine, an effect reversible with 5μM lapatinib. Error bars represent s.d. of measurement replicates (<i>n</i> = 4)</p

Cediranib synergizes with PLX4720 <i>in vivo</i>.

<p>(A) ISTMel1 xenografts were generated in immunodeficient mice (<i>n</i> = 8 per group), which were treated with PLX4720 or control chow and given cediranib or water by oral gavage. ISTMel1 xenografts showed an initial response to PLX4720 treatment alone after approximately two weeks, with some additional but non-significant response to cediranib treatment. Values are shown as mean +/- S.E.M. ranges, with significant differences in mean tumor size of both PLX4720 and PLX4720 and cediranib-treated mice compared to control treatment by ANOVA. However, the combination significantly delayed progression (defined as > 500 mm³ size) beyond this initial response (right, <i>p</i> < 0.002 between PLX4720 and PLX4720 + cediranib arms by log-rank test). (B) In contrast to ISTMel1 xenografts, RPMI7951 xenografts showed a significant difference by ANOVA test in initial response to PLX4720 versus PLX4720 and cediranib treatment after three weeks, and, right, showed a prolonged delay of progression (defined as tumor > 250 mm³) of completely PLX4720-resistant tumors (<i>p</i> < 0.0001 between PLX4720 and PLX4720 and cediranib arms by log-rank test). (C) KDR staining of tumor biopsies from patients entering clinical trials for BRAF with or without MEK inhibitors demonstrated some had strong membrane KDR staining (top) throughout the tumor (inset showing membrane staining, at arrowhead), while others were negative for KDR staining (bottom) except for expected endothelial staining. (D) Comparison of progression-free survival of patients with (<i>n</i> = 6) and without (<i>n</i> = 10) membrane KDR staining showed a significant reduction in PFS (9.3 vs. 3.8 months, <i>p</i> < 0.01 by Student’s t-test) if the patient’s biopsy expressed KDR.</p

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.Fil: Allouche, Jennifer. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Rachmin, Inbal. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Adhikari, Kaustubh. Colegio Universitario de Londres; Reino Unido. The Open University (ou); Reino UnidoFil: Pardo, Luba M.. Erasmus Medical Center; Países BajosFil: Lee, Ju Hee. Yonsei University College of Medicine; Corea del SurFil: McConnell, Alicia M.. Howard Hughes Medical Institute; Estados UnidosFil: Kato, Shinichiro. Nagoya University Graduate School of Medicine; Japón. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Fan, Shaohua. Fudan University; ChinaFil: Kawakami, Akinori. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Suita, Yusuke. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Wakamatsu, Kazumasa. Fujita Health University; JapónFil: Igras, Vivien. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Zhang, Jianming. Shanghai Jiao Tong University School of Medicine; ChinaFil: Navarro, Paula P.. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Lugo, Camila Makhlouta. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Noonan, Haley R.. Howard Hughes Medical Institute; Estados Unidos. Boston Children’s Hospital; Estados UnidosFil: Christie, Kathleen A.. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Itin, Kaspar. Hospital Universitario de Basilea; SuizaFil: Mujahid, Nisma. University of Boston. School of Medicine; Estados Unidos. University of Utah; Estados Unidos. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Lo, Jennifer A.. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Won, Chong Hyun. Ulsan University College of Medicine; Corea del SurFil: Evans, Conor L.. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Weng, Qing Yu. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Wang, Hequn. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Osseiran, Sam. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Lovas, Alyssa. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Németh, István. University of Szeged; HungríaFil: Cozzio, Antonio. Kantonsspital St. Gallen; SuizaFil: Navarini, Alexander A.. Hospital Universitario de Basilea; SuizaFil: Gonzalez-Jose, Rolando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico de Ciencias Sociales y Humanas; Argentin

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes