Identification of Collagen-Suppressive Agents in Keloidal Fibroblasts Using a High-Content, Phenotype-Based Drug Screen.

Keloids are characterized by excessive extracellular collagen and exaggerated scarring. Large-volume lesions can be functionally debilitating, therapeutically intractable, and psychologically devastating. A key barrier to translational momentum for novel antikeloid agents is the lack of a faithful high-content screen. We devised, to our knowledge, a previously unreported phenotype-based assay that measures secreted collagen by keloidal fibroblasts in tissue hypoxic conditions (1% oxygen). Four keloidal fibroblasts and 1 normal dermal fibroblast line were exposed to 199 kinase inhibitors. Of 199 kinase inhibitors, 41 (21%) and 71 (36%) increased and decreased the CI¯norm (mean collagen inhibition normalized to viability) by more than 10%, respectively. The most collagen suppressive agents were CGP60474 (CI¯norm = 0.36), KIN001-244 (CI¯norm = 0.55), and RAF265 (CI¯norm = 0.58). The top candidate, CGP60474, consistently abolished collagens I and VII production, exhibited minimal global toxicity, and induced a fivefold increase in phosphorylated extracellular signal-regulated kinase. This proof-of-concept high-content screen can identify drugs that appear to target critical keloidal pathophysiology-collagen secretion.

The Molecular Context of Vulnerability for CDK9 Suppression in Triple Wild-Type Melanoma.

Approximately half of melanoma tumors lack a druggable target and are unresponsive to current targeted therapeutics. One proposed approach for treating these therapeutically orphaned tumors is by targeting transcriptional dependencies (oncogene starvation), whereby survival factors are depleted through inhibition of transcriptional regulators. A drug screen identified a CDK9 inhibitor (SNS-032) to have therapeutic selectivity against wild-type (wt) BRAFwt/NRASwt melanomas compared with BRAFmut/NRASmut mutated melanomas. We then used two strategies to inhibit CDK9 in vitro-a CDK9 degrader (TS-032) and a selective CDK9 kinase inhibitor (NVP-2). At 500 nM, both TS-032 and NVP-2 demonstrated greater suppression of BRAFwt/NRASwt/NF1wt cutaneous and uveal melanomas than mutant melanomas. RNA sequencing analysis of eight melanoma lines with NVP-2 treatment demonstrated that the context of this vulnerability appears to converge on a cell cycle network that includes many transcriptional regulators, such as the E2F family members. The Cancer Genome Atlas human melanoma tumor data further supported a potential oncogenic role for E2F1 and E2F2 in BRAFwt/NRASwt/NF1wt tumors and a direct link to CDK9. Our results suggest that transcriptional blockade through selective targeting of CDK9 is an effective method of suppressing therapeutically orphaned BRAF/NRAS/NF1 wt melanomas.

Hypoxia and HIF-1α Regulate Collagen Production in Keloids.

Keloids are reactive or spontaneous fibroproliferative dermal tumors characterized by the exaggerated and uncontrolled accumulation of extracellular collagen. Current approaches to mitigate keloidogenesis are largely procedural in nature. However, a better understanding of its biological drivers may lead to novel targeted treatments for keloids. Through whole-genome expression analysis, we found that an HIF-1α transcriptional footprint is preferentially upregulated (activation score = 2.024; P = 1.05E-19) in keloid fibroblasts compared with normal dermal fibroblasts. We verified that HIF-1α protein is more strongly expressed in keloid specimens compared with normal skin (P = 0.035) and that hypoxia (1% O2) leads to increased collagen, especially in the extracellular compartment. Collagen levels were reduced uniformly by selective HIF-1α inhibitor CAY10585. Our results indicate that collagen secretion may be intimately linked to a hypoxic microenvironment within keloid tumors and that HIF-1α blockade could be a novel avenue of treatment for these tumors.

Selective uveal melanoma inhibition with calcium channel blockade.

Uveal malignant melanoma (UMM), the most common primary adult intraocular tumor with a marked metastatic potential, is genetically unique and has unfortunately had few treatment breakthroughs. In this study, we subjected a UMM cell line to high­throughput library screening with 1,018 FDA­approved compounds to identify potential UMM­selective cytotoxic agents. Amlodipine, a dihydropyridine calcium channel blocker (CCB), ranked no. 2 and no. 8 of the most cytotoxic compounds. Thus, we further characterized the differential effects of calcium blockade on UMM and cutaneous malignant melanoma (CMM) lines in vitro using growth inhibition, cell cycle progression, apoptosis and senescence assays. Amlodipine had a significantly higher growth inhibitory potency in UMM (IC50=13.1 µM) than CMM (IC50=15.9 µM, P<0.05) lines. In 3D spherical cell culture, amlodipine treatment significantly impaired tissue volume growth in two UMM lines, but exerted no significant effects among all 3 CMM lines tested. Treatment with 10 and 20 µM amlodipine induced a significant impairment of cell cycle progression and the apoptosis of UMM lines, implicating both of these processes as mediators of the observed growth inhibition in UMM compared to CMM. On the whole, the findings of this study suggest that calcium channel blockade is a potentially effective strategy for selective uveal melanoma targeting.

Growth suppression by dual BRAF(V600E) and NRAS(Q61) oncogene expression is mediated by SPRY4 in melanoma.

The underlying forces that shape mutational patterns within any type of cancer have been poorly characterized. One of the best preserved exclusionary relationships is that between BRAF(V600E) and NRAS(Q61) in melanomas. To explore possible mechanisms which could explain this phenomenon, we overexpressed NRAS(Q61) in a set of BRAF(V600E) melanoma lines and vice versa. Controlled expression of a second activating oncogene led to growth arrest ("synthetic suppression") in a subset of cells, which was accompanied by cell cycle arrest and senescence in several melanoma cell lines along with apoptosis. Through differential gene expression analysis, we identified SPRY4 as the potential mediator of this synthetic response to dual oncogene suppression. Ectopic introduction of SPRY4 recapitulated the growth arrest phenotype of dual BRAF(V600E)/NRAS(Q61) expression while SPRY4 depletion led to a partial rescue from oncogenic antagonism. This study thus defined SPRY4 as a potential mediator of synthetic suppression, which is likely to contribute to the observed exclusivity between BRAF(V600E) and NRAS(Q61R) mutations in melanoma. Further leverage of the SPRY4 pathway may also hold therapeutic promise for NRAS(Q61) melanomas.

Melanocytes are selectively vulnerable to UVA-mediated bystander oxidative signaling.

Long-wave UVA is the major component of terrestrial UV radiation and is also the predominant constituent of indoor sunlamps, both of which have been shown to increase cutaneous melanoma risk. Using a two-chamber model, we show that UVA-exposed target cells induce intercellular oxidative signaling to non-irradiated bystander cells. This UVA-mediated bystander stress is observed between all three cutaneous cell types (i.e., keratinocytes, melanocytes, and fibroblasts). Significantly, melanocytes appear to be more resistant to direct UVA effects compared with keratinocytes and fibroblasts, although melanocytes are also more susceptible to bystander oxidative signaling. The extensive intercellular flux of oxidative species has not been previously appreciated and could possibly contribute to the observed cancer risk associated with prolonged UVA exposure.

Vemurafenib synergizes with nutlin-3 to deplete survivin and suppresses melanoma viability and tumor growth.

PURPOSE: For patients with advanced melanoma, primary and secondary resistance to selective BRAF inhibition remains one of the most critically compelling challenges. One rationale argues that novel biologically informed strategies are needed to maximally cripple melanoma cells up front before compensatory mechanisms emerge. As p53 is uncommonly mutated in melanoma, restoration of its function represents an attractive adjunct to selective BRAF inhibition. EXPERIMENTAL DESIGN: Thirty-seven BRAF(V600E)-mutated melanoma lines were subjected to synergy studies in vitro using a combination of vemurafenib and nutlin-3 (Nt-3). In addition, cellular responses and in vivo efficacy were also determined. We also analyzed changes in the levels of canonical apoptotic/survival factors in response to vemurafenib. RESULTS: Dual targeting of BRAF(V600E) and Hdm2 with vemurafenib and Nt-3, respectively, synergistically induced apoptosis and suppressed melanoma viability in vitro and tumor growth in vivo. Suppression of p53 in melanoma cells abrogated Nt-3's effects fully and vemurafenib's effects partially. A survey of canonical survival factors revealed that both vemurafenib and Nt-3 independently attenuated levels of the antiapoptotic protein, survivin. Genetic depletion of survivin reproduces the cytotoxic effects of the combination strategy. CONCLUSION: These results show preclinical feasibility for overcoming primary vemurafenib resistance by restoring p53 function. Moreover, it identifies survivin as one downstream mediator of the observed synergism and a potential secondary target.

Real-time imaging of novel spatial and temporal responses to photodynamic stress.

Cells subjected to various forms of stress have been shown to induce bystander responses in nontargeted cells, thus extending the stress response to a larger population. However, the mechanism(s) of bystander responses remains to be clearly identified, particularly for photodynamic stress. Oxidative stress and cell viability were studied on the spatial and temporal levels after photodynamic targeting of a subpopulation of EMT6 murine mammary cancer cells in a multiwell plate by computerized time-lapse fluorescence microscopy. In the targeted population a dose-dependent loss of cell viability was observed in accordance with increased oxidative stress. This was accompanied by increased oxidative stress in bystander populations but on different time scales, reaching a maximum more rapidly in targeted cells. Treatment with extracellular catalase, or the NADPH oxidase inhibitor diphenyleneiodinium, decreased production of reactive oxygen species (ROS) in both populations. These effects are ascribed to photodynamic activation of NADPH-oxidase in the targeted cells, resulting in a rapid burst of ROS formation with hydrogen peroxide acting as the signaling molecule responsible for initiation of these photodynamic bystander responses. The consequences of increased oxidative stress in bystander cells should be considered in the overall framework of photodynamic stress.

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: roles of NADPH oxidase, mitochondria, and lipid rafts.

Long wavelength solar UVA radiation stimulates formation of reactive oxygen species (ROS) and prostaglandin E(2) (PGE(2)), which are involved in skin photosensitivity and tumor promotion. High levels of 7-dehydrocholesterol (7-DHC), the precursor to cholesterol, cause exaggerated photosensitivity to UVA in patients with Smith-Lemli-Opitz syndrome (SLOS). Partially replacing cholesterol with 7-DHC in keratinocytes rapidly (<5 min) increased UVA-induced ROS, intracellular calcium, phospholipase A(2) activity, PGE(2), and NADPH oxidase activity. UVA-induced ROS and PGE(2) production were inhibited in these cells by depleting the Nox1 subunit of NADPH oxidase using siRNA or using a mitochondrial radical quencher, MitoQ. Partial replacement of cholesterol with 7-DHC also disrupted membrane lipid raft domains, although depletion of cholesterol, which also disrupts lipid rafts, did not affect UVA-induced increases in ROS and PGE(2). Phospholipid liposomes containing 7-DHC were more rapidly oxidized by a free radical mechanism than those containing cholesterol. These results indicate that 7-DHC enhances rapid UVA-induced ROS and PGE(2) formation by enhancing free radical-mediated membrane lipid oxidation and suggests that this mechanism might underlie the UVA photosensitivity in SLOS.

Recurrent patterns of dual RB and p53 pathway inactivation in melanoma.

Two growth inhibitory hurdles that must be overcome by the evolving cancer cell include pathways regulated by RB and p53. In human melanoma cells, inactivation of a single locus, CDKN2A, can lead to abrogation of both RB and p53 functionality through loss of the two CDKN2A cognate transcripts-p16 and p14ARF, respectively. We thus set out to assess how recurrent patterns of genetic injury at three critical human melanoma loci-CDKN2A, TP53, and CDK4-cooperate to disrupt both RB and p53 pathways. Overall, 77.8% of the melanoma cell lines analyzed showed genetic evidence of dual RB and p53 pathway compromise; this percentage is even higher if protein expression loss is considered. Although homozygous deletion of all three critical CDKN2A exons (exons 1 beta, 1 alpha, and 2) represent the most common mechanism, concurrent loss of CDKN2A(Exon1 alpha) and CDKN2A(Exon1 beta) and simultaneous point mutagenesis of CDK4 and TP53 reflect alternative cassettes of dual inactivation. In cell lines with isolated CDKN2A(Exon2) mutations, coincident alterations in TP53 or deletion of CDKN2A(Exon1 beta) suggest that p16 transcript may be preferentially targeted over the p14ARF transcript as additional p53 pathway lesions are recruited. Moreover, predictive modeling of CDKN2A(Exon2) missense mutations further suggests that the amino acid substitutions in this region negatively impact p16 to a greater extent than p14ARF. Taken together, our data point to a clear need in human melanoma cell lines, as in its murine counterpart, to disrupt both RB and p53 pathways and recurrent mechanisms may play into the unique genetic vulnerabilities of this tumor type.