The main function of the mammalian skin is to protect the organism against various deleterious environmental factors such as UVR. The skin contains specialized pigment producing cells, known as melanocytes, to protect against UVR. However, when melanocytes acquire mutations from excessive exposure to UVR or other factors, they transform into melanoma. Melanoma is the deadliest type of skin cancer that invades neighboring tissue during early tumor development. The BRAF oncogene is mutated in over 50% of human melanoma cases and therapies that target the BRAF signaling are effective at inducing tumor regression. Unfortunately, melanoma tumors acquire resistance and become unresponsive to BRAF therapy because of alterations in various signaling networks. Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In addition, melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. In order to better understand the signaling networks that drive BRAF mutant melanoma, we identified a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant tumors. RhoJ is a small GTPase that cycles between an active-GTP bound state and an inactive-GDP bound state that regulates various physiological mechanisms. Previously we demonstrated that RhoJ is involved in chemoresistance in melanoma and in suppressing ATR activity. Using an autochthonous mouse model of melanoma, we determined that loss of RhoJ significantly prolonged the life of mice bearing BRAFV600E mutant melanomas by delaying tumor initiation and reducing metastasis. Transcriptome analysis revealed that RhoJ deletion in BRAF mutant tumors modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, generating tumors that grow more slowly and metastasize less avidly. PAK inhibitors induce apoptosis in melanoma cells in vitro via a BAD-dependent mechanism and induce the regression of BRAF mutant melanoma tumors in vivo. We also demonstrate that ATR mutant tumors accumulate multiple mutations and alter the immune system by decreasing T-cells to encourage tumor growth. Taken together, these studies identify the RhoJ-BAD signaling network as a therapeutic vulnerability for BRAF mutant tumors. Additionally, these studies also identify a novel mechanism by which melanoma cells modulate the tumor microenvironment to promote survival.

Epidermal melanocytes synthesize melanin within specialized lysosome-related organelles known as melanosomes. These melanosomes are transferred to neighboring keratinocytes where they surround nuclei to protect against ultraviolet-induced UV damage. The synthesis of melanin, a process known as melanogenesis, is subject to complex and intricate regulation at multiple levels. While extensive studies have elucidated many of the transcriptional and regulatory pathways, melanosomal trafficking comparatively far less understood. To further complicate matters, several lysosomal and autophagic components are utilized during these processes, but it is unclear what delineates the specificity of their regulation. To help elucidate these mechanisms, our laboratory conducted a genome-wide siRNA screen, which identified several novel regulators of melanogenesis. Several autophagy components, including the phosphoinositol binding protein WIPI1, were among those implicated in the screen. Through further investigation, it was determined that WIPI1 regulates both melanosomal transcription and trafficking, but the mechanism by which it coordinated both processes was not thoroughly elucidated. WIPI1 binds to three different phosphoinositides, PI(3)P, PI(3,5)P2, and PI(5)P, the latter two of which are synthesized by the PIKfyve kinase complex. PI(3,5)P2 and PI(5)P have been priorly implicated in regulating membrane trafficking and signaling pathways. Furthermore, mutation in select PI(3,5)P2 effectors or in the PIKfyve complex itself has been shown to affect pigment accumulation, though the mechanisms have not been priorly elucidated. In our studies, we have demonstrated that loss of PIKfyve in vivo results in the progressive loss of pigmentation. Disruption of the PIKfyve leads to defects in vesicle trafficking and severe vacuolization. Our in vitro and in vivo studies have identified similar disruption in melanocytes resulting in the inhibition of melanosome maturation and melanosomal acidification. These mechanisms would suggest an important role of PI(3,5)P2 in melanogenesis. In contrast, partial depletion of PIKfyve depleted WIPI1 and resulted in downregulation of these signaling pathways and expression of MITF-M and TYR, while the addition of PI(5)P had the opposite effect. Taken together these studies demonstrate novel regulatory roles of the PIKfyve complex, elucidating distinct roles of PI(3,5)P2 and PI(5)P in melanogenesis. These studies expand upon the fields of knowledge in both melanocyte and phosphoinositide biology and provide significant insight into the regulatory mechanisms of melanocytes.

Melanogenesis, the process by which specialized cells within the epidermis synthesize melanin, is an intricate and complicated process that requires the coordinated action of transcriptional, translational and trafficking processes. Melanin, a complex polymer derived from the amino acid tyrosine, is the primary determinant of hair, skin and eye color in humans. It plays a monumental role in the protection of the eyes and skin from solar ultraviolet-radiation through direct absorption mechanisms. Melanin is aberrantly regulated in melasma and vitiligo, two pigmentary disorders that are characterized by areas of skin that are hyper-pigmented and hypo-pigmented, respectively. Although a variety of treatment options exist for these disorders, many of the treatments are not very effective and have significant side effects. Tyrosinase inhibitors are used to treat melasma, and tyrosinase (TYR) is an enzyme that catalyzes the rate-limiting step in melanin synthesis. In this work, we investigate the role of TYR in regulating pigment production in vivo and examine the suitability of TYR as a therapeutic target for hyperpigmentary disorders. We demonstrate that the partial depletion of Tyr in the mouse was not sufficient to induce significant decreases in melanin accumulation in the mouse hair follicle, suggesting that TYR may not be an ideal target for the treatment of hyperpigmentary disorders. To address the need for novel, effective treatments for pigmentary disorders, a genome-wide siRNA functional genomics approach was employed to identify pharmaceutically tractable, single gene loci that impact melanogenesis. The screen uncovered 92 novel regulators of melanogenesis, including Aldehyde dehydrogenase 1A1 (ALDH1A1), which was one of only three genes able to impact TYR expression in three distinct genetic backgrounds. We discovered that a potent catalytic inhibitor of ALDH1A1, cyanamide, was able to inhibit not only the accumulation of pigment, TYR and MITF mRNAs in melanoma cells, but also melanin synthesis in three-dimensional human skin equivalents, suggesting that inhibitors of this enzyme may be ideal to therapeutically treat hyperpigmentation disorders, including melasma. In this work, we have identified how ALDH1A1 regulates melanogenesis. We show that 9-cis retinoic acid, a molecule important in cellular differentiation and the product of ALDH1A1 catalysis in vitamin A metabolism, stimulates the accumulation of pigment, TYR and MITF mRNAs, and TYR protein in melanoma cells and primary human epidermal melanocytes. As the biological effects of 9-cis retinoic acid are mediated by two classes of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), we searched for and identified a putative RAR/RXR binding site within the promoter of MITF-A, an isoform of MITF not known previously to play a role in melanin synthesis. While rigorous chromatin immunoprecipitation (ChIP) studies demonstrated significant enrichment of both RARα and RXRα upstream of the transcription start site of MITF-A, no enrichment was found in the promoter region of MITF-M, suggesting a novel mechanism in the regulation of melanogenesis. We then went on to show, through luciferase-activity assays, that retinoids can activate the full-length MITF-A promoter, but not a truncated MITF-A promoter that lacks the putative RAR/RXR heterodimer binding site. Lastly, we demonstrate that when used in combination, RARα and RXRα agonists stimulate the accumulation of MITF-A and MITF-M transcripts. Taken together, our data outline a mechanism for how ALDH1A1 regulates melanogenesis. We provide key evidence that targeting this enzyme may be appropriate in the development of novel therapeutics for the treatment of melasma. Finally, we show that retinoids or retinoid-analogs may be ideal in targeting hypopigmentary disorders, including vitiligo.

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Melanomas resist conventional chemotherapeutics in part through intrinsic disrespect of apoptotic checkpoint activation. In this study, using an unbiased genome-wide RNAi screen we identified RhoJ and its effector Pak1, as key modulators of melanoma cell sensitivity to DNA damage. We find that RhoJ activates Pak1 in response to drug-induced DNA damage, which then uncouples ATR from its downstream effectors, ultimately resulting in a blunted DNA damage response (DDR). In addition, ATR suppression leads to the decreased phosphorylation of ATF2, and consequent increased expression of the melanocyte survival gene Sox10 resulting in a higher DDR threshold required to engage melanoma cell death. In the setting of normal melanocyte behavior, this regulatory relationship may facilitate appropriate epidermal melanization in response to UV-induced DNA damage. However, pathological pathway activation during oncogenic transformation produces a tumor that is intrinsically resistant to chemotherapy and has the propensity to accumulate additional mutations. These findings identify DNA damage agents and pharmacological inhibitors of RhoJ/PAK1 as novel synergistic agents that can be used to treat melanomas that are resistant to conventional chemotherapies.