Novel loss-of-function variant in DENND5A impedes melanosomal cargo transport and predisposes to familial cutaneous melanoma

Abstract

Purpose: More than half of the familial cutaneous melanomas have unknown genetic predisposition. This study aims at characterizing a novel melanoma susceptibility gene.

Methods: We performed exome and targeted sequencing in melanoma-prone families without any known melanoma susceptibility genes. We analyzed the expression of candidate gene DENND5A in melanoma samples in relation to pigmentation and UV signature. Functional studies were carried out using microscopic approaches and zebrafish model.

Results: We identified a novel DENND5A truncating variant that segregated with melanoma in a Swedish family and 2 additional rare DENND5A variants, 1 of which segregated with the disease in an American family. We found that DENND5A is significantly enriched in pigmented melanoma tissue. Our functional studies show that loss of DENND5A function leads to decrease in melanin content in vitro and pigmentation defects in vivo. Mechanistically, harboring the truncating variant or being suppressed leads to DENND5A losing its interaction with SNX1 and its ability to transport the SNX1-associated vesicles from melanosomes. Consequently, untethered SNX1-premelanosome protein and redundant tyrosinase are redirected to lysosomal degradation by default, causing decrease in melanin content.

Conclusion: Our findings provide evidence of a physiological role of DENND5A in the skin context and link its variants to melanoma susceptibility.

Keywords: DENND5A; Melanoma; Pigmentation; SNX1; Susceptibility gene.

Fig. 1. Rare DENND5A variants identified in multi-case melanoma families.

a. Pedigrees of multi-case melanoma families with heterozygous DENND5A variants. +/+ and +/− indicate the tested family members as DENND5A wildtypes and heterozygotes, respectively. b. A flowchart showing the variant filtering of exome sequencing applied to Family A. c. Chromatograms of the validated DENND5A variants found in indicated families. The 1 bp insertion (c.2903_2904insG) found in Family A is shown on the left, the 1 bp substitution (c.*680C>T) found in Family B is shown on the right in blue. d. Schematic diagram shows the positions of identified variants in DENND5A mRNA (upper panel), and the predicted size of truncated protein (p.Ser969fs) derived from the frameshift variant. a.a. = amino acid. e. DENND5A amino acid alignments of a variety of species (Human, Mouse, Rat, Zebrafish, Chimpanzee, Goat, Chicken, Sheep) highlighting the conserved region flanking the frameshift area (shown in red). f. Representative western blotting and quantification (g) of DENND5A protein in EBV-Lymphoblastoid cells established from 2 unrelated controls and DENND5A heterozygote (III:1) from Family A. h. Correlation of cutaneous melanoma incidence rates, and the aggregated deleterious variants frequency in DENND5A, MC1R, MITF, CDKN2A, CDK4 and POT1 across the globe, including Southern Asian, Eastern Asian, African, Latino, Non-Finnish Europeans, Finnish and Swedish people. Two-tailed, paired t test (g), Pearson correlation (h),

Fig. 2. DENND5A is variably expressed in familial melanomas and enriched in pigmented melanoma tissue.

a. Western blotting of DENND5A protein expression in tumors from familial melanomas from a Karolinska University Hospital-based registry since 2000. b. Quantification of relative DENND5A protein expression. Red dot represents DENND5A protein expression in melanoma (#355) derived from heterozygote (II:1). A dotted line is drawn across the DENND5A heterozygote as reference of potential DENND5A suppression. c. DENND5A mRNA expression in the Skin Cutaneous Melanoma (SKCM) cohort of The Cancer Genome Atlas is classified into ‘Normal-like’ and ‘Pigmentation’ subtype by applying ‘Pigmentation gene signature’ and then compared. (d-f). Spatial transcriptomic (ST) analysis on a cutaneous malignant melanoma (CMM). d. (left) Hematoxylin & Eosin stained CMM section and (right) higher magnification showing the pigmentation from the section. Scale bar: 1 cm (left), 0.25 cm (right). e. ST microarray on CMM section with barcoded spots of 100 μm diameter and 200 μm center-to-center distance. f. A density plot of DENND5A expression ran by trendsceek in the same section, with the regions of significantly elevated DENND5A expression marked red. g. A matrix of ultraviolet (UV)-related alterations (CC>TT in red; dpC>T, C>T transition at dipyrimidines, in pink) from all the Cancer Genome Atlas (TCGA) skin cutaneous melanomas (SKCM), arising from both chronic (CSD) and non-chronic (non-CSD) sun-exposed body sites (n=38). Sample types (primary or metastatic), body sites (CSD, or non-CSD) and detected DENND5A somatic variants are color-coded and then sorted after DENND5A mRNA expression. h. Proportion of UV signature (CC>TT transitions) from DENND5A^High (top 15) and DENND5A^Low (lowest 15) mRNA are compared and shown as mean ± S.E.M.; Two-tailed, Unpaired t test (c, h).

Fig. 3. DENND5A inhibition leads to melanin reduction, morphological changes and diminished PMEL.

a. Representative western blotting image of DENND5A knockdown efficiency in MNT-1 cells. b. Melanin content of MNT-1 cells before and after knockdown of DENND5A, measured and normalized to corresponding protein yields is shown. c. Phalloidin staining of control (siCON) MNT-1 cells and after silencing DENND5A or SNX1, as indicated. d. Morphological changes are quantified by dividing the length of each cell with its width. e. Melanin samples loaded in a 96-well plate showing the visible difference between MNT-1 parental cells, and DENND5A gene knockout clones (#1, #2) established by CRISPR-Cas9 system. f. Relative melanin content of the parental and DENND5A KO #1 and #2 is quantified and shown. g-i. Brightfield images of (g) wild-type zebrafish (Danio Rerio), (h) Dennd5a-knockdown (Dennd5a^{ATG MO}) and (i) Dennd5a-variant mimic (Dennd5a^{splice-site MO}). Higher magnification images of the ventral medial stripes from each group are shown. j. The ventral medial stripes of zebrafish from each group are quantified (10 zebrafish embryos per group) and compared. k. Representative images of the cyclobutane pyrimidine dimer (CPD) staining in parental MNT-1 cells and its DENND5A KO #1 and #2 after UV exposures (30J/m²). l. The intensities of CPD in each area is first quantified and normalized to the intensities of DAPI in the same area and then compared. Scale bar: 20 μm (c), 2 mm (g, h, i). 50 μm (k). m. Schematic chart of the processing of early stage melanosomal cargo PMEL from the ER, trans-Golgi network, to stage I and stage II melanosomes. Antibody PMEL-C recognizes the C-terminus of full-length PMEL precursor and the processed Mβ fragments, both of which are membrane-bound. Antibody HMB45 recognizes the repeated domain (RPT domain) of the full-length PMEL and detects the luminal fragments Mα and MαC, which are crucial for fibrillar matrix formation that is characteristic of Stage II melanosomes. Western blotting (n) and quantification (o) of melanosome markers from different stages of control (siCON) MNT1 and silencing DENND5A (siDENND5A). Quantification is performed from three independent experiments, normalized to controls (b, f, o), and presented as mean ± S.E.M. Two-tailed, Unpaired t test (b, d, f, j, l, o).

Fig. 4. Defective DENND5A triggers lysosomal degradation of melanosomal cargoes and fails to associate with SNX1-decorated vesicles.

a. Representative confocal microscopy images of control MNT-1 (siCON, upper) and after silencing DENND5A (siDENND5A, lower). LAMP1 (green) and PMEL (PMEL-C antibody, red) labeled MNT-1 cells are labeled and shown in merged channels and with higher magnification on the right-hand side. Co-localization is measured using images from two independent experiments and shown in b. Western blotting (c) and quantification (d) of melanosome markers for control (siCON)-MNT1 and siDENND5A-MNT1 after treatment of Golgi-Stop or lysosome protease inhibitor Leupeptin, respectively. e. DENND5A KO #1-MNT-1 cells were transiently introduced with V5-DENND5A^WT (upper) and V5-DENND5A^mt (lower) respectively. Transfected cells are stained for DENND5A (green) and SNX1 (red). Co-localization co-efficiency is measured using images from two independent experiments and shown in (f). Scale bars: 10 μm (a, e), 1 μm for the magnified panels. Data quantification is presented as mean ± S.E.M. Two-tailed, Unpaired t test (d), Mann-Whitney test (b, f). g. Immunoprecipitation of V5-tag on DENND5A KO #1 cells over-expressing the wild-type (V5-DENND5A^WT) and mutated (V5-DENND5A^mt) constructs. h. Proposed working model showing (left) the homeostasis of melanosome maturation and lysosomal degradation coordinated by DENND5A-SNX1 interaction. (right) C-del DENND5A loses its interaction with SNX1 and triggers mis-sorting of PMEL from en route to melanosomes to degradation subdomain (stage I melanosome), resulting in lysosomal entry and degradation of PMEL.