Endogenous DOPA inhibits melanoma through suppression of CHRM1 signaling

Melanoma risk is 30 times higher in people with lightly pigmented skin versus darkly pigmented skin. Using primary human melanocytes representing the full human skin pigment continuum and preclinical melanoma models, we show that cell-intrinsic differences between dark and light melanocytes regulate melanocyte proliferative capacity and susceptibility to malignant transformation, independent of melanin and ultraviolet exposure. These differences result from dihydroxyphenylalanine (DOPA), a melanin precursor synthesized at higher levels in melanocytes from darkly pigmented skin. We used both high-throughput pharmacologic and genetic in vivo CRISPR screens to determine that DOPA limits melanocyte and melanoma cell proliferation by inhibiting the muscarinic acetylcholine receptor M1 (CHRM1) signaling. Pharmacologic CHRM1 antagonism in melanoma leads to depletion of c-Myc and FOXM1, both of which are proliferation drivers associated with aggressive melanoma. In preclinical mouse melanoma models, pharmacologic inhibition of CHRM1 or FOXM1 inhibited tumor growth. CHRM1 and FOXM1 may be new therapeutic targets for melanoma

Targeting anti-apoptotic pathways eliminates senescent melanocytes and leads to nevi regression

Human melanocytic nevi (moles) result from a brief period of clonal expansion of melanocytes. As a cellular defensive mechanism against oncogene-induced hyperplasia, nevus-resident melanocytes enter a senescent state of stable cell cycle arrest. Senescent melanocytes can persist for months in mice and years in humans with a risk to escape the senescent state and progress to melanoma. The mechanisms providing prolonged survival of senescent melanocytes remain poorly understood. Here, we show that senescent melanocytes in culture and in nevi express high level of the anti-apoptotic BCL-2 family member BCL-W but remain insensitive to the pan-BCL-2 inhibitor ABT-263. We demonstrate that resistance to ABT-263 is driven by mTOR-mediated enhanced translation of another anti-apoptotic member, MCL-1. Strikingly, the combination of ABT-263 and MCL-1 inhibitors results in synthetic lethality to senescent melanocytes, and its topical application sufficient to eliminate nevi in male mice. These data highlight the important role of redundant anti-apoptotic mechanisms for the survival advantage of senescent melanocytes, and the proof-of-concept for a non-invasive combination therapy for nevi removal

Supplementary Figure S7 from LNS8801 inhibits Acute Myeloid Leukemia by inducing the production of reactive oxygen species and activating the endoplasmic reticulum stress pathway

Supplementary Figure S7. LNS8801-induced AML inhibition is independent of classical GPER signaling (A) Annexin V+ flow cytometry analysis of U937 cells treated with 250nM LNS8801, 10uM Forskolin, 100uM EPAC-specific cAMP (8-pCPT-2'-O-Me-cAMP), and 100uM PKA-specific cAMP (N6-benzoyl-cAMP). Cells were incubated for 24 hours. 3 replicates per condition were used. (B) Detection of intracellular calcium upon drug treatment via a spectrofluorimeter assay. Fura2-AM dye was used for detection. 25nM E2, 250nM LNS8801, 1uM Ionomycin, 2uM thapsigargin, and 100uM histamine were used. 3 replicates were used per condition.</p

FIGURE 7 from LNS8801 inhibits Acute Myeloid Leukemia by Inducing the Production of Reactive Oxygen Species and Activating the Endoplasmic Reticulum Stress Pathway

LNS8801 inhibits AML in subcutaneous in vivo model but not systemic model. A, Systemic in vivo experiment using luciferase-expressing MOLM14 cells (MOLM14-Luc). Male NSG mice were used for the experiment. Mice were treated with vehicle or LNS8801 daily through oral gavage. Three animals were used per condition. B, Subcutaneous tumor volume of MOLM14 in NSG mice treated orally with vehicle or 10 mg/kg LNS8801 daily. Graph shows summary of two independent experiments. Conditions for each experiment were identical. Four mice were used per group per experiment, totaling 8 mice per condition. C, Survival curve of NSG mice with subcutaneous MOLM14 tumors. Statistical analysis for the survival curve was done via log-rank (Mantel–Cox) test and Grehan–Breslow–Wilcoxon test (****, P ≤ 0.0001).</p

Supplementary Table S2 from LNS8801 inhibits Acute Myeloid Leukemia by inducing the production of reactive oxygen species and activating the endoplasmic reticulum stress pathway

Supplementary Table S2</p

Supplementary Figure S5 from LNS8801 inhibits Acute Myeloid Leukemia by inducing the production of reactive oxygen species and activating the endoplasmic reticulum stress pathway

Supplementary Figure S5. GPER knockdown in MOLM14 Western blot of GPER knockdown clones of MOLM14 cells.</p

Supplementary Figure S10 from LNS8801 inhibits Acute Myeloid Leukemia by Inducing the Production of Reactive Oxygen Species and Activating the Endoplasmic Reticulum Stress Pathway

Supplementary Figure S10. LNS8801 induces cell death independently of the classical secreted death ligands (A) Time course western blot of U937 cells treated with 250nM LNS8801. (B) Annexin V staining flow analysis of U937 cells treated with either 20ng/mL TNF⍺, 10ug/mL TNFR1 antibody, or 250nM LNS8801. Cells were incubated for 72 hours. 3 replicates were used per condition. (C) Annexin V staining flow analysis of U937 cells treated with LNS8801-treated U937 conditioned media. Cells were incubated in conditioned media for 72 hours. 3 replicates were used per condition. One-way ANOVA was used for statistical analysis (*p=<0.05, **p=<0.01, ***p=<0.001, ****p=<0.0001).</p

Supplementary Figure S11 from LNS8801 inhibits Acute Myeloid Leukemia by Inducing the Production of Reactive Oxygen Species and Activating the Endoplasmic Reticulum Stress Pathway

(A and C) Representative DCF histograms of (A) U937 and (C) MOLM14 cells treated with 250nM LNS8812 or 250nM LNS8801 at indicated time points. (B and D) Summary graph of median fluorescence intensity (MFI) of (B) U937 and (D) MOLM14 cells treated with 250nM LNS8812 or 250nM LNS8801. 3 replicates were analyzed per condition. Statistical analysis was done via one-way ANOVA with multiple comparisons was used for panels B and D (*p=<0.05, **p=<0.01, ***p=<0.001, ****p=<0.0001).</p

Supplementary Table S1 from LNS8801 inhibits Acute Myeloid Leukemia by Inducing the Production of Reactive Oxygen Species and Activating the Endoplasmic Reticulum Stress Pathway

Supplementary Table S1</p

Supplementary Figure S12 from LNS8801 inhibits Acute Myeloid Leukemia by Inducing the Production of Reactive Oxygen Species and Activating the Endoplasmic Reticulum Stress Pathway

(A and B) Western blots of (A) U937 and (B) MOLM14 cells treated with 25nM estradiol (E2) or 50nM thapsigargin (Tg) for indicated time. Western blot quantification was done by normalizing each band to its respective actin band.</p