Abstract Congenital Melanocytic Nevi (CMN) can reach giant sizes, transform to childhood melanoma, and thus trigger pre-emptive surgery inducing profound morbidity. In the prior funding period, we constructed four CMN models to test regression-inducing treatments aimed at avoiding surgery. Three are genetically engineered mice driven by the (floxed)NRASQ61R oncogene, responsible for most human CMN, with melanocyte-targeting via tamoxifen- inducible or constitutive expression from Tyr or Dct promoters (varied strengths). The fourth model is xenografted resected human CMNs on immunodeficient mice. Several models displayed melanoma transformation after long latency. Inducible NRASQ61R clarified the kinetics of proliferative vs senescent nevus phases and verified regressive treatments to succeed in either phase. Tyr-Cre;NRASQ61R exhibited severe human-like CNS and skin lesions whereas the weaker Dct-Cre induced a more typical skin-restricted giant nevi for which inhibitors of MEK, PI3K, or cKIT induced incomplete (~80%) regressions and combinations fully depleted visual nevocytes. Topical treatment with the proinflammatory hapten Squaric Acid Dibutyl Ester (SADBE) induced complete visual CMN regression after 3 doses. This regimen also fully prevented murine melanoma transformation, within all treated nevus skin. We found that topical SADBE requires macrophages (not B, T, or NK cells), so it could be tested in human CMN xenografted onto SHO mice (which lack adaptive immunity but retain macrophages). SADBE recruited murine macrophages into the human xenografts and regressed ~90% of the human nevocytes. Since SADBE is already used elsewhere in dermatology, and has been reported to induce “depigmentation,” we plan to test it clinically. However, since clinical trials with efficacy endpoints are not supported in R01s from NIAMS, we are seeking separate funding to support this clinical trial that already received FDA/IND and IRB approvals. While we are excited by the models and strategies uncovered during this funding period, we believe it is crucial to build on this progress to increase the likelihood of achieving real therapeutic breakthrough for this devastating children’s disease. Accordingly Aims 1-3 use our models to boldly seek further enhanced efficacy and minimized toxicity of treatments: Aim 1 deeply and systematically develops kinase inhibitor combinations. It also tests MEK inhibitors (MEKi) with SADBE, as both exhibit individual efficacy and MEKi was recently shown to promote M1- like (pro-inflammatory) over M2-like macrophage differentiation, a mechanistic feature that may enhance SADBE’s efficacy. Aim 2 seeks localized T cell adaptive immunity recruitment, to complement SADBE’s macrophage induction. We have shown anti-PD1 triggers recruitment of melanocyte (gp100) targeted T-cells when combined with topical SADBE. We will also combine fractional laser treatment plus anti-PD1 which, as we recently published, also triggers local recruitment of...