Neuroblastoma reliance on DNMT1 through amplified MYCN

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Project Summary: MYCN-amplified neuroblastoma is a deadly disease. Despite years of evidence that amplified MYCN drives this dangerous subset of NB, no indirect or direct inhibitors of MYCN have been demonstrated clinically, and thus, new therapies for MYCN-amplified NB remains a high-level priority in pediatric cancer therapy. In this current proposal, we demonstrate that DNA (cytosine-5)- methyltransferase 1 (DNMT1) inhibition is synthetic lethal to MYCN amplified NBs. In addition, we have found DNMT1 inhibitors act as MYCN inhibitors, creating a feedback that can be exploited pharmaceutically. As the DNMT1 inhibitor decitabine is clinically approved, and guadecitabine (SGI- 110) is in clinical trials with our clinical collaborator serving as PI in one of the trials (John Glod, NCI Pediatric Branch), this grant aims to further define the sensitivity of DNMT1 inhibitors in MYCN- amplified NB. Objectives: Our objective is to test the hypothesis that DNMT1 inhibitors are synthetic lethal to amplified MYCN in NB, and that amplified MYCN drives DNMT1 expression to cause global methylation including at key tumor suppressor loci, as well as suppression of a key synthase gene of GD2. As GD2 synthase expression increases rapidly with DNMT1 inhibitors, we propose DNMT1 inhibitors can be rationally combined with the anti-GD2 inhibitor dinutuximab, used routinely in the maintenance phase of MYCN-amplified NBs. Specific Aims: Specific Aim 1. Characterize the sensitivity to, and mechanism of, DNMT1 inhibition in MYCN-amplified neuroblastoma Specific Aim 2: Determine the efficacy and tolerability of DNMT1 inhibitors in diverse mouse models of MYCN- amplified neuroblastoma, alone and in combination with standard maintenance therapy Study Design: We have found that DNMT1 inhibition is effective in MYCN-amplified NB. We will further characterize the role of amplified MYCN in DNMT1 inhibitor sensitivity, using cell culture models where we will manipulate MYCN expression, and verify that the activity we see in both chemically and biologically distinct DNMT1 inhibitors is in fact on-target through DNMT1 genetic studies. We will perform ChIP and other studies to explore whether MYCN directly regulates DNMT1 to effect the methylation of the genome. We will determine more closely what the key DNMT1 targets are in MYCN-amplified NB. Additionally, we will interrogate a collection of MYCN-amplified mouse models, including GEMMS and PDXs, to determine the activity of both decitabine and guadecitabine. Lastly, we have found DNMT1 inhibition affects GD2 synthesis and key retinoic acid (RA) resistant genes, and we will determine whether DNMT1 inhibitors sensitize to the anti-GD2 antibody dinutuximab and to RA in vivo , both of which ire used for maintenance therapy in MYCN-amplified NB.