Project Summary: High-risk neuroblastoma (NB) is responsible for ~13% of pediatric cancer-related deaths. Venetoclax (VEN) is a BCL-2 inhibitor that has revolutionized adult hematological cancer care. Unique among solid tumors, ven has demonstrated promising pre-clinical activity in MYCN-amplified NB, but likely will require rational combinations for clinical activity. In our previous funded work, we have demonstrated VEN makes an effective drug partner across several combination therapies (e.g. Lochmann et al. Science Translational Medicine 2018; Dalton et al. Molecular Cancer Therapeutics 2021). In this renewal application, we focus on two clinically translational combination therapies involving two new classes of drugs, SHP2 inhibitors which target the MEK pathway, and BCL-xL targeting antibody-drug conjugate (ADC). In NB, one of the more intriguing discoveries recently has been the hyperactivation of the MAPK/MEK pathway, particularly in relapsed tumors. However, MEK inhibitors have not proven effective in preclinical studies of RAS/RAF wild-type NB, which makes up more than 95% of cases. Here, we demonstrate that in a high-throughput screen (HTS) of a SHP2 allosteric inhibitor, SHP099, that NB are the most sensitive group among all cancers, while MEK inhibitors are relatively ineffective. We provide preclinical evidence that these inhibitors are favorable to MEK inhibitors through a series of experiments. Importantly, SHP2 inhibitors form a rational combination partner with VEN through modulation of BCL-2 family proteins. Lastly, through efforts like a HTS of the co-BCL-2/BCL-xL inhibitor, navitoclax (Ham et al. Cancer Discovery 2016), we have demonstrated MYCN-amplified NB are among the two most sensitive solid tumor cancers, mirroring sensitivity to the VEN data, but with marked enhancement of sensitivity through dual targeting of BCL-xL. This concept has been previously clinically unattainable due to on-target thrombocytopenia from BCL-xL inhibition in platelets. To circumvent this problem, AbbVie has developed a BCL-xL targeting ADC ABBV-155. We find the antigen, B7-H3 (CD276), is widely expressed in NB, and, as a result, combining ABBV-155 with VEN is effective in MYCN-amplified NB. Specific Aims: Specific Aim 1: Evaluate the efficacy and mechanism of activity of SHP2 inhibition + venetoclax in MYCN- amplified neuroblastoma models Specific Aim 2: Evaluate the efficacy and mechanism of activity of platelet-sparing BCL-xL inhibitors + venetoclax in MYCN-amplified neuroblastoma models Study Design: We will further characterize the high sensitivity of SHP2 inhibition with VEN and ABBV-155 with VEN in vitro and in genomically-annotated patient-derived xenograft (PDX) orthotopic models of MYCN- amplified NB. We will perform BH3 profiling (BP) to further evaluate the role of BCL-2 proteins in sensitivity and investigate whether BP and dynamic BP will help us identify sensitive and resistant NBs to each combination.