Project Summary/Abstract. NUT-fusion oncoproteins, most commonly BRD4-NUT, drive NUT carcinoma (NC), an aggressive, poorly differentiated squamous cancer with a 6.5-month median survival and < 20% 3- year survival. NC predominantly affects adolescents and young adults and, with no routinely effective therapy, has a critical unmet need. BRD4-NUT drives growth by altering histone modifications and chromatin 3D structure to upregulate expression of pro-growth genes. We demonstrated that BET bromodomain inhibitors reverse these epigenetic modifications and block tumor growth. These findings led to the treatment of NC and numerous other malignancies with BET inhibitors. However, the clinical efficacy of BET inhibitor monotherapy is limited, indicating that BET inhibitors alone do not fully address NC biology. The goal of this proposal is to overcome the limitations of BET inhibition in NC and therefore improve patient outcomes by identifying targetable mechanisms of gene regulation that maintain NC growth. Using our combined expertise in translational NC cancer biology (CA French) and chromatin biology (KP Eagen) we will take a two-pronged approach to improve NC treatment through mechanism-based target discovery. Both prongs share the premise that identifying factors that cooperate with BRD4-NUT to promote NC growth will lead to new therapeutic targets. Our first Aim is based upon our recent discovery that repression of tumor suppressor genes by the histone methyltransferase EZH2 complements oncogene activation by BRD4-NUT. Simultaneous inhibition of EZH2 and BRD4-NUT synergistically blocks NC growth in vitro and in vivo. However, the precise mechanism of synergy remains unknown. Additionally, toxicity was observed in some xenograft models, and thus toxicity in humans is likely. Therefore, we propose to identify more precise therapeutic targets by determining key factors that mediate the oncogenic synergy between EZH2 and BRD4-NUT. Our second Aim is motivated by the discovery of a novel MLL1::NUTM1 fusion in a patient. Our proteomics data indicate that MLL1 and its key binding partner MENIN are both present in the BRD4-NUT oncogenic complex providing biochemical evidence that this fusion may reveal new factors that cooperate with BRD4-NUT. Accordingly, inhibiting the interaction between MLL1 and MENIN induces differentiation and blocks growth of NC cells. The combination of MENIN-MLL1 with EZH2 inhibition synergistically blocks NC growth in vitro. This data suggests that combination therapy with MENIN-MLL1 and EZH2 inhibitors could potentially replace BET inhibition, which we will test in pre-clinical models of NC. Aim 1. Determine the mechanism by which EZH2 cooperates with BRD4-NUT to drive NC growth. Aim 2. Elucidate the role of MENIN in BRD4-NUT oncogenic function.

Project Narrative NUT carcinoma is an incurable highly aggressive cancer driven by the BRD4-NUT oncoprotein that has a median survival of 6.5 months with no effective treatment options. We predict the proposed studies will identify novel mechanisms that synergize to drive NC growth while blocking differentiation that will facilitate precise and effective therapeutic targeting. The findings will greatly impact treatment of NC and likely other BRD4-dependent and pediatric fusion-driven cancers.