Final Report Summary - BET(TER) TARGETS (Targeting BET Bromodomains in Cancer –<br/>Mechanisms of Sensitivity and Resistance) Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukemia (AML), BET inhibitors are being explored as promising therapeutic avenue in numerous cancers. While clinical trials are already underway, the mechanisms underlying sensitivity and resistance to these agents remain poorly understood. This project aimed to identify factors involved in primary and acquired BET inhibitor resistance in leukemia using two complementary approaches.Objective 1: Characterization of BRD4 protein complexes in sensitive vs. resistant cells and analyze the functional relevance of differential complex members using advanced RNAi tools. So far we have profiled the composition of the BRD4 complex through affinity purification of endogenous BRD4 followed by quantitative mass spectrometry (MS) in 2 sensitive and 2 resistant leukemia cell lines. This resulted in the significant identification of a large variety of nuclear proteins. 1) We’ve seen great complexity that is highly diverse in all analyzed cell lines, suggesting that the BRD4 interactome is very complex and highly context dependent.(2) With respect to the intended differentiation between sens/resistant cell lines, this complexity poses a challenge for identifying interactions that are specific to sens/res scenarios, which (if existing) can only be identified in a large series of analyses that seems not feasible within the scope of this project.As a backup strategy we profiled in total 18 human cancer cell lines from 3 different tissue contexts via dynamic RNA sequencing with and without a BET inhibitor. This approach allowed us to identify a gene set in leukemia that is differentially expressed between sensitive and resistant cell lines. Following studies using RNAi against a small number of genes in conjunction with a BET inhibitor showed a synthetic lethal relationship. Additional studies with an inhibitor targeting the associated signaling pathway demonstrated a combinatorial effect with BET inhibition. BET inhibition triggers acute repression of MYC in human leukemias regardless of their sensitivity profile, resistant leukemias are uniformly characterized by their ability to rapidly restore MYC transcription. Profiling and functional studies reveal that this process involves the transcriptional activation and recruitment of WNT signaling components, which compensate for the loss of BRD4 and drive resistance to BET inhibition in various cancer models.Objective 2: Identification and validation of mediators of BET inhibitor sensitivity and resistance using multiplexed RNAi screening in vitro and in vivo.We performed a chromatin-focused shRNAmir screen in a sensitive MLL/AF9;NrasG12D driven AML mouse model, and investigated dynamic transcriptional profiles in sensitive and resistant murine and human leukemia. As the top hit, our screen reveals that suppression of the PRC2 complex, contrary to effects in other contexts, renders AML cells resistant to BET inhibition. Suppression of PRC2 does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodeling of regulatory pathways that restore the transcription of key Brd4 targets such as Myc. Objective 3: Further characterization of the identified targets. Depending on the nature of the target we conceive to profile downstream effects of RNA mediated suppression by RNAseq and ChIPseq. In parallel we will validate the hits from Objective 1 and 2 in different in vivo models.Using dynamic ChIP- and STARR-seq enhancer profiling we show that resistant states are characterized by a remodeling of the regulatory landscape, which includes the activation of a focal MYC enhancer element that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signaling as a driver and candidate biomarker of primary and acquired BET inhibitor resistance in leukemia, and implicate the transcriptional rewiring of oncogenic expression programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.Within only three years BET inhibitors have taken an astonishing path from the discovery of BRD4 as a genetic target to first exciting data from clinical phase-I trials. Despite this rapid advance, the cellular mechanisms underlying the vast variability in sensitivities to BET inhibition remain poorly understood, and (despite major efforts in academic and commercial research) there is still no genetic or epigenetic biomarker to predict the response to these agents. Our study tackles this challenging problem using a multifaceted approach involving functional genetic screening, transcriptional and enhancer profiling, and functional-genetic studies in mouse models, human leukemia and primary patient samples. Our study reveals that BRD4 regulates a highly specialized and remarkably diverse set of target genes, even in cancers of similar tissue context and drug sensitivity. Leukemia cells can become resistant to BET inhibition by rewiring the transcriptional regulation of key BRD4 target genes such as MYC. Our study establishes that in myeloid leukemia the BRD4-depency of MYC can be attenuated through compensatory transactivation of distinct signaling pathways, which implicates signaling components as drivers and potential biomarkers of primary and acquired BET inhibitor resistance. From a more general perspective, our results highlight that the heterogeneity and plasticity of transcriptional machinery plays a major role in promoting primary and acquired resistance to chromatin-targeted therapies.