Cell state-dependent chromatin targeting in NUT carcinoma.

Aberrant transcriptional programming and chromatin dysregulation are common to most cancers. Whether by deranged cell signaling or environmental insult, the resulting oncogenic phenotype is typically manifested in transcriptional changes characteristic of undifferentiated cell growth. Here we analyze targeting of an oncogenic fusion protein, BRD4-NUT, composed of two normally independent chromatin regulators. The fusion causes the formation of large hyperacetylated genomic regions or megadomains, mis-regulation of c-MYC , and an aggressive carcinoma of squamous cell origin. Our previous work revealed largely distinct megadomain locations in different NUT carcinoma patient cell lines. To assess whether this was due to variations in individual genome sequences or epigenetic cell state, we expressed BRD4-NUT in a human stem cell model and found that megadomains formed in dissimilar patterns when comparing cells in the pluripotent state with the same cell line following induction along a mesodermal lineage. Thus, our work implicates initial cell state as the critical factor in the locations of BRD4-NUT megadomains. These results, together with our analysis of c-MYC protein-protein interactions in a patient cell line, are consistent with a cascade of chromatin misregulation underlying NUT carcinoma.

Cell state-dependent chromatin targeting in NUT carcinoma.

Aberrant transcriptional programming and chromatin dysregulation are common to most cancers. Whether by deranged cell signaling or environmental insult, the resulting oncogenic phenotype is typically manifested in transcriptional changes characteristic of undifferentiated cell growth. Here we analyze targeting of an oncogenic fusion protein, BRD4-NUT, composed of 2 normally independent chromatin regulators. The fusion causes the formation of large hyperacetylated genomic regions or megadomains, mis-regulation of c-MYC, and an aggressive carcinoma of squamous cell origin. Our previous work revealed largely distinct megadomain locations in different NUT carcinoma patient cell lines. To assess whether this was due to variations in individual genome sequences or epigenetic cell state, we expressed BRD4-NUT in a human stem cell model and found that megadomains formed in dissimilar patterns when comparing cells in the pluripotent state with the same cell line following induction along a mesodermal lineage. Thus, our work implicates initial cell state as the critical factor in the locations of BRD4-NUT megadomains. These results, together with our analysis of c-MYC protein-protein interactions in a patient cell line, are consistent with a cascade of chromatin misregulation underlying NUT carcinoma.

Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior.

Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.

Combination Targeting of the Bromodomain and Acetyltransferase Active Site of p300/CBP.

p300 and CBP are highly related histone acetyltransferase (HAT) enzymes that regulate gene expression, and their dysregulation has been linked to cancer and other diseases. p300/CBP is composed of a number of domains including a HAT domain, which is inhibited by the small molecule A-485, and an acetyl-lysine binding bromodomain, which was recently found to be selectively antagonized by the small molecule I-CBP112. Here we show that the combination of I-CBP112 and A-485 can synergize to inhibit prostate cancer cell proliferation. We find that the combination confers a dramatic reduction in p300 chromatin occupancy compared to the individual effects of blocking either domain alone. Accompanying this loss of p300 on chromatin, combination treatment leads to the reduction of specific mRNAs including androgen-dependent and pro-oncogenic prostate genes such as KLK3 (PSA) and c-Myc. Consistent with p300 directly affecting gene expression, mRNAs that are significantly reduced by combination treatment also exhibit a strong reduction in p300 chromatin occupancy at their gene promoters. The relatively few mRNAs that are up-regulated upon combination treatment show no correlation with p300 occupancy. These studies provide support for the pharmacologic advantage of concurrent targeting of two domains within one key epigenetic modification enzyme.

Inhibition of MEK and ATR is effective in a B-cell acute lymphoblastic leukemia model driven by Mll-Af4 and activated Ras.

Infant B-cell acute lymphoblastic leukemias (B-ALLs) that harbor MLL-AF4 rearrangements are associated with a poor prognosis. One important obstacle to progress for this patient population is the lack of immunocompetent models that faithfully recapitulate the short latency and aggressiveness of this disease. Recent whole-genome sequencing of MLL-AF4 B-ALL samples revealed a high frequency of activating RAS mutations; however, single-agent targeting of downstream effectors of the RAS pathway in these mutated MLL-r B-ALLs has demonstrated limited and nondurable antileukemic effects. Here, we demonstrate that the expression of activating mutant N-Ras G12D cooperates with Mll-Af4 to generate a highly aggressive serially transplantable B-ALL in mice. We used our novel mouse model to test the sensitivity of Mll-Af4/N-Ras G12D leukemia to small molecule inhibitors and found potent and synergistic preclinical efficacy of dual targeting of the Mek and Atr pathways in mouse- and patient-derived xenografts with both mutations in vivo, suggesting this combination as an attractive therapeutic opportunity that might be used to treat patients with these mutations. Our studies indicate that this mouse model of Mll-Af4/N-Ras B-ALL is a powerful tool to explore the molecular and genetic pathogenesis of this disease subtype, as well as a preclinical discovery platform for novel therapeutic strategies.