BET Bromodomain Inhibition Suppresses the Function of Hematopoietic Transcription Factors in Acute Myeloid Leukemia.

The bromodomain and extraterminal (BET) protein BRD4 is a validated drug target in leukemia, yet its regulatory function in this disease is not well understood. Here, we show that BRD4 chromatin occupancy in acute myeloid leukemia closely correlates with the hematopoietic transcription factors (TFs) PU.1, FLI1, ERG, C/EBPα, C/EBPß, and MYB at nucleosome-depleted enhancer and promoter regions. We provide evidence that these TFs, in conjunction with the lysine acetyltransferase activity of p300/CBP, facilitate BRD4 recruitment to their occupied sites to promote transcriptional activation. Chemical inhibition of BET bromodomains was found to suppress the functional output of each hematopoietic TF, thereby interfering with essential lineage-specific transcriptional circuits in this disease. These findings reveal a chromatin-based signaling cascade comprised of hematopoietic TFs, p300/CBP, and BRD4 that supports leukemia maintenance and is suppressed by BET bromodomain inhibition.

Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation.

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in ∼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.

MAP kinase phosphatase-1 deficiency impairs skeletal muscle regeneration and exacerbates muscular dystrophy.

In skeletal muscle, the mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a critical negative regulator of the MAPKs. Since the MAPKs have been reported to be both positive and negative for myogenesis, the physiological role of MKP-1 in skeletal muscle repair and regeneration has remained unclear. Here, we show that MKP-1 plays an essential role in adult regenerative myogenesis. In a cardiotoxin-induced muscle injury model, lack of MKP-1 impaired muscle regeneration. In mdx mice, MKP-1 deficiency reduced body weight, muscle mass, and muscle fiber cross-sectional area. In addition, MKP-1-deficient muscles exhibit exacerbated myopathy accompanied by increased inflammation. Lack of MKP-1 compromised myoblast proliferation and induced precocious differentiation, phenotypes that were rescued by pharmacological inhibition of p38alpha/beta MAPK. MKP-1 coordinates both myoblast proliferation and differentiation. Mechanistically, MyoD bound to the MKP-1 promoter and activated MKP-1 expression in proliferating myoblasts. Later, during myogenesis, MyoD uncoupled from the MKP-1 promoter leading to the down-regulation of MKP-1 and facilitation of promyogenic p38alpha/beta MAPK signaling. Hence, MKP-1 plays a critical role in muscle stem cells and in the immune response to coordinate muscle repair and regeneration.

Role of MKP-1 in osteoclasts and bone homeostasis.

Bone mass is maintained through the complementary activities of osteoblasts and osteoclasts; yet differentiation of either osteoblasts and osteoclasts engages the mitogen-activated protein kinase (MAPK) pathway. The MAPKs are negatively regulated by a family of dual-specificity phosphatases known as the MAPK phosphatases (MKPs). MKP-1 is a stress-responsive MKP that inactivates the MAPKs and plays a central role in macrophages; however, whether MKP-1 plays a role in the maintenance of bone mass has yet to be investigated. We show here, using a genetic approach, that mkp-1(-/-) female mice exhibited slightly reduced bone mass. We found that mkp-1(+/+) and mkp-1(-/-) mice had equivalent levels of bone loss after ovariectomy despite mkp-1(-/-) mice having fewer osteoclasts, suggesting that mkp-1(-/-) osteoclasts are hyperactive. Indeed, deletion of MKP1 led to a profound activation of osteoclasts in vivo in response to local lipopolysaccharide (LPS) injection. These results suggest a role for MKP-1 in osteoclasts, which originate from the fusion of macrophages. In support of these observations, receptor activator for nuclear factor-kappaB ligand induced the expression for MKP-1, and osteoclasts derived from mkp-1(-/-) mice had increased resorptive activity. Finally, receptor activator of nuclear factor-kappaB ligand-induced p38 MAPK and c-Jun NH2-terminal kinase activities were enhanced in osteoclasts derived from mkp-1(-/-) mice. Taken together, these results show that MKP-1 plays a role in the maintenance of bone mass and does so by negatively regulating MAPK-dependent osteoclast signaling.

Novel role for SHP-2 in nutrient-responsive control of S6 kinase 1 signaling.

Amino acids are required for the activation of the mammalian target of rapamycin complex 1 (mTORC1), which plays a critical role in cell growth, proliferation, and metabolism. The branched-chain amino acid leucine is an essential nutrient that stimulates mTORC1 to promote protein synthesis by activating p70 S6 kinase 1 (S6K1). Here we show that the protein tyrosine phosphatase SHP-2 is required for leucine-induced activation of S6K1 in skeletal myoblasts. In response to leucine, S6K1 activation is inhibited in myoblasts either lacking SHP-2 expression or overexpressing a catalytically inactive mutant of SHP-2. Activation of S6K1 by leucine requires the mobilization of intracellular calcium (Ca(2+)), which we show is mediated by SHP-2 in an inositol-1,4,5-trisphosphate-dependent manner. Ectopic Ca(2+) mobilization rescued the S6K1 activation defect in SHP-2-deficient myoblasts. SHP-2 was identified to act upstream of phospholipase C ß4, linking it to the generation of nutrient-induced Ca(2+) release and S6K1 phosphorylation. Consistent with these results, SHP-2-deficient myoblasts exhibited impaired leucine sensing, leading to defective autophagy and reduced myoblast size. These data define a new role for SHP-2 as a nutrient-sensing regulator in skeletal myoblasts that is required for the activation of S6K1.

Analysis of protein tyrosine phosphatases and substrates.

Protein tyrosine phosphorylation is a reversible post-translational modification that is essential for life in eukaryotic cells. The combinatorial action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs) determines the net level of cellular tyrosine phosphorylation. This unit discusses methods to determine the level of protein tyrosine phosphatase activity and methods for discovering novel substrates for protein tyrosine phosphatases.