Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is a common event in human cancer, either through inactivation of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 or activating mutations of p110-A. These hotspot mutations result in oncogenic activity of the enzyme and contribute to therapeutic resistance to the anti-HER2 antibody trastuzumab. The PI3K pathway is, therefore, an attractive target for cancer therapy. We have studied NVP-
Small molecule inhibitors of HER2 are clinically active in women with advanced HER2 positive breast cancer who have progressed on trastuzumab treatment. However, the effectiveness of this class of agents is limited by either primary resistance or acquired resistance. Using an unbiased genetic approach we performed a genome wide loss-of-function shRNA screen to identify novel modulators of resistance to lapatinib, a recently approved anti-HER2 tyrosine kinase inhibitor. Here, we have identified the tumour suppressor PTEN as a modulator of lapatinib sensitivity in vitro and in vivo. In addition, we demonstrate that two dominant activating mutations in PIK3CA (E545K and H1047R), which are prevalent in breast cancer, also confer resistance to lapatinib. Furthermore, we show that PI3K induced lapatinib resistance can be abrogated through the use of NVP-BEZ235, a dual inhibitor of PI3K/mTOR. Our data show that deregulation of the PI3K pathway, either through loss-of-function mutations in PTEN or dominant activating mutations in PIK3CA, leads to lapatinib resistance which can be effectively reversed by NVP-BEZ235.
e One of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight into the poorly recognized function of macroH2A in transcriptional activation and demonstrate its relevance in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells.
Genetic loss-of-function studies on development, cancer and somatic cell reprogramming have suggested that the group of macroH2A histone variants might function through stabilizing the differentiated state by a yet unknown mechanism. Here, we present results demonstrating that macroH2A variants have a major function in maintaining nuclear organization and heterochromatin architecture. Specifically, we find that a substantial amount of macroH2A is associated with heterochromatic repeat sequences. We further identify macroH2A on sites of interstitial heterochromatin decorated by histone H3 trimethylated on K9 (H3K9me3). Loss of macroH2A leads to major defects in nuclear organization, including reduced nuclear circularity, disruption of nucleoli and a global loss of dense heterochromatin. Domains formed by DNA repeat sequences are disorganized, expanded and fragmented, and mildly re-expressed when depleted of macroH2A. At the molecular level, we find that macroH2A is required for the interaction of repeat sequences with the nucleostructural protein lamin B1. Taken together, our results argue that a major function of macroH2A histone variants is to link nucleosome composition to higher-order chromatin architecture.
Histone variants are structural components of eukaryotic chromatin that can replace replication-coupled histones in the nucleosome. The histone variant macroH2A.1.1 contains a macrodomain able to bind NAD+ derived metabolites. Here, we report that macroH2A.1.1 is rapidly induced during myogenic differentiation through a switch in alternative splicing. Importantly, myotubes lacking macroH2A.1.1 display a defect in mitochondrial respiratory capacity. We find that the metabolite-interacting macrodomain is essential for sustaining optimal mitochondrial function, but dispensable for gene regulation. Through direct binding, macroH2A.1.1 inhibits basal poly-ADP ribose polymerase 1 activity and thus reduces nuclear NAD+ consumption. Consequentially, accumulation of the NAD+ precursor NMN allows the maintenance of mitochondrial NAD+ pools critical for respiration.Our data indicate that macroH2A.1.1-containing chromatin regulates mitochondrial respiration by limiting nuclear NAD+ consumption and establishing a buffer of NAD+ precursors in differentiated cells.
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