Charles S. Hemenway

Calcineurin. Structure, function, and inhibition.

Calcineurin is a serine-threonine specific Ca(2+)-calmodulin-activated protein phosphatase that is conserved from yeast to humans. Remarkably, this enzyme is the common target for two novel and structurally unrelated immunosuppressive antifungal drugs, cyclosporin A and FK506. Both drugs form complexes with abundant intracellular binding proteins, cyclosporin A with cyclophilin A and FK506 with FKBP 12, which bind to and inhibit calcineurin. The X-ray structure of an FKPB12-FK506-calcineurin AB ternary complex reveals that FKBP12-FK506 binds in a hydophobic groove between the calcineurin A catalytic and the regulatory B subunit, in accord with biochemical and genetic studies on inhibitor action. Calcineurin plays a key role in regulating the transcription factor NF-AT during T-cell activation, and in mediating responses of microorganisms to cation stress. These findings highlight the potential of yeast genetic studies to define novel drug targets and elucidate conserved elements of signal transduction cascades.

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases

The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.

Dot1a-AF9 Complex Mediates Histone H3 Lys-79 Hypermethylation and Repression of ENaCα in an Aldosterone-sensitive Manner

Aldosterone is a major regulator of epithelial Na+ absorption and acts in large part through induction of the epithelial Na+ channel (ENaC) gene in the renal collecting duct. We previously identified Dot1a as an aldosterone early repressed gene and a repressor of ENaCα transcription through mediating histone H3 Lys-79 methylation associated with the ENaCα promoter. Here, we report a novel aldosterone-signaling network involving AF9, Dot1a, and ENaCα. AF9 and Dot1a interact in vitro and in vivo as evidenced in multiple assays and colocalize in the nuclei of mIMCD3 renal collecting duct cells. Overexpression of AF9 results in hypermethylation of histone H3 Lys-79 at the endogenous ENaCα promoter at most, but not all subregions examined, repression of endogenous ENaCα mRNA expression and acts synergistically with Dot1a to inhibit ENaCα promoter-luciferase constructs. In contrast, RNA interference-mediated knockdown of AF9 causes the opposite effects. Chromatin immunoprecipitation assays reveal that overexpressed FLAG-AF9, endogenous AF9, and Dot1a are each associated with the ENaCα promoter. Aldosterone negatively regulates AF9 expression at both mRNA and protein levels. Thus, Dot1a-AF9 modulates histone H3 Lys-79 methylation at the ENaCα promoter and represses ENaCα transcription in an aldosterone-sensitive manner. This mechanism appears to be more broadly applicable to other aldosterone-regulated genes because overexpression of AF9 alone or in combination with Dot1a inhibited mRNA levels of three other known aldosterone-inducible genes in mIMCD3 cells.

Histone H3 lysine 79 methyltransferase Dot1 is required for immortalization by MLL oncogenes.

Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However, the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1, the only known histone H3 lysine 79 (H3K79) methyltransferase, has been shown to interact with multiple MLL fusion partners including AF9, ENL, AF10, and AF17. In this study, we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9, we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis, suggesting the involvement of Dot1 in survival pathways. In summary, our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target.

MLL fusion partners AF4 and AF9 interact at subnuclear foci

The MLL gene is involved in translocations associated with both acute lymphoblastic and acute myelogenous leukemia. These translocations fuse MLL with one of over 30 partner genes. Collectively, the MLL partner genes do not share a common structural motif or biochemical function. We have identified a protein interaction between the two most common MLL fusion partners AF4 and AF9. This interaction is restricted to discrete nuclear foci we have named ‘AF4 bodies’. The AF4 body is non-nucleolar and is not coincident with any known nuclear structures we have examined. The AF4–AF9 interaction is maintained by the MLL–AF4 fusion protein, and expression of the MLL–AF4 fusion can alter the subnuclear localization of AF9. In view of other research indicating that other MLL fusion partners also interact with one another, these results suggest that MLL fusion partners may participate in a web of protein interactions with a common functional goal. The disruption of this web of interactions by fusion with MLL may be important to leukemogenesis.

The polycomb protein MPc3 interacts with AF9, an MLL fusion partner in t(9;11)(p22;q23) acute leukemias.

Polycomb group (PcG) proteins assemble to form large multiprotein complexes involved in gene silencing. Evidence suggests that PcG complexes are heterogeneous with respect to both protein composition and specific function. MPc3 is a recently described mouse Polycomb (Pc) protein that shares structural homology with at least two other Pc proteins, M33 and MPc2. All three Pc proteins bind another PcG protein, RING1, through a conserved carboxy-terminal C-box motif. Here, data are presented demonstrating that MPc3 also interacts with AF9, a transcriptional activator implicated in the development of acute leukemias. The carboxy-terminus of AF9 is fused to the MLL protein in leukemias characterized by t(9;11)(p22;q23) chromosomal translocations. Importantly, it is the carboxy-terminus of AF9 to which MPc3 binds. The AF9 binding site of MPc3 maps to a central, non-conserved, region of the polypeptide sequence. In contrast to MPc3, data indicate that the Pc protein M33 does not interact with AF9. This finding suggests a potentially unique role for MPc3 in linking a PcG silencing complex to a transcriptional activator protein.

The mixed lineage leukemia fusion partner AF9 binds specific isoforms of the BCL-6 corepressor

The mixed lineage leukemia (MLL) gene at chromosome band 11q23 is commonly involved in reciprocal translocations that are detected in acute leukemias. Evidence suggests that the resulting MLL fusion genes contribute to leukemogenesis. AF9 is a common MLL fusion partner in acute myeloid leukemia. The AF9 protein functions as a transcriptional activator in artificial reporter gene assays and a structurally related protein in yeast, ANC1/TFG3, is a component of the SWI/SNF complex. Apart from these observations, little is known about the biologic function of AF9 in mammals. We have found that a recently described transcriptional repressor, BCL-6 corepressor (BCoR), interacts with the carboxy-terminus of AF9. The interaction of AF9 with BCoR has been confirmed by independent in vitro and in vivo protein-binding studies. The BCoR gene is expressed as several alternatively spliced transcripts. AF9 only binds BCoR isoforms that contain a unique 34 aa sequence located in the mid-portion of the protein. In artificial reporter gene assays, a BCoR isoform that binds AF9 efficiently suppresses AF9 transcriptional activity, while a nonbinding isoform does not. These results indicate that different isoforms of BCoR have unique biologic properties and that cell function may be partly determined by the different isoforms that are present within the cell.

The Bmi-1 oncoprotein interacts with dinG and MPh2 : the role of RING finger domains

Experimentally-induced mutations in the C3HC4 RING finger domain of the Bmi-1 oncoprotein block its ability to induce lymphomas in mice. In this report, the role of the Bmi-1 RING finger in mediating protein-protein interactions is examined using the yeast two-hybrid system. Bmi-1 interacts directly with the RING finger protein dinG/RING1B. Heterodimerization of the two proteins requires the intact RING finger structures of both Bmi-1 and dinG. Although the RING finger domains are necessary for dimerization, they are not sufficient for this process as residues outside the C3HC4 motif are also required. Thus, binding specificity may be partly conferred by residues outside the RING motif. Both Bmi-1 and dinG interact with the Polyhomeotic protein MPh2 through binding domains apart from the RING finger. The data suggest a model whereby Bmi-1, dinG, and MPh2 form a stable heterotrimeric complex in which each protein contributes to the binding of the others.

Immunosuppressant Target Protein FKBP12 Is Required for P-Glycoprotein Function in Yeast

The mammalian P-glycoprotein (Pgp) is a ~170-kDa membrane protein that mediates multidrug resistance in many chemotherapy-resistant tumors by effluxing toxic compounds from the cell. Pgp homologs are expressed in many organisms, from bacteria to yeast and mammals. Previous studies established a model system to analyze the function of murine, human, and Plasmodium falciparum Pgp by heterologous expression in the yeast Saccharomyces cerevisiae. However, such studies have been hampered by the inherent resistance of yeast cells to chemotherapeutic agents. We find that an erg6 mutation, which blocks the final synthetic step of the membrane sterol ergosterol, renders yeast sensitive to anthracyclines and dactinomycin, clinically relevant Pgp substrates. We demonstrate that expression of the murine mdr3 gene confers dactinomycin resistance in both the erg6 mutant yeast strain and in an erg6 rad52 DNA repair mutant yeast strain. Similarly, murine mdr3 expression confers resistance to the immunosuppressants cyclosporin A (CsA) and FK506 in a CsA-FK506-sensitive vph6 mutant yeast strain. CsA and FK506 are known to partially overcome Pgp-mediated drug resistance, suggesting the targets of these drugs might regulate Pgp function. We find that both murine mdr3 and the yeast Pgp homolog STE6 function in yeast mutants lacking the CsA target proteins cyclophilin A and calcineurin. In contrast, murine mdr3 function was severely compromised in yeast mutants lacking the FK506/rapamycin target protein FKBP12. Both wild-type FKBP12 and an F43Y FKBP12 mutant with reduced prolyl isomerase activity supported mdr3 function. Our results support the model that immunosuppressants reverse multidrug resistance by competing with other Pgp substrates but reveal that inhibition of FKBP12-dependent Pgp function may also contribute to reversal of multidrug resistance by FK506 and rapamycin.

The synthetic peptide PFWT disrupts AF4–AF9 protein complexes and induces apoptosis in t(4;11) leukemia cells

The MLL gene at chromosome band 11q23 is commonly involved in reciprocal translocations detected in acute leukemias. A number of experiments show that the resulting MLL fusion genes directly contribute to leukemogenesis. Among the many known MLL fusion partners, AF4 is relatively common, particularly in acute lymphoblastic leukemia in infants. The AF4 protein interacts with the product of another gene, AF9, which is also fused to MLL in acute leukemias. Based on mapping studies of the AF9-binding domain of AF4, we have developed a peptide, designated PFWT, which disrupts the AF4–AF9 interaction in vitro and in vivo. We provide evidence that this peptide is able to inhibit the proliferation of leukemia cells with t(4;11) chromosomal translocations expressing MLL–AF4 fusion genes. Further, we show that this inhibition is mediated through apoptosis. Importantly, the peptide does not affect the proliferative capacity of hematopoietic progenitor cells. Our findings indicate that the AF4–AF9 protein complex is a promising new target for leukemia therapy and that the PFWT peptide may serve as a lead compound for drug development.