Down-regulation of homeobox genes MEIS1 and HOXA in MLL-rearranged acute leukemia impairs engraftment and reduces proliferation.

Rearrangements of the MLL (ALL1) gene are very common in acute infant and therapy-associated leukemias. The rearrangements underlie the generation of MLL fusion proteins acting as potent oncogenes. Several most consistently up-regulated targets of MLL fusions, MEIS1, HOXA7, HOXA9, and HOXA10 are functionally related and have been implicated in other types of leukemias. Each of the four genes was knocked down separately in the human precursor B-cell leukemic line RS4;11 expressing MLL-AF4. The mutant and control cells were compared for engraftment in NOD/SCID mice. Engraftment of all mutants into the bone marrow (BM) was impaired. Although homing was similar, colonization by the knockdown cells was slowed. Initially, both types of cells were confined to the trabecular area; this was followed by a rapid spread of the WT cells to the compact bone area, contrasted with a significantly slower process for the mutants. In vitro and in vivo BrdU incorporation experiments indicated reduced proliferation of the mutant cells. In addition, the CXCR4/SDF-1 axis was hampered, as evidenced by reduced migration toward an SDF-1 gradient and loss of SDF-1-augmented proliferation in culture. The very similar phenotype shared by all mutant lines implies that all four genes are involved and required for expansion of MLL-AF4 associated leukemic cells in mice, and down-regulation of any of them is not compensated by the others.

Regulation of microtubule dynamics by inhibition of the tubulin deacetylase HDAC6.

We studied the role of a class II histone deacetylase, HDAC6, known to function as a potent alpha-tubulin deacetylase, in the regulation of microtubule dynamics. Treatment of cells with the class I and II histone deacetylase inhibitor TSA, as well as the selective HDAC6 inhibitor tubacin, increased microtubule acetylation and significantly reduced velocities of microtubule growth and shrinkage. siRNA-mediated knockdown of HDAC6 also increased microtubule acetylation but, surprisingly, had no effect on microtubule growth velocity. At the same time, HDAC6 knockdown abolished the effect of tubacin on microtubule growth, demonstrating that tubacin influences microtubule dynamics via specific inhibition of HDAC6. Thus, the physical presence of HDAC6 with impaired catalytic activity, rather than tubulin acetylation per se, is the factor responsible for the alteration of microtubule growth velocity in HDAC6 inhibitor-treated cells. In support of this notion, HDAC6 mutants bearing inactivating point mutations in either of the two catalytic domains mimicked the effect of HDAC6 inhibitors on microtubule growth velocity. In addition, HDAC6 was found to be physically associated with the microtubule end-tracking protein EB1 and a dynactin core component, Arp1, both of which accumulate at the tips of growing microtubules. We hypothesize that inhibition of HDAC6 catalytic activity may affect microtubule dynamics by promoting the interaction of HDAC6 with tubulin and/or with other microtubule regulatory proteins.

Mammalian diaphanous-related formin Dia1 controls the organization of E-cadherin-mediated cell-cell junctions.

The formin-homology protein Dia1 is a target of RhoA and a potent activator of nucleation and elongation of actin filaments. Here, we demonstrate that short-hairpin (sh) RNA-mediated downregulation of Dia1 in human MCF7 epithelial cells disrupts adherens junctions, as manifested by the significantly decreased localization of E-cadherin and associated proteins to cell-cell contacts. Expression of mouse Dia1, which is insensitive to the human Dia1-specific shRNA, rescued the junctional integrity. Coexpression of GFP-tagged Dia1 and a constitutively active RhoA mutant, RhoA-V14, resulted in localization of the exogenous GFP-Dia1 to the cell-cell junctions. This localization was accompanied by a strong increase in the width of the adhesion zone and augmentation of the actin, E-cadherin and beta-catenin content of the junctions. A constitutively active Dia1 mutant lacking the N-terminal portion was unable to localize to cell-cell junctions and did not show any junction-strengthening effect. The adherens junction enhancement induced by Dia1 and active RhoA did not require microtubules, but depended on the activity of myosin II. Inhibition of myosin II activity abolished the Dia1-mediated reinforcement of cell-cell junctions and instead induced the formation of numerous actin-rich filopodia at the contact zone. Thus, Dia1 localizes to and controls cadherin-mediated junctions in a RhoA-dependent manner.

The PVT-1 oncogene is a Myc protein target that is overexpressed in transformed cells.

The human PVT-1 gene is located on chromosome 8 telomeric to the c-Myc gene and it is frequently involved in the translocations occurring in variant Burkitt's lymphomas and murine plasmacytomas. It has been proposed that PVT-1 regulates c-Myc gene transcription over a long distance. To get new insights into the functional relationships between the two genes, we have investigated PVT-1 and c-Myc expression in normal human tissues and in transformed cells. Our findings indicate that PVT-1 expression is restricted to a relative low number of normal tissues compared to the wide distribution of c-Myc mRNA, whereas the gene is highly expressed in many transformed cell types including neuroblastoma cells that do not express c-Myc. Reporter gene assays were used to dissect the PVT-1 promoter and to identify the region responsible for the elevated expression observed in transformed cells. This region contains two putative binding sites for Myc proteins. The results of transfection experiments in RAT1-MycER cells and chromatin immunoprecipitation (ChIP) assays in proliferating and differentiated neuroblastoma cells indicate that PVT-1 is a downstream target of Myc proteins.

Assembly and mechanosensory function of focal adhesions: experiments and models.

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.