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

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