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The myelodysplastic syndromes (MDSs) are collections of heterogeneous hematologic diseases characterized by refractory cytopenias as a result of ineffective hematopoiesis. Development of effective treatments has been impeded by limited insights into any unifying pathogenic pathways. We provide evidence that the p38 MAP kinase is constitutively activated or phosphorylated in MDS bone marrows. Such activation is uniformly observed in varied morphologic subtypes of low-risk MDS and correlates with enhanced apoptosis observed in MDS hematopoietic progenitors. Most importantly, pharmacologic inhibition of p38α by a novel small molecule inhibitor, SCIO-469, decreases apoptosis in MDS CD34+ progenitors and leads to dose-dependant increases in erythroid and myeloid colony formation. Down-regulation of the dominant p38α isoform by siRNA also leads to enhancement of hematopoiesis in MDS bone marrow progenitors in vitro. These data implicate p38 MAPK in the pathobiology of ineffective hematopoiesis in lowrisk MDS and provide a strong rationale for clinical investigation of SCIO-469 in MDS.

A DNA gap repair assay was used to determine the effect of mutations in the DNA damage checkpoint system on the efficiency and outcome (crossover/non-crossover) of recombinational DNA repair. In Saccharomyces cerevisiae gap repair is largely achieved by homologous recombination. As a result the plasmid either integrates into the chromosome (indicative of a crossover outcome) or remains extrachromosomal (indicative of a non-crossover outcome). Deletion mutants of the MEC1 and RAD53 checkpoint kinase genes exhibited a 5-fold decrease in gap repair efficiency, showing that 80% of the gap repair events depended on functional DNA damage checkpoints. Epistasis analysis suggests that the DNA damage checkpoints affect gap repair by modulating Rad51 protein-mediated homologous recombination. While in wild-type cells only approximately 25% of the gap repair events were associated with a crossover outcome, Mec1-deficient cells exhibited a >80% crossover association. Also mutations in the effector kinases Rad53, Chk1 and Dun1 were found to affect crossover association of DNA gap repair to various degrees. The data suggest that the DNA damage checkpoints are important for the optimal functioning of recombinational DNA repair with multiple terminal targets to modulate the efficiency and outcome of homologous recombination.

The DNA damage checkpoint pathway and the MAP kinase pathway respond to various forms of environmental as well as endogenous stresses through signal transduction mechanisms involving protein kinases. Both pathways are intertwined in mammalian cells, but potential crosstalk between these two pathways in budding yeast has not been examined yet. We show that the Rad53 checkpoint kinase and the Hog1 MAP kinase of Saccharomyces cerevisiae become phosphorylated upon exposure to hydrogen peroxide, indicative of activation of the DNA damage checkpoint and MAP kinase pathways in response to oxidative stress. Rad53 kinase is equally activated in wild type and in hog1-Delta cells. Likewise, the activation of Hog1 MAP kinase is not dependent on Mec1 kinase, the central checkpoint kinase in budding yeast. Mutants in either pathway are sensitive to hydrogen peroxide and the double mutants exhibit a near perfectly additive phenotype. These data demonstrate that the DNA damage checkpoint pathway and the MAP kinase pathway respond to oxidative stress independently of each other and suggest that these two stress signaling pathways are activated by different types of insults induced by hydrogen peroxide.

DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.

Rad55 protein is one of two Rad51 paralogs in the budding yeast Saccharomyces cerevisiae and forms a stable heterodimer with Rad57, the other Rad51 paralog. The Rad55-Rad57 heterodimer functions in homologous recombination during the assembly of the Rad51-ssDNA filament, which is central for homology search and DNA strand exchange. Previously, we identified Rad55 protein as a terminal target of the DNA damage checkpoints, which coordinate the cellular response to genotoxic stress. Rad55 protein phosphorylation is signaled by a significant electrophoretic shift and occurs in response to a wide range of genotoxic stress. Here, we map the phosphorylation site leading to the electrophoretic shift and show that Rad55 protein is a bona fide direct in vivo substrate of the central DNA damage checkpoint kinase Mec1, the budding yeast equivalent of human ATM/ATR. We provide protocols to monitor the Rad55 phosphorylation status in vivo and assay Rad55-Rad57 phosphorylation in vitro using purified substrate with the Mec1 and Rad53 checkpoint kinases.

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.

The serine-threonine kinase Dun1 contains a forkhead-associated (FHA) domain and functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. It belongs to the Chk2 family of checkpoint kinases, which includes S. cerevisiae Rad53 and Mek1, Schizosaccharomyces pombe Cds1, and human Chk2. Dun1 is required for DNA damage-induced transcription of certain target genes, transient G(2)/M arrest after DNA damage, and DNA damage-induced phosphorylation of the DNA repair protein Rad55. Here we report that the FHA phosphoprotein recognition domain of Dun1 is required for direct phosphorylation of Dun1 by Rad53 kinase in vitro and in vivo. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G(2)/M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.

Despite recent advances in the treatment of multiple myeloma (MM), this disease remains incurable. Accumulating evidence suggest that the bone marrow (BM) microenvironment of MM plays a critical role in tumor growth, survival, and drug resistance. A key aspect of this tumor-supportive environment is elevated levels of cytokines and other soluble factors. Most prominent among these is IL-6, which acts as a survival factor for MM cells and promotes their proliferation, migration, and drug resistance. Other mediators also implicated in the disease are VEGF and TNFa. The p38 MAPK is activated by a multitude of signals, including pro-inflammatory cytokines (e.g., TNFa and IL-1ß) and environmental stress. Furthermore, p38 activation has been shown to be important for the synthesis and secretion of IL-6, VEGF, and TNFa. Consequently, inhibition of p38 is postulated to reduce the production of these factors implicated in MM and to have therapeutic benefit by suppressing the tumor-supportive state of the BM microenvironment. Here, we demonstrate that SCIO-469, a specific and potent inhibitor of p38a MAPK, strongly inhibits MM cell proliferation by affecting MM cells directly as well as the BM microenvironment. SCIO-469 directly inhibits MM cell proliferation in long term culture. Importantly, SCIO-469 potently inhibits IL-6 and VEGF secretion from BM stromal cells (BMSC). To examine the effect of inhibiting BMSC-derived factors important in MM, we measured MM cell proliferation using transwell plates that separate BMSC from MM cells via a porous membrane. In transwell plates containing only MM cells, MM cell proliferation was modest and was inhibited by SCIO-469. In contrast, the presence of BMSC in transwell inserts dramatically increased the proliferation of MM cells over the course of the study. This result suggests that factors (e.g., IL-6) secreted by BMSC greatly stimulate MM cell proliferation. When SCIO-469 was added to these transwell cultures containing BMSC, MM cell proliferation was inhibited significantly. Consistent with these results, we show that levels of IL-6 under these conditions mirror exactly the proliferation of MM cells; IL-6 level is high in vehicle-treated cultures and is suppressed in SCIO-469-treated cultures. Finally, in a mouse xenograft plasmacytoma model of MM, we show that p38 inhibition significantly inhibited the increase in MM tumor volume. Collectively, our data indicate that SCIO-469 is a suppressor of the BM microenvironment and an effective inhibitor of MM cell proliferation in vitro and in vivo. Since SCIO-469 also inhibits secretion of osteoclast-stimulating factors (RANKL, IL-11, and MIP1a) in the microenvironment, SCIO-469 may not only inhibit MM cell survival but may also alleviate bone-related pathologies (bone destruction and osteolytic lesions) commonly associated with MM. Therefore, SCIO-469 may offer great promise for an improved outcome for patients with MM.