Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export

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Block of C/EBPα function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations

Mutations constitutively activating FLT3 kinase are detected in ∼30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal–regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein α (C/EBPα) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells, pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was also observed when C/EBPα mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to aspartate (S21D), which mimics phosphorylation of C/EBPα. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction of constitutively active C/EBPα may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies

PPM1D Mutations Drive Clonal Hematopoiesis in Response to Cytotoxic Chemotherapy.

Clonal hematopoiesis (CH), in which stem cell clones dominate blood production, becomes increasingly common with age and can presage malignancy development. The conditions that promote ascendancy of particular clones are unclear. We found that mutations in PPM1D (protein phosphatase Mn2+/Mg2+-dependent 1D), a DNA damage response regulator that is frequently mutated in CH, were present in one-fifth of patients with therapy-related acute myeloid leukemia or myelodysplastic syndrome and strongly correlated with cisplatin exposure. Cell lines with hyperactive PPM1D mutations expand to outcompete normal cells after exposure to cytotoxic DNA damaging agents including cisplatin, and this effect was predominantly mediated by increased resistance to apoptosis. Moreover, heterozygous mutant Ppm1d hematopoietic cells outcompeted their wild-type counterparts in vivo after exposure to cisplatin and doxorubicin, but not during recovery from bone marrow transplantation. These findings establish the clinical relevance of PPM1D mutations in CH and the importance of studying mutation-treatment interactions. VIDEO ABSTRACT.This work was supported by the Center Prevention and Research Institute of Texas (CPRIT) (RP160451 and R120501) and the NIH (DK092883, DK116428, S10RR024574, AI036211, P30 CA125123, and P30 CA016672). The Welch Foundation (G-0040), MD Anderson’s MoonShot Program, the Baylor Research Advocates for Student Scientists, and the BCM MSTP program also provided support. K.T. is supported by a Khalifa Physician Scientist Award, the Physician Scientist Program at MD Anderson, and a Leukemia SPORE Career Enhancement Award. G.V. is funded by a Cancer Research UK Senior Cancer Research Fellowship (C22324/A23015), the Kay Kendall Leukaemia Fund, Bloodwise, and core funding from the Sanger Institute (WT098051). We also thank the Samuel Waxman Cancer Research Foundation

Activation of WIP1 phosphatase by HTLV-1 Tax mitigates the cellular response to DNA damage.

Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G(1) phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G(1)/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G(1) exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export-3

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export"</p><p>http://www.retrovirology.com/content/4/1/95</p><p>Retrovirology 2007;4():95-95.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2234431.</p><p></p> HA-UB. Cells were either mock (-) or UV irradiated (+) and the ubiquitination status of Tax was examined by immunoblot analysis for Tax

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export-6

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export"</p><p>http://www.retrovirology.com/content/4/1/95</p><p>Retrovirology 2007;4():95-95.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2234431.</p><p></p>mined by immunofluorescent microscopy in transfected 293 cells. Tax is expressed in nuclear foci in the absence of DNA damage (A) and is localized in the cytoplasm following UV irradiation (B). The addition of a UB tag at either the N- (C) or C- (E) termini caused Tax to be localized in the cytoplasm but treatment with LMB (D and F) sequestered UB-Tax and Tax-UB to nuclear foci. All images are shown at a magnification of 63×. The percentage of cells expressing nuclear foci (black bar) or cytoplasmic Tax (gray bar) was scored in five hundred transfected cells, in three independent experiments (G). Western blot showing expression of UB-Tax, Tax-UB, and native Tax proteins (H)

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export-1

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export"</p><p>http://www.retrovirology.com/content/4/1/95</p><p>Retrovirology 2007;4():95-95.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2234431.</p><p></p>or UV irradiated (30 J/m, 30 minute recovery). The immunoprecipitates were analyzed by immunoblot for Tax (left) or HA (right)

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export-4

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export"</p><p>http://www.retrovirology.com/content/4/1/95</p><p>Retrovirology 2007;4():95-95.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2234431.</p><p></p>ed in 293 cells for 48 hrs and subjected to 30 J/mUV irradiation (B, D, F, H, J) or mock irradiated (A, C, E, G, I) and allowed to recover for 30 min. sc35 (left column) and Tax (left center column) were visualized by immunofluorescent microscopy separately, together (Merged, right center column), or together with DAPI (DAPI Merged, right column). All images are shown at a magnification of 63×

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export-2

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export"</p><p>http://www.retrovirology.com/content/4/1/95</p><p>Retrovirology 2007;4():95-95.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2234431.</p><p></p>bsence (black bars) or presence of 30 J/mUV irradiation (gray bars) is shown. Error bars represent the standard error in three independent experiments. (B) Western blot showing expression of each Tax mutant analyzed