Late-onset hemorrhagic cystitis (HC) is a well-known complication of bone marrow transplantation (BMT) that is mainly attributed to infection with BK virus (BKV) and adenovirus (AdV). From 1986 through 1998, 282 patients underwent BMT, and 45 of them developed HC. Urine samples tested positive for AdV in 26 patients, of which 22 showed virus type 11. Among patients who underwent allogeneic BMT, logistic regression analysis revealed acute graft-versus-host disease (grade, > or = 2) to be the most significant predictive factor for HC (P < .0001). In addition, a total of 193 urine samples regularly obtained from 26 consecutive patients who underwent allogeneic BMT were examined for BKV, JC virus (JCV), and AdV by means of polymerase chain reaction. Of patients without HC, approximately 30% of the specimens tested positive for BKV (58 samples) and JCV (55 samples), whereas 5 (3%) tested positive for AdV. Of the 3 samples obtained from patients with HC, the numbers of positive results for BKV, JCV, and AdV were 3, 1, and 1, respectively; the numbers of positive results increased to 14 of 17, 9 of 17, and 10 of 17, respectively, when we added another 14 samples obtained from 14 patients with HC (P < .0001, P = .026, and P < .0001, respectively). In conclusion, there was significant correlation between AdV and HC in the patients we studied.

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Chromosomal translocations and somatic mutations occurring in the 5 0 noncoding region of the BCL6 gene, encoding a transcriptional repressor, are most frequent genetic abnormalities associated with non-Hodgkin B-cell lymphoma and result in deregulated expression of BCL6. However, the significance of deregulated expression of BCL6 in lymphomagenesis and its effect on clinical outcomes of lymphoma patients have remained elusive. In the present study, we established Daudi and Raji B-cell lymphoma cell lines that overexpress BCL6 or its mutant, BCL6-Ala333/343, in which serine residues required for degradation through the proteasome pathway in B-cell receptor-stimulated cells are mutated. BCL6 overexpression did not have any significant effect on cell proliferation, but significantly inhibited apoptosis caused by etoposide, which induced a proteasome-dependent degradation of BCL6. BCL6-Ala333/343 was not degraded after etoposide treatment and strongly inhibited apoptosis. In these lymphoma cell lines, etoposide increased the generation of reactive oxygen species (ROS) and reduced mitochondria membrane potential, both of which were inhibited by the antioxidant N-acetyl-l-cysteine (NAC). NAC also inhibited apoptosis. Furthermore, BCL6 overexpression was found to inhibit the increase in ROS levels and apoptosis in response to etoposide and other chemotherapeutic reagents. These results raise the possibility that deregulated expression of BCL6 may endow lymphoma cells with resistance to chemotherapeutic reagents, most likely by enhancing the antioxidant defense systems.

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4–11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway.

Fms-like tyrosine kinase 3 (Flt3) 2 also known as fetal liver kinase-2 (Flk-2) is a receptor-tyrosine kinase expressed on hematopoietic progenitors and regulates early steps of hematopoietic progenitor cell proliferation, survival, and differentiation (1, 2). Wild-type Flt3 (Flt3-WT) has been shown to be highly expressed in several hematopoietic malignancies including 70 -100% cases of acute myeloid leukemia (AML). Oncogenic internal tandem duplication (ITD) mutations in the juxtamembrane domain of Flt3 (Flt3-ITD) and point mutations within the tyrosine kinase domain, such as the most predominant D835Y mutation, are the most frequent kinase mutations in AML, occurring in 25-30 and 5-10% of cases, respectively, and are associated with poor prognosis (1-3). Flt3-ITD results in ligand-independent autophosphorylation and activation of the receptor with subsequent activation of multiple downstream targets, including the signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase, and Akt pathways. Flt3-ITD has been shown to induce a myeloproliferative disorder in murine models. Flt3-ITD exists partially in an immature, underglycosylated, constitutively phosphorylated form (4). Aberrant intracellular localization and activity of Flt3-ITD generate oncogenic phosphorylation patterns and aberrantly activate signaling cascades (5). Because Flt3 or Flt3-ligand (FL) knock-out mice have only a subtle hematopoietic stem/progenitor cell deficit and show no significant disadvantage in viability, interference with deregulated Flt3 functions appears as a promising treatment option for AML (1-3). Thus, several Flt3 inhibitors are currently under evaluation for their efficacy in AML patients with Flt3-ITD.Recent studies showed that both activated Flt3 and mutant Flt3 are degraded through the proteasome (6 -11). The proteasomal degradation of targeted proteins, including protein kinases, requires polyubiquitination catalyzed by a series of enzymes containing ubiquitin activating enzymes (E1), ubiquitin conjugases (E2), and ubiquitin ligases (E3) (12, 13). E3 ubiquitin ligases confer substrate specificity and are responsible for mediating the transfer of ubiquitin from E2s to the substrates. The Cbl proteins are a highly conserved family of RING finger E3 ubiquitin ligases that regulate signaling by receptor and non-receptor-tyrosine kinases, including EGF receptor, PDGF receptor ␣, and FMS (14). There are three mammalian Cbl proteins encoded by separate genes: c-Cbl, Cbl-b, and Cbl-c. These proteins contain an N-terminal phosphotyrosine binding domain that allows direct interaction with activated receptortyrosine kinases and a RING finger domain that classifies Cbl

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Although the BCR/ABL tyrosine kinase inhibitor imatinib is highly effective for treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia, relapse with emerging imatinib resistance mutations in the BCR/ABL kinase domain poses a significant problem. Here, we show that the multikinase inhibitor sorafenib inhibits proliferation and induces apoptosis at much lower concentrations in Ton.B210 cells when driven by inducibly expressed BCR/ABL than when driven by interleukin-3. The increased sensitivity to sorafenib was also observed in cells inducibly expressing BCR/ABL with the imatinib-resistant E255K or T315I mutation. Sorafenibinduced apoptosis in these cells and Ph+ leukemic cells was synergistically enhanced by rottlerin, bortezomib, or ABT-737 and inhibited by the pan-caspase inhibitor BOC-d-fmk or the overexpression of Bcl-XL. It was further revealed that sorafenib activates Bax and caspase-3 and reduces mitochondrial membrane potential specifically in BCR/ABL-driven cells. Sorafenib also inhibited BCR/ABL-induced tyrosine phosphorylation of its cellular substrates and its autophosphorylation in Ton.B210. It was finally shown that sorafenib inhibits the kinase activity of BCR/ABL as well as its E255K and T315I mutants in in vitro kinase assays. These results indicate that sorafenib induces apoptosis of BCR/ABLexpressing cells, at least partly, by inhibiting BCR/ABL to activate the mitochondria-mediated apoptotic pathway. Thus, sorafenib may provide an effective therapeutic measure to treat Ph+ leukemias, particularly those expressing the T315I mutant, which is totally resistant to imatinib and the second generation BCR/ABL inhibitors. [Cancer Res 2009;69(9):3927-36]