A growing body of evidence shows that Signal Transducer and Activator of Transcription 5 (STAT5) protein, a key member of the STAT family of signaling proteins, plays a pivotal role in the progression of many human cancers, including acute myeloid leukemia and prostate cancer. Unlike STAT3, where significant medicinal effort has been expended to identify potent direct inhibitors, Stat5 has been poorly investigated as a molecular therapeutic target. Thus, in an effort to identify direct inhibitors of STAT5 protein, we conducted an in vitro screen of a focused library of SH2 domain binding salicylic acid-containing inhibitors (∼150) against STAT5, as well as against STAT3 and STAT1 proteins for SH2 domain selectivity. We herein report the identification of several potent (K(i) < 5 μM) and STAT5 selective (>3-fold specificity for STAT5 cf. STAT1 and STAT3) inhibitors, BP-1-107, BP-1-108, SF-1-087, and SF-1-088. Lead agents, evaluated in K562 and MV-4-11 human leukemia cells, showed potent induction of apoptosis (IC(50)'s ∼ 20 μM) which correlated with potent and selective suppression of STAT5 phosphorylation, as well as inhibition of STAT5 target genes cyclin D1, cyclin D2, C-MYC, and MCL-1. Moreover, lead agent BP-1-108 showed negligible cytotoxic effects in normal bone marrow cells not expressing activated STAT5 protein. Inhibitors identified in this study represent some of the most potent direct small molecule, nonphosphorylated inhibitors of STAT5 to date.

DLK1 is an imprinted gene on chromosome 14. Using informative coding single nucleotide polymorphisms, we found DLK1 expression to be monoallelic in normal bone marrow, whereas it was biallelic in 76% of acute myeloid leukemia (AML) overexpressing DLK1 (61% of all AML). Quantitative methylation analysis of 7 cytosine-phosphate-guanosine-rich areas (3 upstream of or within DLK1, the putative intergenic-differentially methylated region and 3 upstream of or within MEG3) revealed a strong association between biallelic DLK1 expression and hypermethylation of a cytosine-phosphateguanosine-rich region 18 kb upstream of DLK1. Allele-specific methylation analysis of this region revealed the alleles to be differentially methylated in normal bone marrow and monoallelic DLK1 AML, whereas there was increased methylation of both alleles in AML with biallelic expression. Moreover, chromatin immunoprecipitation analysis revealed that CCTCbinding factor binds to this region in monoallelic but not biallelic expression samples. Taken

The LYL1 gene encodes a basic helix-loop-helix transcription factor involved in T-cell acute lymphoblastic leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of LYL1 was at higher levels in the majority cases of acute myeloblastic leukemia (AML) or myelodysplastic syndrome when compared to normal bone marrow. Our study also showed that LYL1 was highly expressed in most AML cell lines and in CD34 þ AML cells. To determine whether LYL1 had an affect on the phenotype and behavior of myeloid cells, we introduced full-length LYL1 cDNA into K562 cells using electroporation and U937 cells with retroviral infection. Both of the derivative cell lines with overexpression of LYL1 had an increased growth rate and clonogenecity. Forced expression of LYL1 in K562 cells enhanced spontaneous and hemin-induced erythroid differentiation but blocked spontaneous as well as PMA-induced megakaryocytic differentiation. Overexpression of LYL1 in U937 cells blocked all-trans retinoic acid-induced monocytic differentiation. The LYL1-transfected U937 cells were also more resistant to the cytotoxic drug cytarabine. These results demonstrate that LYL1 may play a role in early hematopoiesis and may be a potential oncogenic factor in AML.

Chromosomal rearrangements of the mixed lineage leukemia (MLL) gene occur in ∼10% of B-cell acute lymphoblastic leukemia (B-ALL) and define a group of patients with dismal outcomes. Immunohistochemical staining of bone marrow biopsies from most of these patients revealed aberrant expression of BCL6, a transcription factor that promotes oncogenic B-cell transformation and drug resistance in B-ALL. Our genetic and ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analyses showed that MLL-AF4 and MLL-ENL fusions directly bound to the BCL6 promoter and up-regulated BCL6 expression. While oncogenic MLL fusions strongly induced aberrant BCL6 expression in B-ALL cells, germline MLL was required to up-regulate Bcl6 in response to physiological stimuli during normal B-cell development. Inducible expression of Bcl6 increased MLL mRNA levels, which was reversed by genetic deletion and pharmacological inhibition of Bcl6, suggesting a positive feedback loop between MLL and BCL6. Highlighting the central role of BCL6 in MLL-rearranged B-ALL, conditional deletion and pharmacological inhibition of BCL6 compromised leukemogenesis in transplant recipient mice and restored sensitivity to vincristine chemotherapy in MLL-rearranged B-ALL patient samples. Oncogenic MLL fusions strongly induced transcriptional activation of the proapoptotic BH3-only molecule BIM, while BCL6 was required to curb MLL-induced expression of BIM. Notably, peptide (RI-BPI) and small molecule (FX1) BCL6 inhibitors derepressed BIM and synergized with the BH3-mimetic ABT-199 in eradicating MLL-rearranged B-ALL cells. These findings uncover MLL-dependent transcriptional activation of BCL6 as a previously unrecognized requirement of malignant transformation by oncogenic MLL fusions and identified BCL6 as a novel target for the treatment of MLLrearranged B-ALL.

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Association between certain surface markers and acute myelogenous leukemia (AML) with t(8;21) has been described. The specificity and the predictive values of these markers have never been assessed. In this study, we aimed, to explore whether a specific pattern could predict for this translocation. Of 405 consecutive AML, 18 (4.4%) had the t(8;21). Patients with this cytogenetic abnormality showed higher frequency of CD34 (P = 0.003), HLA-DR (P = 0.03), Tdt (P = 0.02), CD19 (P < 0.0001), and CD56 (P < 0.0001) and lower CD33 (P = 0.0001). Taken singly, the sensitivity of these markers for AML with t(8;21) ranged between 39 and 100% with CD34+ having the highest and CD33- having the lowest and the positive predictive values (PPV) ranged between 5 and 21% with CD19+ having the highest and HLA-DR+ having the lowest. When combinations of different markers were analyzed by multivariate analysis, the pattern CD34+/HLA-DR+/MPO+ was found to have the highest sensitivity (100%) with a PPV of 14% and the pattern CD34+/CD19+/CD56+ had the highest PPV (100%) with a sensitivity of 67%. We conclude that AML with t(8;21) is better identified by a combination of markers than by a single antigen pattern, the absence of CD34+, HLA-DR+ or MPO+ would preclude and the expression of the pattern CD34+/CD19+/CD56+ is highly predictive and could serve as a screening criteria for the t(8;21).

Prophylaxis with posaconazole in cancer patients with prolonged neutropenia is not only cost effective but also cost saving. The economic benefits were due to the drug's ability to reduce the incidence of high cost fungal infections, particularly Aspergillus species.

Acute myelogenous leukemia with t(8;21) is a distinct clinicopathologic entity in which the malignant myeloblasts display a characteristic pattern of surface antigen expression. Quantitative analysis of surface marker expression in patients with this chromosomal abnormality compared to acute myelogenous leukemia patients with a different karyotype has not been reported. From 305 consecutive newly diagnosed acute myelogenous leukemia patients underwent immunophenotyping and cytogenetic analysis at our center; 16 patients (5.2%) had a t(8;21). Fluorescence intensity values were obtained, using a set of reference microbeads, by conversion of mean channel fluorescence to molecular equivalent of soluble fluorochrome. Patients with t(8;21) displayed higher levels of CD34, HLA-DR and MPO expression (Po0.001 for each) and lower levels of CD13 (P ¼ 0.03) and CD33 (P ¼ 0.02) expression. In order to study the sensitivity, specificity and predictive value of these markers, molecular equivalent of soluble fluorochrome thresholds were statistically determined. The statistically established threshold for each of the individual markers (CD34460.5 Â 10 3 , HLA-DR4176.1 Â 10 3 , MPO4735.1 Â 10 3 , CD13o24.3 Â 10 3 and CD33o17.3 Â 10 3 ) had a sensitivity of 100%, a specificity of 62-92% and a positive predictive value of 7-45%. In multivariate analysis, two quantitative patterns (CD34460.5 Â 10 3 and MPO4176.1 Â 10 3 ; CD33o17.3 Â 10 3 and MPO4176.1 Â 10 3 ) had a sensitivity, specificity and positive predictive value of 100%. These aberrant phenotypic patterns might help identify patients with t(8;21) at diagnosis and could be useful in minimal residual disease monitoring. In patients with acute myelogenous leukemia (AML), t(8;21)(q22;q22) is a relatively frequent structural cytogenetic abnormality. This chromosomal translocation results in an in-frame fusion between the first 5 exons of the AML1 gene and essentially all of the ETO gene producing a chimeric protein. 1 This protein, AML1-ETO, retains the ability of AML1 to heterodimerize with CBFb and to bind DNA as well as allowing ETO to interact with the N-Co-R/Sin3/HDAC complex. 2 This results in its acting as a negative dominant inhibitor of wild-type AML1. 3 The creation of AML1-ETO is necessary, but not sufficient, for leukemogenesis in AML with t(8;21). 4 The recently developed WHO classification of hematologic malignancies recognizes AML with t(8;21) as a distinct clinicopathologic entity. 5 This form of AML is found more frequently in children and young adults 6 and patients are predisposed to extramedullary localization. 7 Complete remission rates and long-term event-free survival are high, particularly when treatment incorporates high-dose cytosine arabinoside. 8 Histopathology characteristically demonstrates frequent type III