Rationale for targeting BCL6 in MLL-rearranged acute lymphoblastic leukemia.

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 MLL-rearranged B-ALL.

Octadecyloxyethyl Adefovir Exhibits Potent in vitro and in vivo Cytotoxic Activity and Has Synergistic Effects with Ara-C in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) continues to be a deadly disease, with only 50-70% of patients achieving complete remission and less than 30% of adults having sustained long-term remissions. In order to address these unmet medical needs, we carried out a high-throughput screen of an in-house library of on- and off-patent drugs with the OCI/AML-2 cell line. Through this screen, we discovered adefovir dipi-voxil (adefovir-DP) as being active against human AML. In addition to adefovir-DP, there are second-generation formulations of adefovir, including octadecyloxyethyl adefovir (ODE-adefovir) and hexadecyloxypropyl adefovir (HDP-adefovir), which were designed to overcome the pharmacokinetic problems of the parent compound adefovir. Given the known clinical benefit of nucleoside analogs for the treatment of AML, we undertook studies to evaluate the potential benefit of adefovir-based molecules. In AML cell lines and patient samples, adefovir-DP and ODE-adefovir were highly potent, whereas HDP-adefovir was significantly less active. Interestingly, ODE-adefovir was remarkably less toxic than adefovir-DP towards normal hematopoietic cells. In addition, ODE-adefovir at a dose of 15 mg/kg/day showed potent activity against human AML in a NOD/SCID mouse model, with a reduction of human leukemia in mouse bone marrow of > 40% in all mice tested within 20 days of treatment. Based on its chemical structure, we hypothesized that the cytotoxicity of ODE-adefovir toward AML was through cell cycle arrest and DNA damage. Indeed, ODE-adefovir treatment induced cell cycle arrest in the S phase and increased levels of pH2Ax, indicating the induction of DNA damage. Furthermore, there was an increase in phospho-p53, transactivation of proapoptotic genes and activation of the intrinsic apoptotic pathway. Subsequent investigation unveiled strong synergism between ODE-adefovir and ara-C, making their coadministration of potential clinical benefit. Expression of MRP4, a nucleoside transporter, appeared to influence the response of AML cells to ODE-adefovir, as its inhibition potentiated ODE-adefovir killing. Taken together, our findings indicate that clinical development of ODE-adefovir or related compounds for the treatment of AML is warranted.

An upstream insulator regulates DLK1 imprinting in AML.

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-phosphate-guanosine-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 CCTC-binding factor binds to this region in monoallelic but not biallelic expression samples. Taken together, our data indicate that an insulator located 18 kb upstream of DLK1 plays an important role in regulating DLK1 imprinting.

Posaconazole versus fluconazole or itraconazole for prevention of invasive fungal infections in patients undergoing intensive cytotoxic therapy for acute myeloid leukemia or myelodysplasia: a cost effectiveness analysis.

INTRODUCTION: Invasive fungal infections (IFI) remain a clinical concern in hematological patients with prolonged neutropenia because they are a major cause of morbidity and mortality. In a recent randomized trial, prophylaxis with posaconazole was associated with fewer IFI and related deaths relative to a fluconazole or itraconazole (Flu/Itra) control group (p < 0.001). In the current study, a cost effectiveness analysis was conducted to estimate the economic value of posaconazole as an alternative to Flu/Itra when used to prevent IFI in this patient population. METHODS: A decision analysis model was developed using clinical and economic data from randomized comparative trials, the economic literature, and from expert opinion. The data were then used to estimate the incremental cost per life year saved with oral posaconazole prophylaxis relative to Flu/Itra from the Canadian provincial health care system perspective. The base case results were then tested with a sensitivity analysis which evaluated extremes in the incidence of IFI as well as variations in their cost of management. RESULTS: Prophylaxis with posaconazole provides increased efficacy and an overall cost savings of approximately $Can4,259 per patient. Despite variations in the base case parameters, the sensitivity analysis suggested stability in the primary findings. Posaconazole was associated with an overall cost savings (range = $Can1,765 to $Can4,505) in all of the scenarios evaluated. Optimal cost effectiveness was obtained because the drug was able to avoid the more resource intensive Aspergillus infections. CONCLUSIONS: 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.

The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells.

INTRODUCTION: There is growing evidence that the Wilms' tumor 1 suppressor gene (WT1) behaves as an oncogene in some forms of breast cancer. Previous studies have demonstrated that the N-terminal domain of WT1 can act as a dominant negative through self-association. In the studies presented here we have explored the potential for the zinc finger domain (ZF) of WT1 to also have dominant-negative effects, and thus further our understanding of this protein. METHODS: Using full-length and ZF-only forms of WT1 we assessed their effect on the WT1 and c-myc promoter using luciferase and chromatin immunoprecipitation assays. The gene expression levels were determined by quantitative real-time RT-PCR, northern blot and western blot. We also assessed the effect of the ZF-only form on the growth of breast cancer cell lines in culture. RESULTS: Transfection with WT1-ZF plasmids resulted in a stronger inhibition of WT1 promoter than full-length WT1 in breast cancer cells. The WT1-ZF form lacking the lysine-threonine-serine (KTS) insert (ZF - KTS) can bind to the majority of WT1 consensus sites throughout the WT1 promoter region, while the ZF containing the insert (ZF + KTS) form only binds to sites in the proximal promoter. The abundances of endogenous WT1 mRNA and protein were markedly decreased following the stable expression of ZF - KTS in breast cancer cells. The expressions of WT1 target genes, including c-myc, Bcl-2, amphiregulin and TERT, were similarly suppressed by ZF - KTS. Moreover, WT1-ZF - KTS abrogated the transcriptional activation of c-myc mediated by all four predominant isoforms of WT1 (including or lacking alternatively spliced exons 5 and 9). Finally, WT1-ZF - KTS inhibited colony formation and cell division, but induced apoptosis in MCF-7 cells. CONCLUSION: Our observations strongly argue that the WT1-ZF plasmid behaves as a dominant-negative regulator of the endogenous WT1 in breast cancer cells. The inhibition on proliferation of breast cancer cells by WT1-ZF - KTS provides a potential candidate of gene therapy for breast cancer.

Intravascular lymphoma presenting with bone marrow involvement and leukemic phase.

We describe the case of a 62-year-old man with recent onset of constitutional symptoms and vague intellectual deficit. The blood showed pancytopenia with blastemia, and bone marrow confirmed an extensive "vacuolated blast-like cell" infiltrate. Initial diagnosis of, and treatment for Burkitt's leukemia/lymphoma was questioned when the "blasts" typed as CD5+ mature B-cells; however, it was revised to intravascular lymphoma (IVL) only after the sinusoidal pattern was confirmed by immunocytochemistry. Literature review indicated that blood and bone marrow involvement in IVL appears to be rare, but a systematic search for this involvement is often not carried out. CD5 expression has been increasingly reported in this disease. The actual frequency and the significance of this expression are still to be defined.

Acute myelogenous leukemia with t(8;21)--identification of a specific immunophenotype.

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).

Small molecule STAT5-SH2 domain inhibitors exhibit potent antileukemia activity.

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.

Multicolor karyotyping and clinicopathological analysis of three intravascular lymphoma cases.

Intravascular lymphoma (IVL) is a rare neoplastic disease characterized by the presence of large malignant lymphoid cells in small vessels. It is often diagnosed at autopsy. Clinical manifestations are typically neurologic and dermatologic. Karyotypic abnormalities have been described in a small number of cases and have revealed complex alterations in the majority of cases. We have identified three cases of IVL with varied clinicopathological findings. Karyotypic analysis was undertaken by standard G-banding and supplemented by multi-colored karyotyping (M-FISH) to decipher the chromosomal content of marker chromosomes and undefined additions. M-FISH clarified the chromosomal abnormalities in two cases and unveiled cryptic translocations der(10)t(10;22), der(17)t(17;22), and balanced t(11;14). Comparison with previously published karyotypes revealed prominent involvement of chromosomes 1, 3, 6, 11, 14, and 18, similar to the pattern of clonal evolution in other B-cell lymphomas. The most frequent alterations seen were -6 or 6q- and +18 or dup(18q), with a minimally deleted region located at 6q21-q23 and a commonly amplified region located at 18q13-q23, respectively. Few differences between the classical and Asian variant of this disease were apparent at the karyotypic level. Cytogenetic analysis of additional cases supplemented by multicolor karyotyping may help identify the full spectrum of genetic alterations associated with IVL and assist in the delineation of the critical mutations associated with initiation and progression of this disease.

Correlation between karyotype and quantitative immunophenotype in acute myelogenous leukemia with t(8;21).

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 (P < 0.001 for each) and lower levels of CD13 (P = 0.03) and CD33 (P = 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 (CD34 > 60.5 x 10(3), HLA-DR > 176.1 x 10(3), MPO > 735.1 x 10(3), CD13 < 24.3 x 10(3) and CD33 < 17.3 x 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 (CD34 > 60.5 x 10(3) and MPO > 176.1 x 10(3); CD33 < 17.3 x 10(3) and MPO > 176.1 x 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.