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The poor outcomes in infant acute lymphoblastic leukemia (ALL) necessitate new treatments. Here we discover that EIF4E protein is elevated in most cases of infant ALL and test EIF4E targeting by the repurposed antiviral agent ribavirin, which has anticancer properties through EIF4E inhibition, as a potential treatment. We find that ribavirin treatment of actively dividing infant ALL cells on bone marrow stromal cells (BMSCs) at clinically achievable concentrations causes robust proliferation inhibition in proportion with EIF4E expression. Further, we find that ribavirin treatment of KMT2A-rearranged (KMT2A-R) infant ALL cells and the KMT2A-AFF1 cell line RS4:11 inhibits EIF4E, leading to decreases in oncogenic EIF4E-regulated cell growth and survival proteins. In ribavirin-sensitive KMT2A-R infant ALL cells and RS4:11 cells, EIF4E-regulated proteins with reduced levels of expression following ribavirin treatment include MYC, MCL1, NBN, BCL2 and BIRC5. Ribavirin-treated RS4:11 cells exhibit impaired EIF4E-dependent nuclear to cytoplasmic export and/or translation of the corresponding mRNAs, as well as reduced phosphorylation of the p-AKT1, p-EIF4EBP1, p-RPS6 and p-EIF4E signaling proteins. This leads to an S-phase cell cycle arrest in RS4:11 cells corresponding to the decreased proliferation. Ribavirin causes nuclear EIF4E to re-localize to the cytoplasm in KMT2A-AFF1 infant ALL and RS4:11 cells, providing further evidence for EIF4E inhibition. Ribavirin slows increases in peripheral blasts in KMT2A-R infant ALL xenograft-bearing mice. Ribavirin cooperates with chemotherapy, particularly L-asparaginase, in reducing live KMT2A-AFF1 infant ALL cells in BMSC co-cultures. This work establishes that EIF4E is broadly elevated across infant ALL and that clinically relevant ribavirin exposures have preclinical activity and effectively inhibit EIF4E in KMT2A-R cases, suggesting promise in EIF4E targeting using ribavirin as a means of treatment.

Background Infant acute lymphoblastic leukemia (ALL) has never occurred in families except for the ∼100% concordant cases in monozygous twins attributed to twin-to-twin metastases. We report the first kindred with infant ALL in non-twin siblings. The siblings were diagnosed with MLL-rearranged (MLL-R) ALL 26 months apart. The second affected sibling had an unaffected dichorionic monozygous co-twin. Both had fatal outcomes. Procedures Translocations were characterized by karyotype, FISH, multiplex FISH, and MLL breakpoint cluster region (bcr) Southern blot analysis. Breakpoint junctions and fusion transcripts were cloned by PCR. TP53 mutation and NADPH quinone oxidorecuctase 1 (NQO1) C609T analyses were performed, and pedigree history and parental occupations were ascertained. The likelihood of chance occurrence of infant ALL in non-twin siblings was computed based on a binomial distribution. Zygosity was determined by single nucleotide polymorphism (SNP) array. Results The translocations were not related or vertically transmitted. The complex karyotype of the proband's ALL had chromosome 2, 3, 4, and 11 abnormalities causing a 5′-MLL-AFF1-3′ fusion and a non-productive rearrangement of 3′MLL with a chromosome 3q intergenic region. The affected twin's ALL exhibited a simple t(4;11). The complex karyotype of the proband's ALL suggested a genotoxic insult, but no exposure was identified. There was no germline TP53 mutation. The NQO1 C609T risk allele was absent. The likelihood of infant ALL occurring in non-twin siblings by chance alone is one in 1.198 × 109 families. Conclusions Whether because of a deleterious transplacental exposure, novel predisposition syndrome, or exceedingly rare chance occurrence, MLL-R infant ALL can occur in non-twin siblings. The discordant occurrence of infant ALL in the monozygous twins was likely because they were dichorionic.

SUMMARYCryptosporidium, a parasite known to cause large drinking and recreational water outbreaks, is tolerant of chlorine concentrations used for drinking water treatment. Human laboratory-based surveillance for enteric pathogens detected a cryptosporidiosis outbreak in Baker City, Oregon during July 2013 associated with municipal drinking water. Objectives of the investigation were to confirm the outbreak source and assess outbreak extent. The watershed was inspected and city water was tested for contamination. To determine the community attack rate, a standardized questionnaire was administered to randomly sampled households. Weighted attack rates and confidence intervals (CIs) were calculated. Water samples tested positive for Cryptosporidium species; a Cryptosporidium parvum subtype common in cattle was detected in human stool specimens. Cattle were observed grazing along watershed borders; cattle faeces were observed within watershed barriers. The city water treatment facility chlorinated, but did not filter, water. The community attack rate was 28·3% (95% CI 22·1–33·6), sickening an estimated 2780 persons. Watershed contamination by cattle probably caused this outbreak; water treatments effective against Cryptosporidium were not in place. This outbreak highlights vulnerability of drinking water systems to pathogen contamination and underscores the need for communities to invest in system improvements to maintain multiple barriers to drinking water contamination.

Survival in infants younger than 1 year who have acute lymphoblastic leukemia (ALL) is inferior whether MLL is rearranged (R) or germline (G). MLL translocations confer chemotherapy resistance, and infants experience excess complications. We characterized in vitro sensitivity to the pan-antiapoptotic BCL-2 family inhibitor obatoclax mesylate in diagnostic leukemia cells from 54 infants with ALL/bilineal acute leukemia because of the role of prosurvival BCL-2 proteins in resistance, their imbalanced expression in infant ALL, and evidence of obatoclax activity with a favorable toxicity profile in early adult leukemia trials. Overall, half maximal effective concentrations (EC50s) were lower than 176 nM (the maximal plasma concentration [Cmax] with recommended adult dose) in 76% of samples, whether in MLL-AF4, MLL-ENL, or other MLL-R or MLL-G subsets, and regardless of patients' poor prognostic features. However, MLL status and partner genes correlated with EC50. Combined approaches including flow cytometry, Western blot, obatoclax treatment with death pathway inhibition, microarray analyses, and/or electron microscopy indicated a unique killing mechanism involving apoptosis, necroptosis, and autophagy in MLL-AF4 ALL cell lines and primary MLL-R and MLL-G infant ALL cells. This in vitro obatoclax activity and its multiple killing mechanisms across molecular cytogenetic subsets provide a rationale to incorporate a similarly acting compound into combination strategies to combat infant ALL.

Summary Infant leukaemia is an embryonal disease in which the underlying MLL translocations initiate in utero. Zebrafish offer unique potential to understand how MLL impacts haematopoiesis from the earliest embryonic timepoints and how translocations cause leukaemia as an embryonal process. In this study, a zebrafish mll cDNA syntenic to human MLL spanning the 5′ to 3′ UTRs, was cloned from embryos, and mll expression was characterized over the zebrafish lifespan. The protein encoded by the 35-exon ORF exhibited 46·4% overall identity to human MLL and 68–100% conservation in functional domains (AT-hooks, SNL, CXXC, PHD, bromodomain, FYRN, taspase1 sites, FYRC, SET). Maternally supplied transcripts were detected at 0–2 hpf. Strong ubiquitous early zygotic expression progressed to a cephalo-caudal gradient during later embryogenesis. mll was expressed in the intermediate cell mass (ICM) where primitive erythrocytes are produced and in the kidney where definitive haematopoiesis occurs in adults. mll exhibits high cross species conservation, is developmentally regulated in haematopoietic and other tissues and is expressed from the earliest embryonic timepoints throughout the zebrafish lifespan. Haematopoietic tissue expression validates using zebrafish for MLL haematopoiesis and leukaemia models.

We prospectively observed a child exposed to intensive multimodality therapy for metastatic neuroblastoma from emergence of a MLL translocation to disease diagnosis. The t(4;11)(p12;q23) was detected in the marrow 17 months after starting treatment following topoisomerase II poisons, alkylating agents, local radiation, hematopoietic stem cell transplantation, anti-GD2 monoclonal antibody with granulocyte macrophage–colony-stimulating factor, and a high cumulative dose of oral etoposide. Reciprocal genomic breakpoint junctions and fusion transcripts joined MLL with FRYL, the Drosophila melanogaster protein homologue of which regulates cell fate. Etoposide metabolites induced topoisomerase II cleavage complexes that could form both breakpoint junctions. Cells harboring the translocation replaced the marrow without clinical evidence of leukemia and differentiation appeared unaffected for 37 months. Subsequent bilineage dysplasia and increased blasts in addition to the translocation fulfilled criteria for MDS. The MEIS1 target gene of typical MLL fusion oncoproteins was underexpressed before and at MDS diagnosis. These results are consistent with repair of topoisomerase II cleavage from etoposide metabolites as the translocation mechanism, whereas other agents in the regimen may have contributed to progression of the clone with the translocation to MDS. MLL-FRYL did not increase MEIS1 expression, conferred a proliferative advantage without altering differentiation, and had protracted latency to disease.

Infant acute lymphoblastic leukemia (ALL) has never occurred in families except for the ∼100% concordant cases in monozygous twins attributed to twin-to-twin metastases. We report the first kindred with infant ALL in non-twin siblings. The siblings were diagnosed with MLL-rearranged (MLL-R) ALL 26 months apart. The second affected sibling had an unaffected dichorionic monozygous co-twin. Both had fatal outcomes.Translocations were characterized by karyotype, FISH, multiplex FISH, and MLL breakpoint cluster region (bcr) Southern blot analysis. Breakpoint junctions and fusion transcripts were cloned by PCR. TP53 mutation and NADPH quinone oxidorecuctase 1 (NQO1) C609T analyses were performed, and pedigree history and parental occupations were ascertained. The likelihood of chance occurrence of infant ALL in non-twin siblings was computed based on a binomial distribution. Zygosity was determined by single nucleotide polymorphism (SNP) array.The translocations were not related or vertically transmitted. The complex karyotype of the proband's ALL had chromosome 2, 3, 4, and 11 abnormalities causing a 5'-MLL-AFF1-3' fusion and a non-productive rearrangement of 3'MLL with a chromosome 3q intergenic region. The affected twin's ALL exhibited a simple t(4;11). The complex karyotype of the proband's ALL suggested a genotoxic insult, but no exposure was identified. There was no germline TP53 mutation. The NQO1 C609T risk allele was absent. The likelihood of infant ALL occurring in non-twin siblings by chance alone is one in 1.198 × 10(9) families.Whether because of a deleterious transplacental exposure, novel predisposition syndrome, or exceedingly rare chance occurrence, MLL-R infant ALL can occur in non-twin siblings. The discordant occurrence of infant ALL in the monozygous twins was likely because they were dichorionic.

Panhandle PCR techniques to amplify known sequence flanked by unknown sequence have been useful for MLL genomic breakpoint junctions and fusion transcripts because MLL has a large number of partner genes. However, genomic panhandle PCR approaches are impeded when the restriction fragment that contains the breakpoint junction is too large to amplify. We devised new panhandle PCR approaches for MLL genomic breakpoint junctions that create the template from BglII restriction fragments by attaching MLL sequence to a BglII site in the partner gene. This leads to the annealing of MLL and its complement in the handle and creates an intrastrand loop containing the breakpoint junction sequence for amplification with primers all from MLL. BglII panhandle PCR for der(11) breakpoint junctions was accomplished by ligating a phosphorylated oligonucleotide containing a BglII overhang and sequence complementary to MLL exon 7 to the 3′ ends of BglII digested DNA, and forming the template from the sense strand of DNA. In BglII reverse panhandle PCR for der(other) breakpoint junctions, a phosphorylated oligonucleotide containing a BglII overhang and the complement of antisense sequence in MLL exon 10 was ligated to the 3′ ends of BglII digested DNA, and the template was formed from the antisense strand of DNA. These approaches amplified 5′-MLL-MLLT4-3′ and 5′-AFF1-MLL-3′ breakpoint junctions. The former is significant because few t(6;11) genomic breakpoint junctions have been sequenced. BglII panhandle PCR approaches increase the possibilities for cloning MLL genomic breakpoint junctions where there is heterogeneity in partner genes and breakpoint locations. © 2006 Wiley-Liss, Inc.

Because treatment regimens for breast cancer commonly include gemcitabine, we evaluated two promising combinations in preclinical studies: gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) with either ionizing radiation or docetaxel (Taxotere; Aventis Pharmaceuticals, Inc, Parsippany, NJ). In breast cancer cell lines that expressed either wild-type p53 (MCF-7) or mutant p53 (MCF-7/Adr), sensitivity to the cytotoxic effects of gemcitabine during a 24-hour incubation was similar (IC50 values 80 and 60 nmol/L in MCF-7 and MCF-7/Adr, respectively). Both cell lines were well radiosensitized by gemcitabine at the corresponding IC50, with radiation enhancement ratios of 1.6 to 1.7. Although the MCF-7 cells accumulated nearly twice as much gemcitabine triphosphate compared with the MCF-7/Adr cells, a similar reduction in 2′-deoxyadenosine 5′-triphosphate pools was observed. While the number of dying cells, as measured by sub-G1 DNA content or S-phase cells unable to replicate DNA, differed between the wild-type p53 or mutant p53-expressing cell lines, neither parameter correlated with radiosensitization. Docetaxel was a more potent cytotoxic agent than gemcitabine in MCF-7 cells (IC50 = 1 nmol/L). Strong synergistic cytotoxicity was observed in cells treated with gemcitabine (24 hours) followed by docetaxel (24 hours) or the reverse sequence. However, simultaneous addition of the two drugs was antagonistic. To determine whether synergy with radiation or docetaxel was mediated by increased DNA damage, DNA double-strand breaks (double-strand breaks) were measured by immunostaining for phosphorylated H2AX. Ionizing radiation produced more double-strand breaks than gemcitabine alone, while no significant double-strand breaks formed with docetaxel alone. The addition of docetaxel or ionizing radiation to gemcitabine-treated cells did not increase H2AX foci formation. These results show that the combination of gemcitabine with ionizing radiation or docetaxel produces strong, schedule-dependent synergy in breast cancer cells that is not mediated through increasing DNA double-strand breaks.

Capillary zone electrophoresis (CE) under conditions of reversed polarity is used in conjunction with electrochemical detection (EC) at carbon fiber microcylinder electrodes for the selective and sensitive determination of uric acid in human blood serum. Comigration of anions with the electroosmotic flow is accomplished with reversed polarity and the buffer additive cetyltrimethylammonium bromide (CTAB) in a 2-(N-morpholino)ethanesulfonic acid (MES) buffer system, giving rise to rapid and sensitive analyses. Optimal buffer conditions (pH 7.0), detection potential (0.80 V vs. Ag/AgCl), and electrokinetic injection are employed to allow for maximal resolution and signal intensity. Amperometric end-column detection with a carbon fiber microcylinder electrode results in lower limits of detection for uric acid of about 25 nM (ca. 140 amol injected) without the need for decoupling. Linear calibration plots using uric acid standards in water and serum are obtained over a linear range from 5.00 x 10 - 4 M to 2.50 x 10 - 7 M. Uric acid concentrations obtained for human sera using the CE-EC approach described here are shown to compare favorably to the accepted laboratory values.

Ganciclovir (GCV) is widely used as a prodrug for selective activation in tumor cells expressing herpes simplex virus thymidine kinase (HSV-TK) because of its ability to induce multi-log cytotoxicity to HSV-TK-expressing as well as nonexpressing bystander cells. We now report that another substrate for HSV-TK, D-carbocyclic 2'-deoxyguanosine (CdG), induces multi-log cytotoxicity in HSV-TK-expressing and bystander cells at concentrationsor=3 microM. We have compared the cytotoxicity and cell cycle effects of CdG to that observed with GCV in two human tumor cell lines. The results demonstrated that cytotoxicity of CdG was similar to that of GCV in both U251 glioblastoma and SW620 colon carcinoma cells that stably expressed HSV-TK. In addition, CdG induced a potent bystander effect in both cell types in co-cultures consisting of HSV-TK-expressing and nonexpressing bystander (lacZ-expressing) cells at ratios of 50:50 or 10:90. Selectivity for HSV-TK-expressing compared to lacZ-expressing cells was similar for CdG and GCV in the U251 cells, however CdG was less selective than GCV in the SW620 cell lines. Despite their ability to induce multi-log cytotoxicity at similar concentrations, CdG and GCV exhibited differential effects on cell cycle progression. Cells incubated with 1 microM CdG for 24 hr accumulated in S-phase and G(2)/M after drug washout, and the majority of cells died prior to cell division. This contrasts with the delayed effects of 1 microM GCV that were not evident until after cell division when cells attempted S-phase for the second time. Thus, CdG is a potent cytotoxic agent that merits further investigation to determine whether it will be therapeutically effective in enzyme-prodrug therapy with HSV-TK.

Gene expression profiling was performed on 97 cases of infant ALL from Children's Oncology Group Trial P9407. Statistical modeling of an outcome predictor revealed 3 genes highly predictive of event-free survival (EFS), beyond age and MLL status: FLT3, IRX2, and TACC2. Low FLT3 expression was found in a group of infants with excellent outcome (n = 11; 5-year EFS of 100%), whereas differential expression of IRX2 and TACC2 partitioned the remaining infants into 2 groups with significantly different survivals (5-year EFS of 16% vs 64%; P < .001). When infants with MLL-AFF1 were analyzed separately, a 7-gene classifier was developed that split them into 2 distinct groups with significantly different outcomes (5-year EFS of 20% vs 65%; P < .001). In this classifier, elevated expression of NEGR1 was associated with better EFS, whereas IRX2, EPS8, and TPD52 expression were correlated with worse outcome. This classifier also predicted EFS in an independent infant ALL cohort from the Interfant-99 trial. When evaluating expression profiles as a continuous variable relative to patient age, we further identified striking differences in profiles in infants less than or equal to 90 days of age and those more than 90 days of age. These age-related patterns suggest different mechanisms of leukemogenesis and may underlie the differential outcomes historically seen in these age groups.

Abstract 2600 Background AF9 is a transcription factor that plays an essential role in hematopoiesis and embryonic development. The alteration of AF9 is principally associated in acute myeloid leukemia as fusion partner of human MLL (mixed-lineage leukemia) gene rearrangements. Zebrafish is an excellent model organism to study embryonic development and hematopoiesis. We have previously shown that zebrafish af9 is expressed within the intermediate cell mass (ICM), a site of primitive hematopoiesis in zebrafish. Here we study the loss of af9 in zebrafish development to further understand how af9 modulates early hematopoietic and embryonic development. Methods and results Two morpholino antisense oligos (MOs), designed to block translation and inhibit pre-mRNA splicing of af9, were co-injected in embryos at 1–2 cell stage. To control for off-target effects, two morpholino mismatch oligos were designed and co-injected. Efficacy of MOs was demonstrated by Western blot analysis and RT-PCR in controls and MO-injected embryos (morphants). In vivo monitoring of both morphants and control embryos was carried out by microscopy. Effects of af9 depletion on vasculature and erythropoiesis were evaluated in Tg(fli1:eGFP) and Tg(gata1:DsRed) transgenic lines, respectively. Whole-mount in situ hybridization of known hematopoietic markers was used to decipher the developmental time-points in which af9 regulates blood development. Following injection of two MOs at 1–2 cell stage, we compared the morphological features of the morphants with control embryos at about 24 hours post-fertilization (hpf). The af9 morphants showed small head and eyes, disruption of tail development and pronounced swelling in the posterior ICM. Circulating blood cells were reduced from 26 hpf to later stages of development. At 48 hpf the heart was enlarged, showed a paucity of blood-cells and pericardial edema. Decreased number of blood cells in morphant embryos was further confirmed by o-dianisidine staining at 48 hpf and 72 hpf and in living af9-knockdown gata1:DsRed transgenic animals, suggesting that the differentiation of erythroblasts remains insufficient or impaired. Concordant with this observation, we examined the expression of specific markers for early hematopoiesis (scl, lmo2 and gata2) and primitive erythropoiesis (gata1, hbbe, and band3) using whole-mount in situ hybridization (WISH). At the 5-somite stage, the early hematopoietic precursor marker gata2 was markedly increased while scl and lmo2 remained unaffected in af9 morphants. Interestingly, by 24 hpf gata2 was found to be specifically over-expressed in ICM while no change was observed for scl and lmo2 markers. Besides, the erythroid progenitors and mature erythrocyte markers gata1, band3 and hbbe displayed nearly normal expression. To further confirm the role af9 in early hematopoiesis, we examined its expression in moonshine, a mutant zebrafish with defects in erythroid maturation due to deficiency of tif1γ, a key regulator of hematopoietic gene expression. WISH analysis in moonshine showed loss of af9 expression in the ICM at 24 hpf, suggesting that af9 functions genetically downstream of tif1γ in normal erythroid cell development. To determine the effect of af9 on endothelial and vascular development, we performed knockdown of af9 in fli1:eGFP transgenic line. By 24 hpf, these morphants showed significant increase of fluorescence intensity in the posterior ICM and a clear perturbation in the inter-segmental vessels (ISV) of the trunk at 30 hpf, indicating that af9 is required for early steps in hemangioblast specification and vascular pattern formation in zebrafish. Conclusion af9 regulates gata2 expression during early hemangioblast specification and vascular pattern formation in zebrafish. af9 may also be involved in caudal segment morphogenesis. Taken together, these data provide the initial framework of a pathway that can be used to further integrate the molecular events regulating hemangioblast differentiation. Disclosures: No relevant conflicts of interest to declare.

Translocations and other rearrangements of the MLL gene at chromosome band 11q23 are biologically and clinically important molecular abnormalities in infant acute leukemias, leukemias associated with chemotherapeutic topoisomerase II poisons and, less often, acute leukemias in adults or myelodysplastic syndrome. Depending on the disease and the regimen, MLL-rearranged leukemias may be associated with inferior prognosis, and MLL rearrangements with some of the more than 60 known MLL-partner genes confer especially adverse effects as response to treatment (Blood 108:441-451, 2006). MLL rearrangements are usually evident as overt balanced chromosomal translocations by conventional cytogenetic analysis but up to one-third are cryptic rearrangements and occur in leukemias with del(11)(q23), a normal karyotype, or trisomy 11, the latter two of which sometimes are associated with partial tandem duplications of MLL itself (Proc Natl Acad Sci USA 97:2814-2819, 2000; Proc Natl Acad Sci USA 94:3899-3902, 1997). In addition, subsets of MLL rearrangements are complex at a cytogenetic level and/or molecular level, and fuse MLL with two different partner genes. Rapid and accurate methods to identify and characterize genomic breakpoint junctions and fusion transcripts resulting from the many types of MLL rearrangements are essential for risk group stratification, treatment protocol assignments, new partner gene discovery, understanding leukemia etiology and pathogenesis, and elucidating the impact of less common MLL-partner genes on biology and prognosis. Due to the vast heterogeneity in partner genes, typical gene-specific PCR based methods are not practical, especially when cytogenetics are normal or do not suggest involvement of a known partner gene of MLL. We have advanced seven different panhandle PCR based methods for cloning 5'-MLL-partner gene-3' and 5'-partner gene-MLL-3' genomic breakpoint junctions and identifying 5'-MLL-partner gene-3' fusion transcripts, all of which employ a stem-loop template shaped schematically like a pan with a handle and amplify the template without knowledge of the unknown partner sequence using primers all derived from MLL alone.

Translocations and other rearrangements of the MLL gene at chromosome band 11q23 are biologically and clinically important molecular abnormalities in infant acute leukemias, leukemias associated with chemotherapeutic topoisomerase II poisons and, less often, acute leukemias in adults or myelodysplastic syndrome. Depending on the disease and the regimen, MLL-rearranged leukemias may be associated with inferior prognosis, and MLL rearrangements with some of the more than 60 known MLL-partner genes confer especially adverse effects as response to treatment (Blood 108:441-451, 2006). MLL rearrangements are usually evident as overt balanced chromosomal translocations by conventional cytogenetic analysis but up to one-third are cryptic rearrangements and occur in leukemias with del(11)(q23), a normal karyotype, or trisomy 11, the latter two of which sometimes are associated with partial tandem duplications of MLL itself (Proc Natl Acad Sci USA 97:2814-2819, 2000; Proc Natl Acad Sci USA 94:3899-3902, 1997). In addition, subsets of MLL rearrangements are complex at a cytogenetic level and/or molecular level, and fuse MLL with two different partner genes. Rapid and accurate methods to identify and characterize genomic breakpoint junctions and fusion transcripts resulting from the many types of MLL rearrangements are essential for risk group stratification, treatment protocol assignments, new partner gene discovery, understanding leukemia etiology and pathogenesis, and elucidating the impact of less common MLL-partner genes on biology and prognosis. Due to the vast heterogeneity in partner genes, typical gene-specific PCR based methods are not practical, especially when cytogenetics are normal or do not suggest involvement of a known partner gene of MLL. We have advanced seven different panhandle PCR based methods for cloning 5'-MLL-partner gene-3' and 5'-partner gene-MLL-3' genomic breakpoint junctions and identifying 5'-MLL-partner gene-3' fusion transcripts, all of which employ a stem-loop template shaped schematically like a pan with a handle and amplify the template without knowledge of the unknown partner sequence using primers all derived from MLL alone.

Chemotherapy resistance from imbalanced apoptosis regulation may contribute to poor outcome in leukaemias with t(4;11). Anti-apoptotic BCL-2 expression and target modulation were characterized in cell lines with t(4;11) and BCL-2 expression was examined in MLL and non-MLL infant/paediatric leukaemia cases by Western blot analysis and/or real-time polymerase chain reaction. Cytotoxicity of Genasensetrade mark (Oblimersen Sodium, G3139) alone or combined with cytotoxic drugs was assessed by MTT [(3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays of the cell lines, applying pharmacostatistical response surface modelling of drug interactions. Apoptosis and cell cycle were evaluated by flow cytometry in RS4:11 cells. Primary leukaemias and cell lines with t(4;11) expressed abundant BCL2 mRNA and protein. Variable, sometimes substantial BCL2 mRNA was detected in other leukaemia subtypes. G3139 reduced BCL2 mRNA and protein in RS4:11 cells. The most sensitive cell line to single-agent G3139 was RS4:11. Low G3139 concentrations sensitized RS4:11 and MV4-11 cells to select anti-leukaemia cytotoxic drugs. In RS4:11 cells, combining G3139 with doxorubicin (ADR) increased active caspase 3 and TUNEL staining compared to ADR alone, indicating greater apoptosis, and G3139 increased S-phase progression. The abundant BCL-2 affords a molecular target in leukaemias with t(4;11). G3139 exhibits preclinical activity and synergy with select cytotoxic agents in RS4:11 and MV4-11 cells, and these effects occur through apoptosis.

Radiation sensitization by 2′,2′-difluoro-2′-deoxycytidine (dFdCyd) has correlated with dATP depletion [dFdCDP-mediated inhibition of ribonucleotide reductase (RR)] and S-phase accumulation. We hypothesized that radiosensitization by dFdCyd is due to nucleotide misincorporations in the presence of deoxynucleotide triphosphate pool imbalances, which, if not repaired, augments cell death following irradiation. The ability of dFdCyd to produce misincorporations was measured as pSP189 plasmid mutations in hMLH1-deficient [mismatch repair (MMR) deficient] and hMLH1-expressing (MMR proficient) HCT116 cells. Only MMR-deficient cells showed a significant increase in nucleotide misincorporations (2- to 3-fold increase; P ≤ 0.01) after radiosensitizing concentrations of dFdCyd ± 5 Gy radiation, which persisted for at least 96 h. dFdCyd (10 nmol/L) did not radiosensitize MMR-proficient HCT116 or A549 cells, but following small interfering RNA–mediated suppression of hMLH1, this concentration produced excellent radiosensitization (radiation enhancement ratios = 1.6 ± 0.1 and 1.5 ± 0.1, respectively; P < 0.05) and a 2.5-fold increase in mutation frequency in A549 cells. Cytosine arabinoside (1-β-d-arabinofuranosylcytosine), which can be incorporated into DNA but does not inhibit RR, failed to radiosensitize MMR-deficient cells or increase mutation frequency in the MMR-deficient and MMR-proficient cells. However, the RR inhibitor hydroxyurea radiosensitized MMR-deficient cells and increased nucleotide misincorporations (≥5-fold increase; P < 0.05), thus further implicating the inhibition of RR as the mechanism underlying radiosensitization by dFdCyd. These data showed that the presence and persistence of mismatched nucleotides is integral to radiosensitization by dFdCyd and suggest a role for hMLH1 deficiency in eliciting the radiosensitizing effect. [Mol Cancer Ther 2007;6(6):1858–68]

Gemcitabine [2',2'-difluoro-2'-deoxycytidine (dFdCyd)] is a potent ionizing radiation sensitizer in solid tumor cells in vitro and in vivo. Previously, we have demonstrated (Shewach et al., Cancer Res., 54: 3218-3223, 1994) a strong correlation between depletion of dATP (caused by dFdCyd diphosphate-mediated inhibition of ribonucleotide reductase) and radiosensitization. In addition, we and others (Latz et al., Int. J. Radiat. Oncol. Biol. Phys., 41: 875-882, 1998; Ostruszka and Shewach, Cancer Res., 60: 6080-6088, 2000) have shown that the accumulation of cells in S phase prior to irradiation is also important for radiosensitization with dFdCyd. This led us to hypothesize that the incorporation of incorrect nucleotides because of the dATP pool imbalance was important for radiosensitization with dFdCyd, and, therefore, cells deficient in mismatch repair (MMR) would exhibit greater radiosensitization. We tested this hypothesis by evaluating the ability of HCT116 colon carcinoma cell lines, which differ in MMR proficiency, to be radiosensitized by dFdCyd. The MMR-proficient cell line (HCT116 + ch3) was more sensitive to dFdCyd alone than were the MMR-deficient cell lines (HCT116, HCT116 + ch2, and HCT116 p53(-/-)). Interestingly, the MMR-proficient cells could not be radiosensitized at concentrations of dFdCydor=IC(90), although extremely high concentrations of dFdCyd (IC(96)) enhanced cell killing with radiation. In contrast, the MMR-deficient cells were radiosensitized at concentrations of dFdCydor=IC(50), with radiation enhancement ratios of approximately 1.5. Cell cycle analysis, using dual parameter flow cytometry, demonstrated that all of the cell lines accumulated in S phase after dFdCyd treatment, and, shortly after irradiation, a prominent but transient G(2)-M block was observed. In the MMR-deficient cells, the IC(50) for dFdCyd produced aor=80% decrease in dATP within 4 h after drug addition, and this low dATP level was maintained for another 12-20 h. Although the IC(50) of dFdCyd was unable to sustain a80% decrease in the dATP level in the MMR-proficient cells, the IC(90) did achieve this level of dATP depletion; however, it was unable to radiosensitize the MMR-proficient cells. Similar results were obtained with HCT116 cells, in which the MMR deficiency was corrected by transfection with a vector containing the hMLH1 cDNA. In addition, the deletion of p53 did not increase radiation enhancement ratios. These results demonstrate that MMR deficiency promotes radiosensitization with dFdCyd. We suggest that dATP depletion produces errors of replication in MMR-deficient cells, which, if left unrepaired, enhances cell death by ionizing radiation.

Capillary zone electrophoresis (CE) under conditions of reversed polarity is used in conjunction with electrochemical detection (EC) at carbon fiber microcylinder electrodes for the selective and sensitive determination of uric acid in human blood serum. Comigration of anions with the electroosmotic flow is accomplished with reversed polarity and the buffer additive cetyltrimethylammonium bromide (CTAB) in a 2-(N-morpholino)ethanesulfonic acid (MES) buffer system, giving rise to rapid and sensitive analyses. Optimal buffer conditions (pH 7.0), detection potential (0.80 V vs. Ag/AgCl), and electrokinetic injection are employed to allow for maximal resolution and signal intensity. Amperometric end-column detection with a carbon fiber microcylinder electrode results in lower limits of detection for uric acid of about 25 nM (ca. 140 amol injected) without the need for decoupling. Linear calibration plots using uric acid standards in water and serum are obtained over a linear range from 5.00 x 10(-4) M to 2.50 x 10(-7) M. Uric acid concentrations obtained for human sera using the CE-EC approach described here are shown to compare favorably to the accepted laboratory values.

Abstract 2417 Leukemia is the commonest malignancy in infants, the most frequently occurring form of which is infant ALL. When ALL occurs in infants the disease is clinically aggressive and associated with poor outcome. MLL gene rearrangements producing transforming fusion oncoproteins are found in 75% of infant ALL and they are adverse prognostic factors. Infant ALL has never occurred in families except in monozygous twins, where concordance in leukemia occurrence is nearly 100%. Identical but non-germline genomic breakpoint junction sequences have pointed to an in utero origin of MLL gene rearrangements in these twin cases, where it is believed that metastasis of cells with the rearrangement occurs from one twin to the other via the placental circulation. Here we describe two highly novel siblings both deceased from precursor B cell infant ALL (ages at diagnosis: proband 160 d, sibling 121 d). Remarkably, the second of these two decedents is survived by a now 3 year-old monozygous twin who is as yet unaffected. MLLrearrangements in the leukemia blasts of both affected siblings were characterized by conventional cytogenetics and/or FISH, M-FISH, high resolution Illumina 610K Bead Chip SNP array and Southern blot analysis. MLL genomic breakpoint junction sequences and fusion transcripts were defined using panhandle PCR approaches, PCR with gene-specific primers and reverse transcriptase PCR. The twins were confirmed to be monozygous using the genotype calls from SNP array analysis of the peripheral blood and bone marrow of the unaffected and affected twins, respectively. Quantitative real-time PCR analysis of leukemia DNA was used to determine the allele specific sequences of the NQO1 (NADPH quinone oxidorecutase 1) gene, an inactivating polymorphism in which previously was implicated as a risk factor for MLL-rearranged infant ALL. The complex karyotype in the leukemia cells of the proband was 46, XX, der(2) t(2;3) (q3?;?), der(3) ?t(3;4;11), del(4) (q21), der(11) ?del(11) (p11.2) t(3;11) (?;q23).ish der(3) (5'MLL+), der(11) (3'MLL+) [14]/46, XX[8], suggesting extensive damage to the genome. Consistent with a three-way t(3;4;11) translocation, two alternately spliced 5'-MLL-MLLT2(AF-4)-3' fusion transcripts were identified, indicating disruption of the chromosome band 4q21 partner gene MLLT2. Also consistent with the three-way translocation, reverse panhandle PCR detected a 5'-partner-MLL-3' genomic breakpoint junction fusing 3' MLL to the upstream region of a highly novel chromosome 3 gene encoding a nucleotidyltransferase fold protein C3ORF31 (Accession no. NM_138807; Kuchta, 2009). Unlike in the proband, the ALL of the affected twin exhibited a simple t(4;11)(q21;q23) translocation, the reciprocal genomic breakpoint junctions of which fusing MLL and MLLT2 also have been characterized. Similar to non-familial infant ALL, the genomic breakpoint junctions in both infants contained sequence features of nonhomologous end joining DNA repair. The different MLL gene rearrangements in the leukemia cells in the affected siblings indicate that the translocations were not hereditary. Even though the NQO1 inactivating polymorphism is one genetic risk factor for infant ALL, the NQO1 genotype was wild-type in both affected siblings. Further studies in this uniquely afflicted family with two siblings who succumbed to infant ALL and a monozygous twin of one of the decedents surviving beyond infancy unaffected, will provide a one-time opportunity to capture novel mutations predisposing to the development of, and cooperating with, MLL translocations in infant ALL. The MLLT2 involvement in both cases has even further implications for the knowledge to be gained because the MLL-MLLT2 rearrangement occurs in 50% of infant ALL and adversely impacts outcome. Disclosures: Felix: Children's Hospital of Philadelphia: Methods and Kits for Analysis of Chromosomal Rearrangements Associated with Leukemia - U.S. Patent # 6,368,791 issued April 9, 2002.

Abstract 2757 Introduction: The outcome of infants with acute lymphoblastic leukemia (ALL) remains poor because of the association of frequently occurring MLL translocations with drug resistance and vulnerability of the very young to treatment complications. The two most common MLL partner genes in infant ALL, AF4 (AFF1) and ENL (MLLT1), are associated with particularly poor survival. Better therapies are urgent. One candidate is obatoclax (GeminX Biotechnologies, Inc.), which targets interactions of pan-anti-apoptotic BCL-2 family proteins with BH3 proteins and is now in a Phase I trial for relapsed/refractory pediatric cancers (COG ADVL0816). Previously we showed potent single agent in vitro activity of obatoclax against MLL-rearranged infant ALL (Zhang ASH 2008). Here we evaluate correlations of obatoclax activity with MLL translocation status and gene expression profiles in a large number of cases of infant ALL to define molecular determinants of sensitivity. Methods: Bone marrow, peripheral blood or apheresis samples from the time of diagnosis in 54 infants (age 1–365 d, median 168 d; WBC count 15–1230×103/μL, median 445×103/μL) with ALL (n=52) or bilineal acute leukemia (n=2) were examined, 48 of which were from the COG P9407 trial. By molecular/cytogenetic classification, the cases were MLL-AF4+ (n= 28), MLL-ENL+ (n= 11), other MLL rearrangement positive (other MLL+) (n= 8) or MLL germline (MLL-) (n= 7). Single agent IC50 values from MTT assays after 72 h obatoclax exposures were determined in all cases (including 13 previously tested; Zhang ASH 2008) by plotting the surviving fractions. IC50s in the MLL-AF4+ group were compared to those in each of the other 3 molecular/cytogenetic groups by Wilcoxon's test. Gene expression profiling was performed on Affymetrix HG_U133 Plus2.0 arrays in 47 of the 48 COG P9407 cases. Spearman test was used to identify correlation between log2 expression levels for each probeset and IC50 values across subjects. A heatmap of significant probesets (p≤0.001) was generated by transforming expression levels to z-scores and ordering rows and columns by complete linkage hierarchical clustering. Ingenuity pathway analysis was applied to all probesets with p≤0.01 to identify pathways significantly correlated with IC50. Additional MTT assays were initiated to test sensitivity to agents targeting these pathways. Results: Even though most cases in all 4 groups were sensitive to obatoclax as indicated by IC50s within a clinically achievable range, MLL translocation status still had a significant effect on IC50. MLL-AF4+ cases were least sensitive and MLL-ENL+ cases were most sensitive to obatoclax. Respective IC50 ranges across all 54 cases were: MLL-AF4+, 26–918 nM; MLL-ENL+, 13–294 nM; other MLL+ 10–356 nM; MLL−, 31–488. Compared to MLL-AF4+, the IC50s in MLL-ENL+ cases were significantly lower (p=0.047), IC50s in other MLL+ cases were lower but the difference did not achieve significance (p=0.10), and IC50s in MLL- cases were not significantly different (p=0.64). In the 47 COG P9407 cases studied by MTT assay and gene expression profiling, 450 probesets defined a cluster of 16 cases with higher IC50s, which were predominantly MLL-AF4+ (68.7%). Ingenuity analysis identified significant correlations of the following canonical pathways with the IC50 in the same 47 cases: glycolysis/gluconeogenesis, mTOR signaling, regulation of eIF4 and p70S6K signaling, EIF2 signaling, and fructose and mannose metabolism. In preliminary analyses, cell lines with t(4;11) exhibited time and dose-dependant sensitivity to the eIF4e inhibitor ribavirin. Conclusions: In infant ALL, obatoclax has broad-spectrum activity and there is pan-sensitivity across MLL translocation subtypes and MLL− cases. Still specific MLL partner genes have a strong effect on obatoclax IC50 and there is exquisite sensitivity in MLL-ENL+ cases. This result is important because MLL-ENL is associated with particularly poor survival when conventional therapies are used. The association of differentially expressed genes in canonical cell signaling and metabolism pathways with differences in obatoclax sensitivity forms the basis to combine obatoclax with targeted agents directed at restoring these pathways to enhance responsiveness even further. Disclosures: Felix: None: Patent not licensed.

Abstract 412 ALL arising in infants is a highly refractory disease. Overall event-free survival (EFS) remains poor and infants with MLL rearrangements (MLL-R) or those 90 days of age (in the overall cohort and in the MLL-AF4 cases). Specifically evaluating the impact of patient age treated as a continuous variable revealed a striking transition in expression profiles at 90 days with a differential expression pattern involving many genes encoding histone-related, heat shock family, or immune response regulators (including HLA-DRB4, IL1R2, HSPA1A///1B). These distinctive profiles may reflect different transformed stem/precursor cells or susceptibilities to leukemic transformation at different patient ages, altered marrow microenvironments, or altered immune status; high expression of the heat shock proteins in particular among the youngest infants may reflect a more limited immune surveillance capacity. Given the rarity of infant ALL, this study represents one of the largest uniformly treated groups of infant leukemia to undergo gene expression profiling. In these studies we have identified genes that are highly predictive of outcome at diagnosis, in all infant ALL and in MLL-AF4 cases. Further analysis of these expression profiles, coupled with validation studies in other infant ALL cohorts, may allow for the identification of novel therapeutic targets among the genes discovered herein and ultimately for the development of more effective therapies. Disclosures: Felix: None: Patent not licensed.

Abstract 907 Introduction: MLL translocations are the most pervasive molecular abnormalities in infant ALL and are poor prognostic factors; however, interrelationships between specific MLL partner genes, event-free survival (EFS) and clinical covariates have been elusive due to several factors. Infant ALL is a rare disease with limited cases within MLL translocation subsets, specialized methods are needed to identify the spectrum of MLL partner genes, and most prior clinical trials defined MLL status by karyotype and analyses of only common MLL partner genes. P9407, a COG pilot study testing the utility of intensified induction therapy, developed a detailed molecular cytogenetic classifier of MLL status and partner genes to investigate relationships with EFS and correlations with clinical covariates. Methods: MLL status was defined by karyotype, FISH, Southern blot analysis, RT-PCR analysis of MLL translocations with AF4, ENL or AF9, and cDNA or genomic panhandle PCR. Kaplan-Meier plots were used to estimate EFS as a function of MLL status (rearranged, R; germline, G), MLL partner gene and, within MLL status and partner gene subsets, as a function of age at diagnosis. Fisher's exact test was used to assess correlations between MLL partner genes and age, WBC (K/ml3) or CNS status at diagnosis. Results: MLL status was determined in 210 of 221 total cases [95%; 158 R, 75%; 52 G, 25%]. Partner genes were definitively determined in 133 of the 158 MLL-R cases: AF4/78/49.4%; ENL/33/20.1%; AF9/15/9.5%; EPS15/4/2.5%; AF10/1/0.6%; ASAH3/1/0.6%; ACTN4/1/0.6%. The ACTN4 partner gene was identified in a new complex 3-way MLL/ACTN4/RYR1 rearrangement. In the other 25 MLL-R cases, RT-PCR with gene specific primers excluded AF4 (n=5) or AF4, AF9 and ENL (n=13) or MLL status was not defined beyond MLL-R or MLL-G (n=7). Analyses of 202 eligible subjects within the cohort of 210 in whom MLL status was assigned, indicated a 5-year EFS rate of 45.9±4.6%, which was worse in MLL-R than MLL-G cases (38.8±5.1% v. 66.2±9.3%, P=0.0003). By category, 5-year EFS rates were: ENL (29.0±10.0%), AF4 (34.2±7.4%), other partner genes (45.0±14.9%), AF9 (67.7±17.2%), MLL-G (66.2±9.3%) (P=0.0003). Within MLL-R, AF4 was associated with worse EFS than other partner genes including AF9 but excluding ENL (P=0.043), and ENL with worse EFS than other partner genes including AF9 but excluding AF4 (P=0.024). Also, EFS was worse when AF4 and ENL subsets were combined, compared to any other partner gene (P=0.025). Conversely, the higher EFS with AF9 than with any other partner gene became less significant when AF4 and/or ENL were excluded from the other MLL-R category. EFS in the AF9 subset was not different from that for MLL-G cases. Five-year EFS was worse in patients aged Conclusions: MLL translocations, young age and high WBC have correlated with poor outcome in infant ALL before, but this MLL classifier in the largest cohort of infant ALL with detailed molecular characterization reported to date, adds new elements to the impact of MLL partner genes on outcome and their connection to prognostic factors. AF4 and ENL not only negatively impact EFS, but this effect is larger in the younger infants. Regardless of age, AF9 is a favorable prognostic factor, but the other partner genes associated with better outcome occur more often in older infants. Relationships between high WBC and AF4, and low WBC and AF9 were also discovered. The newfound associations of specific MLL partner genes with age and WBC indicate how disease biology and the classical prognostic factors in this disease are integrally connected. Disclosures: No relevant conflicts of interest to declare.

Abstract 3655 Poster Board III-591 Introduction Infant leukemia is an embryonal hematopoietic cancer because the characteristic translocations disrupting the MLL transcription factor initiate in utero. Zebrafish afford a unique model organism in which to understand MLL-regulated targets in intact animals and at the earliest developmental timepoints not accessible in mammals. We investigated consequences of loss of mll in zebrafish embryos to mimic the microenvironment in which MLL leukemia initiates in infants. Since disruption of one allele by the translocation creates haploinsufficiency, and failed differentiation and profound multi-lineage cytopenias are features of MLL disease, we hypothesized that the zebrafish embryo mll depletion model would uncover new links of MLL to developmental control of hematopoietic targets. Methods Two independent splice blocking morpholino (MO) antisense oligonucleotides titrated to no effect concentrations of mismatch controls were employed to deplete mll and verify specificity. External phenotypes were monitored and whole embryos were studied by TUNEL staining and hemoglobin staining with o-dianisidine. Effects on expression of candidate hematopoietic targets were quantified in sorted GFP+ precursor/myeloid and GFP− non-myeloid hematopoietic cells from control or mll morphant Tg(spi1:EGFP) embryos by Q-RT-PCR. Blood cell morphologic changes were visualized on cytospins. Spatio-temporal changes in hematopoietic gene expression were studied in whole embryos by WISH. Fluorescence intensity changes were measured in live Tg reporter lines with blood cell gene promoters driving expression of fluorescent protein markers. Q-RT-PCR analysis of RNAs from unsorted Mll+/+, Mll+/− and Mll−/− murine embryoid bodies (EBs) was used for validation. Results Head and jaw defects in both morphants phenocopied the neuronal and craniofacial defects of Mll−/− mice; small eyes, the eye defect of mice deficient for the Mll cofactor Meis1; and small size and delayed development, the small size of Taspase1−/− mice from cell cycle impairments when Mll is not cleaved by Taspase1. Increased apoptosis and decreased hemoglobin staining phenocopied other features of Mll deficient mice. Depleting mll also increased or decreased hoxa9a expression in GFP+ or GFP− cells from Tg(spi1:EGFP), respectively, and increased ccna and ccne cyclin gene expression in the GFP+ fraction. Moreover, deregulated gene expression in both fractions identified new mll-regulated hematopoietic targets. In the GFP+ precursor/myeloid fraction, lmo2, scl, cmyb and ikaros were overexpressed, and mll depletion accelerated myeloid (spi1, mpx, lcp1) gene expression. Paradoxically, an entire erythroid program (gata1, hbae1, slc4a1, alas2, sptb, epo, epoR) was upregulated in the myeloid fraction, while erythroid gene downregulation in the GFP− (erythroid) cells would explain the anemia from mll depletion. Conversely, myeloid (runx1, mpx) genes were overexpressed in the GFP− cells. lmo2 and ikaros expression were also increased in this fraction. These gene expression changes manifest as profound blood cell dysmorphologies. WISH showed that mll depletion in wild type embryos increased gata1 and lmo2 expression. Increases in DsRed fluorescence in Tg(gata1:DsRed) and Tg(lmo2:DsRed) mll morphant embryos further substantiated direct or indirect mll regulation of gata1 and lmo2 promoters. Q-RT-PCR demonstrated precocious Gata1 expression in Mll deficient murine EBs relative to their wild type counterparts. Conclusions The external phenotype, increased apoptosis and anemia in the morphants are critical evidence that zebrafish mll depletion phenocopies depletion of mammalian Mll and interacting factors. Prior murine models demonstrated that Mll maintains Hox expression; this work adds new information that mll has differential effects on hoxa9a in myeloid and non-myeloid cells. These studies lead to the conclusion that zebrafish mll orchestrates profound multi-lineage functions as a master regulator in the developmental control of the hematopoietic system, and has cell-type specific effects on newfound stem cell and myeloid targets and an entire erythroid program. The Gata1 expression patterns in Mll deficient murine EBs validate that Mll also directly or indirectly regulates erythropoiesis in mammals. Zebrafish are a relevant in vivo model to identify MLL-dependent pathways in blood cell development. Disclosures: No relevant conflicts of interest to declare.

In infant ALL a constellation of features (young age, hyperleukocytosis, extramedullary/CNS disease, CD10− phenotype, MLL translocation, slow early response to treatment) are associated with poor outcome. Despite the frequent MLL translocations, the biologic basis for these features is unknown. In this study, gene expression profiles of leukemia cells from diagnosis were determined in MLL-rearranged infant ALL and relationships were sought between gene expression and clinical covariates. Patients and Methods: Seventeen infants treated on COG protocol CCG 1953 were studied. Features at diagnosis were: age, 0–333 d, median 121 d; WBC count, 10.2–1,286 ×103/μL, median 229 ×103/μL; CD10−, 14, CD10+, 2, CD10 unknown, 1; CNS1 status, 11, CNS2 status, 2, CNS3 status, 4. Eight were alive and 9 were dead at survival times from 41–3384 d. MLL translocations were identified using karyotype, FISH, Southern blot and PCR. Gene expression profiling was performed with Affymetrix HG_U133 Plus2.0 arrays. Pearson’s correlation coefficients and significance levels were computed between age and log-2 transformed expression levels of each probeset. To find associations between gene expression and MLL partner genes with the age effect controlled, linear regression was run using gene expression as the dependent variable and age and partner gene (AF4 v. other) as the independent variable. Similar linear regressions were run for WBC count, CNS status (excluding CNS2) and survival status. Results: PCR identified the MLL partner genes AF4, ENL, AF9, AF10 and EPS15 in 6, 4, 2, 1 and 1 cases, respectively. In 1 case each with t(4;11)(q21;q23) or t(11;19)(q23;p13.3) partner genes were assigned based on karyotype. Another case harbored a t(6;9;11) (q21;p22;q23). Gene expression was affected more by age at diagnosis than any other factor. Remarkably, gene expression analysis by age at diagnosis as a continuous variable showed correlations of 2072 probesets, all but one of which were positive (p Conclusions: Within infant ALL there are profound intrinsic biologic differences in leukemia cells due to age at diagnosis. MLL-AF4 translocations in infant ALL have variably been associated with more inferior outcomes than other MLL translocations. This analysis indicates that the different MLL translocations are biologically dissimilar and that cases with MLL-AF4 are distinct from cases with MLL-ENL or other partner genes. The study also shows that a specific gene expression program directs the clinical/biologic property of CNS disease, which is overlapping with the effect of AF4 involvement. The complex correlations between clinical covariates, MLL translocations and gene expression in infant ALL merit further study. It will also be important to determine if a similar gene expression profile is associated with CNS disease in other leukemia subtypes.

Infants with acute leukemia, especially neonates, comprise a unique high-risk population prone to chemotherapy resistance and treatment complications. Basal levels of expression of cell death and cell survival factors vary greatly in different types of cancer and are key determinants of response to treatment. Until now, expression of these factors had not been evaluated in detail in acute leukemia of infants. In this study the basal expression of pro-death and pro-survival genes in the intrinsic apoptosis pathway, apoptosis execution genes and mitochondrial maintenance genes was quantified in primary leukemia cells in a large cohort of infants, and relationships between gene expression and MLL status and clinical covariates were analyzed. Methods: Bone marrow or peripheral blood samples from the time of diagnosis were examined in 103 infants (age 1–363 d, median 177 d) with acute leukemia (63 ALL, 40 AML). White blood cell (WBC) counts ranged from 4.4–1676×103/μL (median 127.6×103/μL). MLL status was characterized by standard cytogenetics, FISH, Southern blot analysis, reverse transcriptase PCR and/or panhandle PCR approaches. The ALL cases were classified as MLL-AF4+ (n= 31), other MLL rearrangement positive (other MLL+) (n= 22) or MLL rearrangement negative (MLL−) (n= 10). Due to heterogeneity in partner genes, the AML cases were classified as MLL+ (n= 22) or MLL− (n= 18). Quantitative real time PCR analysis of total RNAs was performed using a custom TaqMan® low-density array with one or more assays, each in quadruplicate, for 35 cell death regulatory genes and 3 housekeeping genes, and ΔCT values (average cycle threshold values normalized to 18S rRNA) were computed. The Behrens-Fisher nonparametric multiple comparison test in the npmc package for R was used to determine the significance of the difference in median −ΔCT values among the three ALL groups and between the two AML groups (p Results: In ALL, expression of the following genes was greater in MLL-AF4+ than MLL− cases: anti-apoptotic BCL-2 and MCL-1; pro-apoptotic BAX and BID; apoptosis execution APAF1, CASP3 and CASP9; and mitochondrial maintenance COX5A, CYC1, UQCR1 and UQCR. For some of these genes, expression was also greater in other MLL+ than MLL− cases. In AML, expression of pro-apoptotic BAK and BIK was higher in MLL+ than MLL− cases. For the sample set as a whole, expression of the following genes was positively correlated with WBC count (r>0.3; p Conclusions: These results suggest that infant ALL and AML differ with respect to the expression of genes that regulate cell death. Furthermore, cell death regulatory gene expression is affected more by MLL translocations in infant ALL than infant AML. This finding may explain the greater impact of MLL translocations on prognosis in infant ALL than AML. The abundance of specific pro- as well as anti-apoptotic BCL-2 family mRNAs in MLL+ infant ALL is also relevant to the identification of novel therapeutic targets in this pathway. The positive correlation between anti-apoptotic BCL-2 expression and WBC count is biologically plausible and consistent with the aggressive nature of acute leukemia in infants.

Introduction: Zebrafish enable studies of early embryogenesis and hematopoiesis like no other animal models. Many zebrafish orthologs of hematopoietic genes have been identified, and zebrafish models of leukemia are emerging but the zebrafish ortholog of MLL, a critical oncogene disrupted by leukemogenic translocations, has not yet been studied. We cloned the complete zmll cDNA and characterized its temporal expression as a framework for further studies of MLL where current knowledge is still incomplete. Methods: Bioinformatic tools were employed to interrogate the existence and relationship of a zmll ortholog and synteny with human MLL. Degenerate RT-PCR was used to determine whether MLL amino acid sequences in domains highly conserved across species could identify the orthologous zebrafish transcript. Cross-species Southern blot analysis was performed to determine if a predicted zmll gene from restriction map simulations of a projected genomic sequence could be detected with a human cDNA probe for the MLL breakpoint cluster region (bcr). The full-length zmll cDNA was obtained using a combination of 5′ RACE PCR, long-range and conventional PCR. The corresponding protein was analyzed in a phylogram tree. The temporal pattern of zmll RNA expression was examined using quantitative (Q) RT-PCR. Results: Bioinformatic analysis using the human MLL protein as the reference sequence identified two putative “similar to MLL proteins” and predicted two proximal transcript sequences on zebrafish chromosome 15. Gene prediction tools suggested a single genomic structure matching both protein sequences. A conserved block of synteny containing several linked genes surrounded the predicted zmll. Furthermore, zmll and human MLL were in the same map order in an uninterrupted segment with the gene for ubiquitination factor E4A. Degenerate RT-PCR analysis of wild-type adult zebrafish RNA based on cross-species amino acid sequences from highly conserved PHD and SET domains amplified the predicted transcript. Cross-species Southern blot analysis with the human probe detected the projected zmll genomic fragments corresponding to the MLL bcr in adult zebrafish genomic DNA. RT-PCR analysis of wild-type adult zebrafish RNA determined that the two predicted “similar to MLL proteins” were from a single gene. The full length 12657 bp, 35-exon zmll cDNA cloned from wild-type 24 hpf zebrafish embryo RNA predicted a 4218 amino acid protein with CXXC, PHD, Bromodomain, FYRN, FYRC, and SET domains and taspase cleavage sites with 45.8% sequence identity and 57.3% similarity to human MLL. Phylogram tree analysis suggested evolutionary divergence of mammals from teleosts but nonetheless conservation of critical functional domains. QRT-PCR demonstrated maternally supplied zmll mRNA during the earliest embryonic developmental timepoints, and expression of zygotic zmll mRNA during embryogenesis and in the zebrafish adult. Conclusions: These results indicate that there is a single zmll gene with highly conserved functional similarity to human MLL. The temporal pattern of expression, including maternal supply of transcript to the embryo, indicates that zmll is important from early embryogenesis through the entire lifespan of the fish. The high evolutionary conservation of critical domains creates the framework to use zebrafish for studying MLL in hematopoiesis and leukemia.

Introduction: Patients with birth defects have a higher incidence of cancer; however, an association of amniotic band syndrome (vascular disruption syndrome) with congenital leukemia has never been reported. We characterized a case of MLL-rearranged leukemia in a neonate affected with this birth defect. Methods: The MLL translocation was characterized in peripheral AML blasts by karyotype analysis, multicolor FISH, Southern blot analysis and genomic and cDNA panhandle PCR. NQO1 genotype was determined by real-time PCR. Clinical history and results: The infant was born at 38 weeks gestation by C-section for suboccipital encephalocele to a 21 year-old gravida 3 para 2 mother with a history of cigarette, marijuana, cocaine and opiate use, and antidepressant, antipsychotic, barbiturate, caffeine and proton pump inhibitor treatment during pregnancy. Drug screen at delivery was positive for opioids and barbiturates. In addition to the encephalocele, a circular constriction of the right arm, consistent with amniotic band syndrome, and blueberry muffin lesions were noted at delivery. The CBC showed mild thrombocytopenia that resolved the next day. Congenital infection was excluded. The encephalocele was repaired. At 19–20 days of age the infant became septic and hepatosplenomegaly and hyperleukocytosis were observed. The peripheral smear and flow cytomery revealed acute myeloid leukemia with monocytic differentiation (CD45+ population positive for CD4, CD14, CD33, CD38, HLA-DR). Karyotype analysis showed a complex structural abnormality disrupting chromosomes 4, 11 and 19 involving MLL. M-FISH showed insertion of chromosome 11 material into a chromosome 19 and translocation between chromosomes 11q and 4q. The infant received cytosine arabinoside and daunomycin but succumbed to AML, sepsis and multi-organ failure within 4 days. Autopsy showed marrow, viscera, brain and skin infiltration with AML and Chiari type III brain malformation. Southern blot analysis detected two MLL bcr rearrangements. Panhandle PCR demonstrated fusion of MLL intron 6 and intron 1 of ELL from band 19p13.1. Short sequence homologies at the breakpoint junction suggested DNA damage resolution by NHEJ repair. The corresponding transcript joined MLL exon 6 to ELL exon 2. The infant was wild-type at NQO1 C609T. Conclusions: This is the first association of amniotic band syndrome and congenital AML, both of which are rare conditions. Although the cause(s) are unknown, both conditions originate in utero and maternal exposures during pregnancy may be relevant. There was a history of maternal fetal loss, which is a risk factor for leukemia in infants. The NQO1 substrate p-benzoquinone in cigarette smoke is a topoisomerase II poison, but the infant did not harbor the NQO1 variant that predisposes to leukemia. Cocaine is an in utero exposure implicated in amniotic band syndrome. The occurrence of amniotic band syndrome and congenital AML in this infant raises questions about potential host predisposition or gene-environment interactions common to both conditions. Alternatively, both rare conditions may have occurred by chance alone in the setting of the many in utero exposures.

Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

Background: Chemotherapy resistance from reduced apoptosis may contribute to poor outcome in infant leukemias with t(4;11). G3139 has clinical activity against refractory adult leukemias with minimal toxicity. Methods: We quantified anti-apoptotic BCL-2 and pro-apoptotic BAX mRNAs in 56 leukemias cases (55/56 from infants; 41 ALL, 15 AML; 24 t(4;11), 32 non-MLL), the ALL cell lines RS4:11 and SEM-K2, the AML cell line MV4-11, all of which have t(4;11), and normal CD34+ cells. BCL-2 and BAX mRNA levels were normalized to β-actin and examined by the comparative CT method. The in vitro cytotoxicity and pro-apoptotic activity of G3139 was investigated in the cell lines. Cytotoxicity was assessed by MTT after exposing log phase cells for 6 days to single agent G3139, or to fixed-concentration G3139 combined with anti-leukemia cytotoxic drugs over a range of concentrations; transfection reagent was not used for G3139 delivery. Pharmacostatistical response surface modeling was performed to determine synergy. Apoptosis was assessed by flow cytometry analysis of Caspase-3 activation and a FACS TUNEL assay. Results: BCL-2 and BAX mRNAs were abundant in the leukemias and cell lines with t(4;11), whereas CD34+ cells showed lower BCL-2 expression. The difference in normalized expression ratios of BCL-2:BAX relative to CD34+ cells approached significance when t(4;11) leukemias and cell lines were compared to non-MLL leukemias (p=0.07). The approximate IC50 of single agent G3139 was 10 μM in RS4:11 cells, 100 μM in MV4-11, and 180 μM in SEM-K2. Low, biologically achievable G3139 concentrations (5 μM, 1 μM) sensitized RS4:11 cells to the cytotoxicity of doxorubicin, 6-thioguanine, etoposide and cytosine arabinoside, but not methotrexate or dexamethasone. Despite the higher IC50 of G3139 alone in MV4-11 cells, synergy also was suggested when G3139 (50 μM, 10 μM) was combined with etoposide and 6-thioguanine since response surface modeling showed ability to achieve lower effective doses than projected from either single agent. When RS4:11 cells were assayed for proof of principle that the observed cytotoxicity was due to apoptosis, exposure to G3139 and doxorubicin together increased active Caspase-3 and TUNEL staining in a time and dose dependent manner. Conclusions: The imbalanced BCL-2/BAX expression suggests that an anti-apoptotic genotype forms the basis for the chemotherapy resistance in infant leukemias with t(4;11). These in vitro studies indicate that G3139 has pre-clinical activity, that select G3139-cytotoxic agent combinations are synergistic against cell lines with t(4;11), and that the observed activity occurs through apoptosis. Further studies are warranted to determine the pre-clinical in vivo effects of similar combinations against leukemias with t(4;11), with the goal of advancement to the clinic if results are promising.

Background: Imatinib has become standard front-line therapy for CML. In ~3% of patients treated with imatinib, abnormalities including trisomy 8 and/or monosomy 7 occur in Ph’ negative subclones and dysplasia occasionally is present, but transformation to AML is rare. We describe the first known case of AML with an MLL translocation during an imatinib-induced molecular remission of CML. Methods: The Ph’ and the t(11;19)(q23;p13.1) were detected and monitored in sequential marrows using cytogenetic and FISH analyses. The BCR-ABL1 fusion transcript was traced by RT-PCR. The MLL translocation was identified and characterized in the AML blasts by Southern blot analysis, panhandle PCR-based methods and conventional PCR. Clinical History and Results: The patient presented with chronic phase CML at age 53 and was treated with hydroxyurea for 6 weeks, IFN-α for 20 months and imatinib for 32 months. Complete cytogenetic response was achieved at 10 months. Molecular remission, as indicated by absence of the BCR-ABL1 transcript, occurred after 15 months of imatinib (37 months from CML diagnosis). Mild dysgranulopoiesis was noted 6 months after imatinib was started and increased on subsequent studies. After 28 months of imatinib (50 months from CML diagnosis), there was marked trilineage dysplasia and the karyotype showed t(11;19)(q23;p13.1) in cells that were Ph’ negative and negative for the BCR-ABL1 fusion transcript. FAB M5b AML was diagnosed four months later. The patient succumbed to AML despite aggressive management with chemotherapy and allogeneic stem cell transplantation. Southern blot analysis of the AML revealed two MLL bcr rearrangements. The reciprocal breakpoint junctions on the der (11) and der (19) chromosomes indicated a translocation of intron 8 of MLL and intron 1 of the known MLL partner gene ELL, which encodes a transcription elongation factor. The involved region of MLL was more 5′ than the secondary leukemia translocation breakpoint hotspot. The der (11) fusion transcripts joined MLL exon 7 to ELL exon 2, which is consistent with alternative splicing, and the der (19) fusion transcript joined ELL exon 1 to MLL exon 9. Conclusions: Secondary leukemias with MLL translocations have been associated with topoisomerase II poisons, but not with the agents administered to this patient. The entity that we describe is distinct from blast crisis, in which Ph’ positive subclones evolve to acquire additional alterations. This case establishes that persistent clonal abnormalities and/or dysplasia in Ph’ negative cells following imatinib therapy may not be benign and may herald AML transformation. With effective and selective molecular eradication of the Ph’ positive clone, the emergence of leukemia with independent abnormalities may become more common. This is a highly concerning clinical complication to consider with BCR-ABL targeted agents.

Epipodophyllotoxins, anthracyclines and other chemotherapeutic DNA topoisomerase II poisons are associated with MLL-rearranged leukemias and, less often, leukemias with different balanced translocations. Every MLL translocation identified so far in patients has been associated with leukemia and, where tested, murine models have established that the der(11) protein products are leukemogenic. We here describe a highly novel t(4;11)(p12;q23) that emerged in the bone marrow that confers a proliferative advantage but not a leukemia phenotype in a patient with stage 4 neuroblastoma (NB) following N8 therapy, which consisted of 3 cycles of cyclophosphamide/doxorubicin/vincristine, 2 cycles of cisplatin (P)/etoposide (VP), surgery, local radiation, anti-GD2 monoclonal antibody (3F8), myeloablative therapy (thiotepa, carboplatin, topotecan) plus autologous PBSC transplant (harvested after 1 cycle of PVP), followed by 7 more cycles of 3F8 plus GM-CSF, 4 cycles of oral VP, and 5 cycles of accutane. Cumulative doses of doxorubicin and VP were 225 mg/m2 and 5.4 g/m2 (4.2 g/m2 orally), respectively. Seventeen months after NB diagnosis (11.5 months after transplant) at completion of oral VP, surveillance FISH showed t(4;11)(p12;q23) in 10.5% of marrow cells. The patient is now 32 months from NB diagnosis and 1 year off all therapy. Although both karyotype and FISH showed t(4;11) in 82–100% cells in 7 serial subsequent marrows, all had normal morphology and there is no clinical evidence of leukemia. Molecular analyses of sequential bone marrow samples were undertaken to characterize the translocation. Southern blot analysis of the MLL bcr in BamHI and BglII digested DNAs showed two rearrangements consistent with both derivative chromosomes. A new BglII-based reverse panhandle PCR identified the der(4) breakpoint junction sequence in a 3.2 kb BglII genomic rearrangement and provided the sequence to isolate the der(11) genomic breakpoint junction by clonotypic PCR. The partner DNA was a novel gene localized to chromosome band 4p12. The translocation fused intron 8 of MLL with intron 8 in the new partner gene, and occurred with a loss of 8–13 bases from MLL and 31–36 bases from the partner gene. Both breakpoint junctions contained short homologous sequences suggesting NHEJ. By clonotypic nested PCR (sensitivity 1 cell in 104–105 cells) the translocation was absent at NB diagnosis, and was first detectable at the completion of oral VP (16 months after neuroblastoma diagnosis). RT-PCR revealed in-frame der(11) and der(4) fusion transcripts. Real-time PCR analysis of known MLL target genes and differentially expressed genes in MLL-rearranged leukemias revealed low levels of HOXA4, A5, A7, A9, FLT3, MEIS1, and PBX3 compared to other treatment-related leukemias. Temporal emergence of this translocation after the high total VP dose is of interest because others recently found that cumulative doses of VP >6 g/m2 carry an especially high risk of leukemia, raising concern about the role of the oral VP in the genesis of the translocation. This study indicates that the different partner genes of MLL do not confer the same leukemia potential. Because the t(4;11)(p12;q23) confers a proliferative advantage but is insufficient for leukemia, we designated the new partner gene MIFL (MLL Insufficient for Leukemia), however it remains to be determined whether any relevant secondary alteration will result in leukemic transformation.