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Eleven-nineteen leukemia (ENL) is a chromatin reader present in complexes stimulating transcriptional elongation. It is fused to mixed-lineage leukemia (MLL) in leukemia, and missense mutations have been identified in Wilms tumor and acute myeloid leukemia. Here we demonstrate that ENL overcomes polycomb silencing through recruitment of PAF1 via the conserved YEATS domain, which recognizes acetylated histone H3. PAF1 was responsible for antirepressive activities of ENL in vitro, and it determined the transforming potential of MLL-ENL. MLL-ENL target loci showed supraphysiological PAF1 binding, hyperubiquitination of histone H2B and hypomodification with H2AUb, resulting in accelerated transcription rates. YEATS mutations induced a gain of function, transforming primary hematopoietic cells in vitro and in transplantation assays through aberrant transcription and H2B ubiquitination of Hoxa9 and Meis1 Mechanistically, H3 and PAF1 competed for ENL interaction, with activating mutations favoring PAF1 binding, whereas the MLL moiety provided a constitutive PAF1 tether allowing MLL fusions to circumvent H3 competition.

Introduction: The bone marrow stromal niche can serve as a protective environment in hematological malignancies such as AML. Intensive research of the bidirectional interactions between leukemic blasts and stromal cells already highlighted numerous mechanisms how malignant cells are capable of shaping their local milieu in an opportunistic fashion. However, the complex mechanisms remain to be fully elucidated. Previously, we found two small intracellular calcium-sensing molecules, S100A8 and S100A9, among the top upregulated genes in primary AML blasts upon stromal contact in an unbiased analysis. They are members of the S100 protein family that can modulate cellular responses such as proliferation, migration, inflammation, and differentiation. Dysregulation of S100 protein expression is described as a common feature in several human cancers. Particularly, expression of S100A8 in leukemic blasts predicts poor survival in de novo AML patients. Thus, we aimed to decipher the underlying pathways of the stroma-mediated S100A8/A9 upregulation as well as its functional consequences. Methods: We cultured AML cell lines in absence or presence of bone marrow stroma cell-conditioned medium. Subsequent, AML cells were analyzed for alterations in intracellular signaling pathways and metabolic characteristics by means of flow cytometry, fluorescence microscopy and quantitative real-time PCR. Additionally, AML cells were analyzed for their resistance towards chemotherapy under the very same culture conditions. Moreover, matched pairs of patient-derived AML samples from the peripheral blood and the bone marrow were compared for key findings from the in vitro analyses. Results: The previously seen upregulation of S100A8 and S100A9 in primary AML blasts upon stromal contact could be confirmed in AML cell lines both on the mRNA and the protein level and proved to be contact-independent and reversible. Interleukin 6-mediated triggering of the Jak/STAT3 signaling was shown to be one major pathway of this induction. The S100A8/A9high subpopulation is characterized by profound metabolic alterations, and by increased surface levels of maturation markers (i.e. CD11b and CD14) both conferring chemoresistance. Strikingly, both differentiation and chemoresistance could be reversed by inhibition of Jak/STAT signaling or metabolic interference. Ultimately, elevated frequencies of S100A8/A9high AML blasts with increased surface levels of maturation markers could be detected in bone marrow-derived specimen compared to matched samples from the peripheral blood in the same patient. Conclusion: We could demonstrate that bone marrow stroma-induced S100A8/A9high AML cells show increased maturation and chemoresistance which both can be targeted by metabolic or oncogenic signaling inhibition representing additional possible targets for further pre-clinical exploration. Disclosures Bruns: Morphosys AG: Research Funding.

Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79–specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.

Pediatric acute lymphoblastic leukemia (ALL) is characterized by preleukemic recurrent chromosomal translocations that emerge in utero. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell precursor patients. A previous study suggested that ETV6-RUNX1-positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1-positive ALL in children (1/10,000) by a factor of 100. This finding strongly indicated that secondary cooperating oncogenic hits were necessary for development of overt leukemia. However, later studies could not confirm this high frequency. To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR-based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based retrospective screening of 300 cord blood samples from Danish newborns. The GIPFEL method is capable of detecting the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In contrast to previously used RNA-based methods, it relies on DNA as sample material, which is more stable than RNA. In the case of ETV6-RUNX1-positive leukemia GIPFEL exploits the unique presence of a genomic fragment joining material from chromosome 12 and 21. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. The ligation site is independent of the translocation point within the individual DNA circle. Digestion of the breakpoint regions of the ETV6 and RUNX1 gene with the restriction enzyme SacI generates fragments smaller than 50 kb. Primer pairs amplify the complete set of theoretically predicted circularized fragments requiring 37 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from mononuclear cells from cord blood samples of 300 newborns that were cryopreserved within 24 h (median 12 h) from birth. After B cell enrichment and column purification of DNA, the DNA was subjected to SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. Samples that screened positive underwent one further demultiplexed PCR, agarose gelelectrophoresis and Sanger sequencing to validate the result and to identify the breakpoint region. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In previously published proof-of-principle blinded studies we tested 61 samples obtained from ETV6-RUNX1-positive ALL patients. Without any unspecific result, 64% for ETV6-RUNX1 fusion genes were detected in that sample set. The sensitivity of the technique was estimated to be 10-4, i.e. one translocation carrying cell within 10,000 normal cells can theoretically be detected. Within the analyzed cohort of 300 healthy newborns 6 screened positive for the ETV6-RUNX1 translocation (2%) (Table 1). Further 700 cord blood samples are currently screened. Table 1: 6 of 300 cord blood samples from healthy newborns screened positive for the ETV6-RUNX1 translocation using the GIPFEL technique (Fueller E*, Schaefer D* et al. PloS One 2014, 9(8): e104419). Number of the positively tested healthy newborn within the cohort, used primers, and introns of RUNX1 and ETV6 affected by the translocation are presented. Our results indicate that the actual incidence of ETV6-RUNX1-positive cells in healthy newborns might be even higher than previously assumed, potentially due to instability of the ETV6-RUNX1 RNA transcript in preserved cord blood samples. This would hint at a comparably low penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and further strengthen the importance of secondary environmentally caused or spontaneously occurring cooperating oncogenic lesions for ETV6-RUNX1-positive childhood leukemia to emerge. Table Table. Disclosures No relevant conflicts of interest to declare.

Chronic lyhocytic leukemia displays a strong dependency on tumor-stroma interactions. CLL cells were found to rapidly die when cultivated in vitro, whereas co-cultivation with mesenchymal stromal cells promotes cell proliferation and ensures leukemia cell survival. It is therefore of great interest to obtain a thourough picture of the exact molecular players involved in this critical interplay. While a number of protagonists are already well described, there are still unresoved issues concerning the effect of stromal cells on the metabolism of CLL cells. In this study we co-cultivated for one week primary CLL cells obtained from peripheral blood with mesenchymal cells (hMSC). Subsequently, hMSC were lysed and fractionated to obtain cytoplamic, nuclear and secreted proteins. To allow comparative analyses, B-cells from healthy individuals and hMSC were co-cultivated. Mass spectrometry-based proteomic analysis resulted in the identification and relative quantification of more than 3700 proteins. Comparative analyses between co-cultures with CLL and healthy B-cells showed that already at a ratio of 0.25% CLL cells could induce a significant proteomic alteration in hMSC. Furthermore, hMSC were found to undergo a metabolic change upon co-cultivation with CLL cells, an effect which was most pronounced in the secreted protein fraction. Annotation analyses according to gene ontology revealed that proteins related to "generation of precursor metaboites" were significantly up-regulated in hMSC upon co-culture with CLL cells. This indicated that these stromal cells "feed" the leukemia cells by producing and secreting high energy metabolite, satisfying the elevated metabolic demands of the CLL cells. Revealing the metabolic interactions between CLL cells and surrounding stromal cells by mass spetrometry-based methods should contribute to the development of innovative treatment strategies. Disclosures No relevant conflicts of interest to declare.

Chromosomal rearrangement of the HRX (MLL, ALL-1, Htrx) gene situated at chromosome band 11q23 is one of the most frequent genetic changes in infant leukemias of myeloid and lymphoid lineage and in treatment-induced secondary leukemias. The HRX gene codes for a predicted 431-kD protein that shows significant homology to the Drosophila trithorax protein, an Hox epigenetic regulator. Typically, the region encoding the HRX gene is rearranged, mostly in reciprocal translocations with a number of partners, resulting in a range of fusion genes. However, this is not the only abnormality affecting HRX because partial duplication of the gene, as well as interstitial deletions, can occur. Despite extensive studies of HRX at the genetic level, the protein products of the HRX gene and their patterns of expression in normal and leukemic cells remain uncharacterized. In this study we analyzed the distribution and localization of HRX proteins in cell lines and human tissues, using both polyclonal and monoclonal antibodies. The specificity of these reagents was confirmed using cells transfected with the HRX-ENL fusion gene. Western blot analyses of protein extracts from cells carrying the t(11; 19) and t(4; 11) translocations showed HRX chimeric proteins whose migrations corresponded to the sizes predicted from analyses of translocation-induced fusion mRNAs expressed by the derivative 11 chromosomes. Immunocytochemical analysis showed a punctate distribution of wild-type and chimeric HRX proteins within cell nuclei, suggesting that HRX localizes to nuclear structures in cells with and without 11q23 translocations. Nuclear staining was found in the majority of tissues studied with the strongest reactivity in cerebral cortex, kidney, thyroid, and lymphoid tissues. Thus, HRX is widely expressed in most cell types including hematopoietic cells, a finding that precludes an immunocytochemical approach for diagnosis of leukemias bearing 11q23 structural abnormalities.

HOX proteins are widely involved in hematopoietic development. These transcription factors combine a conserved DNA-binding homeobox with a divergent N-terminus that mediates interaction with variable cofactors. The resulting combinatorial diversity is thought to be responsible for mammalian HOX specificity. Contrasting this proposed mechanism for normal HOX function, here we demonstrate that, in the context of hematopoietic immortalization and leukemogenesis, individual HOX properties are governed almost exclusively by the homeodomain. Swap experiments between HOXA1 and HOXA9, 2 members of nonrelated paralog groups, revealed that gene expression patterns of HOX transformed cells in vitro are determined by the nature of the homeodomain. Similar results were seen in vivo during HOX-mediated leukemogenesis. An exchange of the homeodomains was sufficient to convert the slow, low-penetrance phenotype of HOXA1-induced leukemia to the aggressive fast-acting disease elicited by HOXA9 and vice versa. Mutation and deletion studies identified several subregions within the DNA binding domain responsible for paralog specificity. Previously defined binding sites for PBX cofactors within the exchangeable, nonhomeobox segment were dispensable for in vitro oncogenic HOX activity but affected in vivo disease development. The transcriptional activator domain shared by HOXA1 and HOXA9 at the very N-terminus proved essential for all transformation.

HOX homeobox genes are important regulators of normal and malignant hematopoiesis. Abdominal-type HOXA genes like HOXA9 are highly leukemogenic. However, little is known about transformation by anterior HOXA genes. Here we performed a comprehensive assessment of the oncogenic potential of every HOXA gene in primary hematopoietic cells. With exception of HOXA2 and HOXA5, all HOXA genes caused a block or delay of hematopoietic differentiation and cooperated with Meis1. No evidence for the alleged tumor-suppressor function of HOXA5 could be found. Whereas all active HOXA genes immortalized mixed granulocytic/monocytic populations, HOXA13 preferentially specified monocytoid development. The anterior HOXA genes HOXA1, HOXA4, and HOXA6 transformed cells, generating permanent cell lines, although they did so less potently than HOXA9. Upon transplantation these lines induced myeloproliferation and acute myeloid leukemia in recipient animals. Kinetic studies with inducible HOX derivatives demonstrated that anterior HOXA genes autonomously contributed to cellular transformation. This function was not mediated by endogenous Hoxa9, which was persistently expressed in cells transformed by anterior HOX genes. In summary our results demonstrate a hitherto unexpected role of anterior HOXA genes in hematopoietic malignancy.

Abdominal-type HoxA genes in combination with Meis1 are well-documented on-cogenes in various leukemias but it is unclear how they exert their transforming function. Here we used a system of conditional transformation by an inducible mixed lineage leukemia-eleven-nineteen leukemia (MLL-ENL) oncoprotein to overexpress Hoxa9 and Meis1 in primary hematopoietic cells. Arrays identified c-Myb and a c-Myb target (Gstm1) among the genes with the strongest response to Hoxa9/Meis1. c-Myb overexpression was verified by Northern blot and quantitative reverse transcription-polymerase chain reaction (RT-PCR). Also MLL-ENL activated c-Myb through up-regulation of Hoxa9 and Meis1. Consequently, short-term suppression of c-Myb by small inhibitory RNA (siRNA) efficiently inhibited transformation by MLL-ENL but did not impair transformation by transcription factor E2A-hepatic leukemia factor (E2A-HLF). The anti c-Myb siRNA effect was abrogated by coexpression of a c-Myb derivative with a mutated siRNA target site. The introduction of a dominant-negative c-Myb mutant had a similar but weaker effect on MLL-ENL-mediated transformation. Hematopoietic precursors from mice homozygous for a hypo-morphic c-Myb allele were more severely affected and could be transformed neither by MLL-ENL nor by E2A-HLF. Ectopic expression of c-Myb induced a differentiation block but c-Myb alone was not transforming in a replating assay similar to Hoxa9/Meis1. These results suggest that c-Myb is essential but not sufficient for Hoxa9/Meis1 mediated transformation. (Blood. 2006;108:297-304)

Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent chromosomal translocations that frequently occur in utero in preleukemic cells. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell ALL. A previous study suggested that ETV6-RUNX1 positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1 positive ALL in children by a factor of 100. This finding indicated that secondary, but relatively rare cooperating oncogenic hits are necessary for the development of overt leukemia. However, later studies could not confirm this high incidence of preleukemic cells in newborns. To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based screening of ≈1000 cord blood samples from healthy newborns. The GIPFEL method is capable to detect the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In the case of ETV6-RUNX1 positive leukemia, GIPFEL exploits the unique presence of a genomic fragment joining material from chromosomes 12 and 21 in the translocation-positive cells. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. Importantly, the ligation site is independent of the translocation point within the individual DNA circle. The published breakpoint regions of the ETV6 and RUNX1 genes involved in the translocation were analyzed in silico for restriction sites that allow digestion of all possible translocation events to yield fragments smaller than approximately 50 kb. This condition was met for ETV6-RUNX1 breakpoints by digestion with SacI. Primer pairs were designed amplifying the complete set of theoretically predicted circularized fragments requiring 36 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from cell lines, diagnostic specimens from ALL patients, peripheral blood from healthy donors and cord blood samples from newborns by column purification. The equivalent of approximately 4x105 cells (2.5 µg DNA) was subjected to the SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In a first proof-of-principle study employing the ETV6-RUNX1 translocation positive cell line REH, process optimization close to the theoretical limits was carried out. Cell dilution and mixing studies revealed that under optimal conditions approximately 40 translocation positive cells (=10-4) present in the input DNA are sufficient to produce a reliable output signal. The method was next tested in a blinded study with 60 samples obtained from ETV6-RUNX1 diagnostic ALL samples. ETV6-RUNX1 samples positive at 10-4, being diluted from these diagnostic samples, still gave a reliable output signal. There was no false positive result. Detection coverage (=sensitivity) was 64%. This method was then applied to a retrospective sample set of cryopreserved anonymized cord blood samples of ≈1000 healthy newborns to determine frequency and levels of translocation-positive cells. First results will be presented. In conclusion this population-based study will allow an estimate of the actual incidence of ETV6-RUNX1 positive preleukemic cells in healthy newborns. The results will enable us to evaluate the penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and will provide a basis for the assessment of potential secondary environmental or spontaneously occurring cooperating oncogenic lesions in ETV6-RUNX1 positive childhood leukemia. Disclosures No relevant conflicts of interest to declare.

Increased expression of Musashi 2 (also known as MSI2), a mRNA binding protein, is reported to trigger progression of chronic myeloid leukemia (CML) from chronic phase (CP) to blast crisis (BC), which is frequently fatal. The data imply that MSI2 is upregulated by HOXA9 leading to disruption of the critical hematopoietic stem cell (HSC) fate decisions via the NUMB-NOTCH signaling pathway. These events lead to HSC proliferation, impaired myeloid differentiation and are associated with worse prognosis in CML and acute myeloid leukemia (Kharas, et al. Nat Med 2010; 16:903; Ito, et al. Nature 2010; 466:765). Previously, we confirmed increased MSI2 levels in CML patients in blast crisis (BC) compared with those in CP, irrespective of lymphoid or myeloid transformation (Kaeda, et al. Blood. 2011;118;supplement). To further elucidate this regulatory pathway we assessed whether HOXA9 expression correlated with increased MSI2mRNA levels and upregulation of NOTCH. Because of the rarity of BC samples and finding MSI2 increased in lymphoid and myeloid BC CML patients, we studied lymphoid [n=12 (Lymphoma:8; Myeloma:3; ALL:1)] and myeloid [n=12 (AML:4; CML:6; HES:1; ET:1)] cells lines. We also included 29 BCR-ABL positive (e1a2: 18; e13a2: 6; e14a2: 5) diagnostic samples from acute lymphoblastic leukaemia (ALL) patients (M:18; F:11); median age 50 years (range: 26-74). We quantified MSI2 and HOXA9 mRNA transcripts by quantitative PCR and expressed the data as % ratio of the control gene, GUSβ.Western blotting of cell line protein extracts was performed to assess expression of NOTCH 1, 2, 3 receptors and delta like ligand 3. We found MSI2 [median: 7.10% (range:0.06-38.71)] and HOXA9 [median: 2.40% (range:0.00-51.13)] expression was generally restricted to myeloid cell lines. Of the 12 lymphoid cell lines, MSI2 was only detectable in SUPB15 with 8.44%. The latter is a B-lymphocyte ALL cell line, known to express BCR-ABL e1a2 transcript. Similarly, HOXA9 [median: 0.00 (range:0.00-27.0%)] expression ranged from undetectable to 0.08%, among this group, apart from HD-MY-Z with 27.0%. In contrast, HOXA9 [median: 0.02% (range:0.01-2.18)] and MSI2 [median: 3.58% (range: 1.24-22.38)] transcripts were detectable in all 29 BCR-ABL positive ALL patients, irrespective of the transcript type expressed. Among the ALL samples 7 (24%) had increased MSI2 levels, i.e. >6.7% (Kaeda, et al. Blood. 2011;118;supplement) and of these 6 expressed e1a2 transcript and the other e13a2. In summary, upregulated MSI2 expression was observed in 17 (32.0%) of the 53 samples screened. But only 4 [KG1, EOL1, HEL and MEG-01 (all myeloid cell lines)] of the 17 also had increased HOXA9 levels. Remarkably, 5 (62%) of the 8 myeloid cell lines with increased MSI2 are known to express BCR-ABL. NOTCH1 receptor was detectable in all the lymphoid cell lines. But, NOTCH expression was highly variable in myeloid cell lines. Overall, an upregulated MSI2 mRNA expression was not reflected in the NOTCH receptor levels nor in the HOXA9levels. Our observations are consistent with MSI2 being limited to myeloid linage. However, in contrast to earlier reports our data imply that MSI2 functions via a pathway other than NOTCH signaling and is not regulated by HOXA9 alone. But the cell lines and ALL patients’ data provide further evidence of correlation between MSI2 and BCR-ABL expression, suggesting they interact, directly or indirectly, to arrest cell differentiation and trigger BC. These findings, together with our reported data, show increased MSI2 levels to be an important biomarker of poor prognosis and are likely to have an impact in optimizing clinical management. It also represents a potential novel therapeutic target, especially for the BCR-ABL positive stem cells resistant to tyrosine kinase inhibitors. Disclosures: Kaeda: Novartis: Research Funding. le Coutre:Novartis: Research Funding.

Chromosomal rearrangement of the HRX (MLL, ALL-1, Htrx) gene situated at chromosome band 11q23 is one of the most frequent genetic changes in infant leukemias of myeloid and lymphoid lineage and in treatment-induced secondary leukemias. The HRX gene codes for a predicted 431-kD protein that shows significant homology to the Drosophila trithorax protein, an Hox epigenetic regulator. Typically, the region encoding the HRX gene is rearranged, mostly in reciprocal translocations with a number of partners, resulting in a range of fusion genes. However, this is not the only abnormality affecting HRX because partial duplication of the gene, as well as interstitial deletions, can occur. Despite extensive studies of HRX at the genetic level, the protein products of the HRX gene and their patterns of expression in normal and leukemic cells remain uncharacterized. In this study we analyzed the distribution and localization of HRX proteins in cell lines and human tissues, using both polyclonal and monoclonal antibodies. The specificity of these reagents was confirmed using cells transfected with the HRX-ENL fusion gene. Western blot analyses of protein extracts from cells carrying the t(11;19) and t(4;11) translocations showed HRX chimeric proteins whose migrations corresponded to the sizes predicted from analyses of translocation-induced fusion mRNAs expressed by the derivative 11 chromosomes. Immunocytochemical analysis showed a punctate distribution of wild-type and chimeric HRX proteins within cell nuclei, suggesting that HRX localizes to nuclear structures in cells with and without 11q23 translocations. Nuclear staining was found in the majority of tissues studied with the strongest reactivity in cerebral cortex, kidney, thyroid, and lymphoid tissues. Thus, HRX is widely expressed in most cell types including hematopoietic cells, a finding that precludes an immunocytochemical approach for diagnosis of leukemias bearing 11q23 structural abnormalities.

Abstract 3499 MicroRNAs (miRNAs), a class of small, non-coding RNAs, are important for posttranscriptional gene regulation in both health and disease. Expression of miRNAs is under stringent regulation at both transcriptional and post-transcriptional levels. Disturbance at either level could cause dysregulation of miRNAs. However, though altered expression of many miRNAs has been reported in various cancers, including acute myeloid leukemia (AML), their dysregulational mechanisms and pathologic functions remain less well understood. Here we report that mature miR-150 level is significantly downregulated in most AML samples, including those with rearrangements of the mixed lineage leukemia (MLL) gene. Strikingly, we found that despite the over 75% decrease of mature miR-150, its primary and precursor transcript abundance is increased to 2∼4 fold in human MLL-associated AML, relative to normal controls. Interestingly, we show that while MLL fusion proteins can bind to the promotor region of miR-150 and promote its primary transcription, they also negatively regulate the maturation process of miR-150 through the MYC/LIN28 functional axis. MiR-150 has been implicated as either an oncogene or a tumor suppressor in various types of solid tumors. However, its function in the pathogenesis of AML is unknown. Here we showed that ectopic expression of miR-150 dramatically inhibited cell growth and promoted apoptosis of human MLL-associated leukemic cells. Furthermore, using colony-forming/replating assays, we found that co-transduction of miR-150 and MLL-AF9 (a fusion gene resulting from t(9;11)) into mouse bone marrow (BM) progenitor cells, caused a significant reduction in colonies (down to 1∼10%; p Previous studies have shown that there is an autoregulatory feedback loop between FLT3/MYB and HOXA9/MEIS1, and the latter two are critical downstream targets of MLL fusion proteins. In addition, FLT3 has been identified as an upstream regulator of MYC, while MYC is also a downstream target of MLL fusion proteins and an upstream regulator of Lin28. These previous findings together with the data we reported above suggest that there is a critical MLL-fusion/MYC/LIN28-miR-150-FLT3/MYB/HOXA9/MEIS1 regulatory circuit in MLL-associated leukemia (see Fig. 1). In this circuit, MLL fusion proteins function as the driver, and their presence leads to the significant up-regulation of all six downstream genes, MYC, LIN28, FLT3, MYB, HOXA9, and MEIS1, as well as the primary transcription of miR-150. The up-regulation of MYC/LIN28 results in the blockade of the miR-150 maturation process. This in turn leads to the release of miR-150 inhibition on FLT3 and MYB expression, which would enhance the expression of HOXA9, MEIS1, MYC, and LIN28, and further enhance/maintain the blockade of miR-150 maturation. As a result, the cells reach and maintain high levels of MYC/LIN28/FLT3/MYB/HOXA9/MEIS1, and thereby transform the cells and lead to leukemogenesis. Our further systematic studies confirmed the existence/fidelity of this regulatory circuit in MLL-associated leukemia. Taken together, we revealed a previously unappreciated regulatory circuit. Our findings may advance our understanding of the complex molecular mechanisms underlying the development and maintenance of MLL-associated leukemia, and may also provide new strategies to treat MLL-associated leukemia, a disease that is presently treatment resistant, and likely also other subtypes of AML (as miR-150 is down-regulated in all subtypes of AML), or even other types of cancer that also utilize at least part of the signaling circuit we have described herein. Disclosures: No relevant conflicts of interest to declare.

Abstract 1393 The oncoprotein E2A-PBX1 is produced by t(1;19)(q23;p13) in pre-B acute lymphoblastic leukemia (ALL). Perplexingly, in vitro and in vivo models of E2A-PBX1 leukemogenesis to date generate either myeloid or T-lymphoblasts, neither of which are representative of the human disease. Furthermore, we have observed recently that lineage-depleted (lin-) murine bone marrow cells infected with an E2A-PBX1 retrovirus selectively fail to repopulate the B-lymphoid compartment on transplantation into irradiated recipients. Therefore, we have now begun to investigate the mechanism by which enforced expression of E2A-PBX1 antagonizes B-lymphopoiesis. We show that E2A-PBX1-transduced lin- fetal liver progenitor cells (FLPs) fail to differentiate to committed CD45R+ pro-B-cells when cultured with IL-7 in the presence of OP9 stromal cells. Instead these cells manifest a relatively immature immunophenotype (CD45R-, CD11b-, CD117+), are murine stem cell factor (SCF) dependent, and undergo myeloid differentiation upon stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) or transplantation into irradiated mice. Amino acid substitutions in E2A-PBX1 that impair co-activator recruitment or DNA binding completely rescue the differentiation block, implying a critical role for target-gene regulation by E2A-PBX1. E2A-PBX1-transduced cells fail to induce the B-lymphoid commitment genes Ebf1 and Pax5, and chromatin immunoprecipitation (ChIP) analysis shows aberrant persistence of the Polycomb silencing mark H3K27me3 at these promoters. Previous studies have identified the Polycomb gene Bmi1 as a candidate E2A-PBX1 target gene. However, enforced expression of Bmi1 neither blocked induction of Ebf1 or Pax5 nor impaired B-lymphopoiesis, indicating that Bmi1 is not sufficient to mediate blocked differentiation by E2A-PBX1. Gene expression microarray analysis identified cytokines (ex., Csf2, IL1a, Il6) and transcription factors (HoxA9, Tal1) up-regulated in E2A-PBX1-transduced FLPs that are known to antagonize B-lymphoid development. In summary, our results support the notion that enforced expression of E2A-PBX1 in early hematopoietic progenitors blocks B-lymphopoiesis, to an important degree, by inducing the transcription of genes whose products antagonize early steps of B-lymphoid commitment, including induction of Ebf1 and Pax5. HoxA9 is particularly interesting as a mediator of E2A-PBX1 effects in view of its extreme induction ratio in our system (at least 1000-fold relative to controls), its propensity to promote myeloid over lymphoid differentiation, and its well-established role in leukemogenesis. Studies to investigate a potential requirement for HoxA9 and other candidates in mediating the E2A-PBX1-driven B-lymphoid differentiation block are ongoing. Disclosures: No relevant conflicts of interest to declare.

Abstract 2443 MLL fusion proteins are potent leukemogenic agents that are created by chromosomal translocations. These events join the N-terminus of the histone methyltransferase MLL to a variety of different fusion partners. Some time ago we showed that a subgroup of frequently occurring MLL fusion partners, including ENL, assemble a novel transcriptional elongation complex termed EAP [1,2] (elongation assisting proteins, later also called AEP for AF4-ENL proteins [3] or SEC for super elongation complex [4]). Aberrant recruitment of EAP to targets normally under control of MLL leads to abnormal stimulation of transcriptional elongation through the activity of pTEFb (positive transcription elongation factor b). This is accompanied by enhanced H3K79 methylation catalyzed by the histone methyltransferase DOT1L that also interacts directly with EAP. As a consequence MLL-targets, like the clustered Hox-homeobox genes, are ectopically expressed and contribute to hematopoietic transformation. Paradoxically, we also observed a persistent copurification of EAP with members of the polycomb repressive complex 1 (PRC1). PRC1 inhibits gene expression and ubiquitinylates H2A. Here we confirm the association of EAP with PRC1 and we show that interaction of ENL and CBX8 (chromobox 8) is necessary for EAP to neutralize the repressive function of PRC1 as prerequisite for efficient Hox expression and transformation. Mass spectrometry and immunological detection verified that ENL is also present in biochemical preparations of PRC1. A study of the architecture of this interaction demonstrated that a CBX8-ENL dimer bridges the PRC1 core to EAP. Interestingly, an analysis of the binding interfaces revealed that EAP would be able to home in on PRC1 engaged chromatin whereas the reverse was not possible because Dot1l and CBX8 could not bind ENL simultaneously. This also suggested that the activity of these two chromatin modifying activities is mutually exclusive. Indeed CBX8 counteracted MLL-ENL in vivo. Introduction of CBX8 into MLL-ENL expressing cells opposed transformation, reduced H3K79 methylation and induced H2A ubiquitinylation at key targets for MLL fusions like the Hox-locus. Deletion of the ENL interaction domain from CBX8 but preserving its PRC1 dependent functions created a “super-repressor” indicating that EAP recruitment via ENL is able to attenuate PRC1 activity. This seems to be a normal function of EAP that exists independently of the MLL fusion context as overexpression of ENL lead to derepression of polycomb controlled genes also in embryonic stem cells. In agreement with these results an elongation reporter system revealed that EAP can completely offset PRC1 repression but only if direct interaction between ENL and CBX8 is possible. During hematopoietic differentiation genes involved in the decision between self renewal and differentiation are often controlled by transcriptional elongation. PRC1 has been found to play a role in this setting and obviously MLL fusion proteins have found yet another method to interfere with this process. Disclosures: No relevant conflicts of interest to declare.

Abstract 60 Next to their function in embryogenesis the clustered Hox-homeobox genes are also master regulators of hematopoietic differentiation and development. Perturbed HOX expression can be found in a significant percentage of acute leukemia. Recently we have shown that several members of the HOXA cluster transform primary hematopoietic cells and induce leukemia in vivo [1]. In an attempt to define Hox downstream targets important for leukemogenesis we determined the gene expression pattern in hematopoietic precursor cells transformed by conditional HOXA1 and HOXA9 derivatives. Interestingly, amongst the several hundred genes controlled by each HOX protein individually we could identify a significant overlap of genes that responded to both. This group included c-Myb that has been shown before to be important for Hox-mediated leukemogenesis indicating that this approach is able to identify a candidate set of genes important for transformation by HOX proteins. Within this collection the gene for the guanine exchange factor Vav2 caught our interest because it had been originally identified in a screen for bona fide oncogenes. Moreover it is known that Vav2 controls activity of the small Rho-type GTPase Rac1 that in turn is an important mediator of (leukemic) stem cell mobility and engraftment. ChIP experiments confirmed HOXA9 binding in the putative vav2 promoter region. In addition a luciferase reporter placed under control of the vav2 promoter showed Hox-responsive behavior indicating that vav2 is a direct Hox-target. Concomitant with vav2-levels also Rac1 activity was under strict control of Hox factors. This was true also for HOX+Meis double transformed cells. To investigate the importance of vav2 for Hox-mediated leukemogenesis hematopoietic cells from vav2 knock-out mice were investigated in detail. vav2−/−cells could be transformed by HOXA9 (+Meis) in vitro with identical efficiency as their wild-type counterparts. Colony formation in replating assays, the capability to induce outgrowth of permanent myeloid precursor lines and the differentiation state of these lines did not differ significantly amongst the wt and vav2−/− samples. However, no active Rac1 could be detected in vav2−/− cell lines transformed by HOXA9. Importantly, retroviral re-expression of vav2 could rescue part of this defect. Obviously, Vav2 constitutes the major GEF controlling Rac1 activity in myeloid precursors despite the presence of the homologous proteins Vav1 and Vav3. Because Rac1 has been implicated in surface receptor recycling and in stem-cell homing we checked the level of the SDF1 “homing-cytokine” receptor CxCR4 (CD184) on Hox-transformed wt and vav2−/−cells and could confirm a complete absence of CD184 on the latter. Again reintroduction of vav2 rescued part of this phenotype. Transplantation experiments are underway and preliminary results indicate that loss of vav2 is accompanied by an impaired ability for extramedullary hemopoiesis, an import determinant of leukemia aggressiveness. Our results explain how Hox genes that are highly expressed in stem- and precursor cells can regulate the important process of stem cell homing. Rac1 inhibitors are known and these substances have been tested [2] in mice with special success on MLL fusion induced leukemias that are a paradigma for Hox-induced leukemogenesis. Therefore our results do not only provide the molecular framework for this observation but they are also able to identify a potential patient collective that may benefit from pharmacological Rac1 inhibition. Disclosures: No relevant conflicts of interest to declare.

Abstract 365 Background: Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemia (AML) and in mixed phenotype acute leukemia in infancy, a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type MLL carries histone methyltransferase activity and affects specific target genes, such us HOXA cluster genes. While the more than three dozen MLL fusion proteins known today exert different specific functions, they finally induce transcription of individual target genes. Consequently, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit typical gene expression profiles including high-level expression of HOXA cluster genes. Aim of this study was to find a correlation between the MLL mutational status and tumor suppressor gene methylation/expression in acute leukemia cell lines. Results: Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. 1.8/24 TSG were methylated in MLLmu AML cells, 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the tumor suppressor genes (TSG) BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu acute myeloid leukemia (AML) cell lines. MLL wild-type (MLLwt) AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3 confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene. Conclusion: These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins. Disclosures: No relevant conflicts of interest to declare.

Abstract 1801 Poster Board I-827 The microenvironment of tumor cells in the bone marrow was demonstrated to contribute to tumor promotion and survival. The role of bone marrow fibroblasts (BMFs) in supporting the malignant plasma cell clone in multiple myeloma (MM) has been established, but it remains unclear to which extent the BM microenvironment in general and BMFs in particular are involved in the progression of monoclonal gammopathy of undetermined significance (MGUS) to MM. Therefore we performed proteomics studies on the secretome of BMFs isolated from healthy donors, patients suffering from MGUS and patients suffering from MM. Compared to normal background, BMFs derived from MGUS secreted elevated levels of proteins indicating mitogenic activity and moderate inflammation. These proteins included periostin, IL-6, CXCL5 and CSF-1. Insulin-like growth factor II, which is normally not expressed by normal BMFs, was secreted by BMF cells derived from MGUS as well as from MM. In addition to those and other proteins, BMF cells derived from MM were found to specifically secrete stem cell growth factor, MMP-28 and stanniocalcin-1. These data indicate a step-wise alteration of BMF secretion activity related to the stage of the underlying plasma cell dyscrasia. Therefore BMF might support the progression from MGUS to MM. In order to correlate the secretion performance of BMF with blood serum levels of candidate marker proteins, Luminex assays are employed. Based upon these results, it is our aim to identify serum biomarkers which allow to assess the functional state of BMF and thus the risk for the progression of MGUS to MM. Disclosures No relevant conflicts of interest to declare.

Abstract 2962 Poster Board II-938 HOX homeobox genes are important regulators of normal and malignant hematopoiesis and abdominal-type HOXA-cluster genes, in particular HOXA7 to HOXA10, are highly leukemogenic. However, little is known about the transforming abilities of anterior HOXA genes HOXA1 to HOXA6 despite a high prevalence of anterior HOX-expression in acute leukemia. Here we performed a comprehensive assessment of the oncogenic potential of every HOXA gene in primary hematopoietic cells. With exception of HOXA2 and HOXA5 all other HOXA genes caused a block or delay of hematopoietic differentiation in serial replating assays and all HOX genes also cooperated with Meis1. No evidence for the alleged tumor-suppressor function of HOXA5 could be found. Whereas all other active HOXA genes induced the outgrowth of mixed granulocytic/monocytic cells, HOXA13 preferentially specified the development of monocytes/macrophages. Albeit more weakly than HOXA9 also the anterior HOXA genes HOXA1, HOXA4, and HOXA6 transformed cells and generated permanent myelomonocytic cell lines arrested at various stages of differentiation. HOX-RNA profiling revealed that each of these lines also transcribed HOXA9. However, kinetic studies with inducible HOX-derivatives showed that HOXA9 expression was not under control by anterior HOXA proteins. Cellular differentiation was induced immediately after loss of anterior HOX activity and HOXA9 down regulation was a secondary event during maturation. This proves that anterior HOX proteins are able to transform hematopoetic cells through an individual contribution independent of HOXA9. In summary our results explain how HOXA9 can predominate in hematologic malignancies while simultaneously it is not necessarily required for transformation. Disclosures: No relevant conflicts of interest to declare.

The mixed lineage leukemia (MLL) family of histone methyltransferases has become notorious for the participation of the founding member MLL in fusion proteins that cause acute leukemia. Despite a high overall structural conservation no other MLL homolog has been found involved in leukemogenic fusion proteins. This surprising fact might be either due to a relative genomic stability of the MLL2 locus that prevents chromosomal translocations or MLL2 might be altogether incapable of contributing to oncogenic fusions because of functional constraints. Here we demonstrate the latter to be true for fusion proteins based on MLL2, the closest relative of MLL. A MLL2-ENL protein constructed in analogy to the highly leukemogenic MLL-ENL was incapable of transforming primary hematopoietic cells. Elaborate swap experiments identified the zinc binding “CxxC”- region of MLL2 and an adjacent “post-CxxC” stretch of basic amino acids as the essential determinants for the observed difference between MLL and MLL2. Surprisingly gel shift experiments indicated that the CxxC and post-CxxC domains of MLL and MLL2 conferred almost indistinguishable in vitro DNA binding properties. However, in vivo these motifs guided MLL-ENL and MLL2-ENL to a largely non-overlapping gene repertoire. Divergent nuclear localization, a distinct potency to activate a MLL model promoter in reporter assays, and measurements of homeobox gene levels in primary cells expressing MLL and MLL2 fusion proteins all argued for a separate target site selection of the two fusions. Therefore, the CxxC domain appears to be largely responsible for target finding and as a consequence this domain is a promising object for therapies aimed at MLL fusion proteins without affecting the general function of other MLL family members.

The chromosomal translocation t(7;12) was described recently in MLL negative acute myeloid leukemias of infancy associated with a very poor clinical outcome. The rearrangement involves the genes HLXB9 (7q36) and ETV6 (12p13) with a fusion transcript of exon1/HLXB9 and exon3/ETV6. An alternative in frame splicing variant of exon1/HLXB9 to exon2/ETV6 is also detectable. Leukemic bone marrow samples of 42 infants, diagnosed in Germany with AML, were screened for the fusion transcript HLXB9/ETV6 with RT-PCR. Inclusion criteria were diagnosis of AML, age Conclusion: The HLXB9/ETV6 fusion transcript can be found in 17% of infants with MLL-negative AML in a German cohort. HLXB9/ETV6 positive leukemias show no increased expression on HOXA9 and MEIS1, but coexpression of T-cell markers and inhibition of the non-canonical NF-kappaB pathway on protein level. HLXB9/ETV6 did not induce malignant transformation in murine hematopoietic precursor cells.

Background and Aims: Molecular profiling identifies proteins characteristically deregulated in malignant diseases. Characteristic biomarkers may be useful to support diagnosis and patient stratification, while the recognition of aberrant cell activities and cell survival strategies may lead to the development of specifically designed pharmacologic strategies. We therefore performed proteomic profiling of primary human multiple myeloma (MM) cells in order to define the impact of protein expression abnormalities in MM. Methods: Plasma cells were isolated from bone marrow of patients with MM or MGUS and forwarded to proteome analysis based on 2D gel electrophoresis in addition to shotgun analysis by nano-LC-MS/MS. Erythrocytes, platelets and plasma as well as quiescent and activated lymphocytes, monocytes, endothelial and dendritic cells from healthy donors were processed in an identical manner for comparative analysis. The resulting data were interpreted with the aid of a homemade SQL database. Results: Among about thousand proteins identified in MM cells, we found aberrant expression of proteins involved in fatty acid beta-oxidation (VLCAD), unfolded protein response/ER-stress (ARMET protein, cytosol aminopeptidase), oxidative stress (ste20/oxidant stress responsive kinase 1), interferon response (MX1), iron uptake (transferrin receptor/CD71), DNA modification (transforming protein ERG, methyltransferase-like protein 7A), apoptosis and survival (apoptosis-inducing factor 1, hsp75), protein synthesis (ribosome-binding protein, Unc-13 D), cell adhesion (CD9/tetraspanin-29, LYRIC/metastatic adhesion protein), signaling (sts-1) and cell-cell interaction (Cystatin F, basigin/collagenase stimulatory factor, stem cell growth factor, small inducible cytokine B7, PD-ECGF/Gliostatin). Conclusion: The presently applied proteome analysis strategy, based on the systematic investigation of purified primary human cells, allowed us to find characteristic alterations in MM cells. Several proteins directly relate to known aberrant cell activities such as elevated protein synthesis and secretion resulting in ER-stress, which makes the cells sensitive to proteasome inhibitor treatment. Work is in progress to use quantitative assessment of such proteins for patient stratification, identification of response predictors, and biomarkers for the distinction of MM and MGUS.