ALBERT

Description: Background: About 10% of acute leukemia (AL) patients harbor MLL-r(earrangements). MLLr acute myeloid leukemia (AML) mainly occurs in young-to-middle-aged adults whereas MLLr acute lymphoblastic leukemia (ALL) mainly occurs in patients younger than 1 year at diagnosis. AML with MLL fusion to MLLT3 via t(9;11)(p22;q23) predicts intermediate prognosis whereas MLL fusion to other partners predicts adverse prognosis. By contrast, in infants with ALL MLLr invariably confers poor prognosis. Much efforts have been made to identify and target proteins required for initiation and maintenance of MLLr AL, with an aim to improve the prognosis of this aggressive AL subtype. Multiple writers, erasers, and readers of histone post-translational modifications (PTMs) have been identified to be fundamental for the initiation and maintenance of MLLr AL. Small molecular inhibitors of some of these chromatin-associated proteins have been identified, such as EPZ004777 against DOT1L, JQ1 and I-BET151 against BRD4, and so on which are also under clinical trials for AL treatment. Among histone modification erasers essential for MLLr AL, JMJD1C and KDM4C that share Jumonji catalytic domain are fundamental for MLLr AL maintenance. Histone H3 lysine 9 (H3K9) demethylase JMJD1C is one of the most promising MLLr AL targets. Multiple independent studies identified JMJD1C as required for MLLr AML, RUNX1(AML1)/RUNX1T1(ETO) AML and even chronic myeloid leukemia and lymphoma cells but not normal hematopoiesis. KDM4C Is essential for Initiation and maintenance of MLLr AL transcriptional profiling of which is dependent on KDM4C. Moreover, pharmacological inhibition of KDM4C blocks leukemia development in syngeneic mouse model and human AML xenograft model. Although a large number of special inhibitors of histone demethylases have been developed, no special inhibitors against KDM3 family member like JMJD1C have been reported. Results: Here we focused on Jumonji domain that is responsible for enzymatic activities of histone demethylases for identifying potential small molecule modulators of histone demethylases. We selected Jumonji domain of histone H3 lysine (H3K9) demethylase JMJD1C with KDM4C as reference to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components through virtual screening method. Although identified independently from each other, compound #4 and #12 both share a common structural backbone and surface plasmon resonance analysis showed that #4 and #12 bind to JMJD1C, KDM3 family member KDM3B, and KDM4 family member KDM4C with modest affinity. In vivo demethylation assay showed that #4 induces global increase of H3K9 methylation. In vitro demethylation assay showed that #4 is able to reverse H3K9 demethylation conferred by KDM3B and KDM4C. We thus named #4 and #12 as JI-4 and JI-12 (JI, Jumonji inhibitor). Cell proliferation and colony formation assays showed that JI-4 and JI-12 predominantly kill MLLr AL. To increase evidence, multiple similar compounds to JI-4 and JI-12 were tested for cell proliferation repression and JI-16 was found to show superior killing activities against hematopoietic malignant cells compared to JI-4 and JI-12. Mechanistically, JI-16 not only induces apoptosis but also differentiation of MLLr AL cells. Transcriptome analysis and quantitative PCR (QPCR) showed that JI-16 induced gene expression profiling is especially enriched in gene sets involved in metabolism. Conclusion: To sum up, we identified potential pan-inhibitors of the Jumonji domain of histone demethylases. Binding in-vivo is followed by selective killing of MLLr AL cells. Disclosures. No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

Description: Anaplastic large cell lymphomas (ALCLs) are highly proliferating tumors that commonly express the AP-1 transcription factor JunB. ALK fusions occur in approximately 50% of ALCLs, and among these, 80% have the t(2;5) translocation with NPM-ALK expression. We report greater activity of JunB in NPM-ALK–positive than in NPM-ALK–negative ALCLs. Specific knockdown of JUNB mRNA using small interfering RNA and small hairpin RNA in NPM-ALK–expressing cells decreases cellular proliferation as evidenced by a reduced cell count in the G2/M phase of the cell cycle. Expression of NPM-ALK results in ERK1/2 activation and transcriptional up-regulation of JUNB. Both NPM-ALK–positive and –negative ALCL tumors demonstrate active ERK1/2 signaling. In contrast to NPM-ALK–negative ALCL, the mTOR pathway is active in NPM-ALK–positive lymphomas. Pharmacological inhibition of mTOR in NPM-ALK–positive cells down-regulates JunB protein levels by shifting JUNB mRNA translation from large polysomes to monosomes and ribonucleic particles (RNPs), and decreases cellular proliferation. Thus, JunB is a critical target of mTOR and is translationally regulated in NPM-ALK–positive lymphomas. This is the first study demonstrating translational control of AP-1 transcription factors in human neoplasia. In conjunction with NPM-ALK, JunB enhances cell cycle progression and may therefore represent a therapeutic target.

Description: Primary effusion lymphoma (PEL) is a rare, aggressive form of B-cell lymphoma. With a median survival time of around six months the prognosis for PEL patients is poor. Therefore, there is a medical need for novel therapeutic strategies. We performed expression array analysis to find potential targets for antibody-based therapy. Unsupervised clustering analysis revealed that PEL cell lines grouped separate from cell lines derived from other B-non Hodgkin lymphoma (B-NHL) entities. Notably, PEL and Hodgkin Lymphoma (HL) cell lines clustered on one arm, separate from all cell lines representing less-differentiated B-NHL variants. PEL and HL cell lines were characterized by a set of common up- and downregulated genes. Typical for PEL and HL was the expression of CCND2 and the absence of Brutons tyrosine kinase and of B-cell markers including CD19, CD20, CD79A and CD79B. Highly expressed in PEL - but not in HL - were CD138, IL-10, SLAMF7 and PRDM1. PRDM1/BLIMP1 is a master regulator of terminal B-cell differentiation. Originally described as repressor, BLIMP1 can also enhance transcription of SLAMF7 in multiple myeloma (MM) and of IL-10 in type 1 regulatory T-cells. Thus, coexpression of the three genes suggests a causal relationship between transcriptionally active PRDM1/BLIMP1 and its targets SLAMF7 and IL-10 also in PEL. Expression of SLAMF7 in PEL is especially noteworthy because a monoclonal antibody targeting SLAMF7 (elotuzumab) has been approved for treatment of patients with MM. We observed that SLAMF7 is comparably expressed in PEL and in MM cell lines. If the results on cell lines can be translated to primary PEL, i.e. if PEL tumor cells express SLAMF7, the patients might benefit from an antibody-based targeted therapy against this antigen. Disclosures No relevant conflicts of interest to declare.

Description: Background: Acute leukemia (AL) originates from both genetic and epigenetic changes that can be targeted to cure AL. Dysregulated DNA methylation has been shown to be associated with AL and demethylating agents 5-azacytidine and decitabine show favored improvement in secondary leukemia. Deficient histone acetylation has also been reported in AL and can be corrected to relieve leukemia. Histone methylation harbors more structural complexities compared to DNA methylation and histone acetylation and is broadly involved in AL. In particular, histone H3 lysine 9 (H3K9) methylation has been associated with AL. Di-methylation of H3K9 is reportedly involved in human hematopoietic stem cell lineage commitment. Moreover, tri-methylation of H3K9 predicts AML survival. H3K9 demethylation is catalyzed by exclusive KDM3 family members (KDM3A, KDM3B, and JMJD1C) that catalyze mono- and di-demethylation of H3K9, non-exclusive KDM4 family members (KDM4A, KDM4B, KDM4C, and KDM4D) that catalyze both H3K9 and H3K36 di- and tri-demethylation, KDM1A (LSD1) that catalyzes H3K4 and H3K9 mono- and di-demethylation, and PHF8 that catalyzes H3K9 mono- and di-demethylation and H4K20 demethylation. Among these, KDM3B, JMJD1C, KDM4C, LSD1, and PHF8 have been reported to be associated with AL in an enzymatic activity-dependent way. Furthermore, small molecular inhibitors of KDM4C and LSD1 have been developed for treatment of AML. H3K9 demethylase KDM3B is located at chromosome 5 band 31, a region frequently deleted or lost in acute myeloid leukemias (AML) and myelodysplasias (MDS). Different from other H3K9 demethylases that are usually responsible for leukemia maintenance, KDM3B harbors potential tumor-suppressive activities in acute myeloid leukemia and myelodysplastic syndromes. However, small molecular antagonists and agonists are lacking for KDM3B. Results: We aim to identify small molecular modulators of KDM3B. We focused on crystal structure of KDM3B Jumonji domain that catalyzes histone demethylation for virtual screening. From approximately 200,000 natural products and Chinese medicine components, we identified a potential KDM3B modulator, namely compound #7. Surface plasmon resonance technology showed that compound #7 binds to KDM3B with favorable affinity. In vitro and in vivo demethylation assay showed that compound #7 is able to increase H3K9 demethylating activity of KDM3B. We thus named compound #7 as KA-7 (KDM3B agonist #7). Interestingly, the identified KDM3B agonist KA-7 is able to selectively repress MLL-rearranged AL in cell proliferation and colony formation assays. Considering that KA-7 targets KDM3B that is located at chromosome 5q, a frequently deleted region in AML and MDS, we explored if KA-7 collaborates with Lenalidomide, an FDA approved drug for treating MDS with deletion at 5q where KDM3B is located. KA-7 was found to be able to synergistically increase the selective killing of AL cells by Lenalidomide. Conclusion: To sum up, physiologic H3K9 demethylase activity of KDM3B can be enhanced by a small molecular modulator KA-7 and causes selective killing against MLL-arranged AL cells. Disclosures. No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: Patients with acute leukemias carrying MLL rearrangements have a poor prognosis. The tumor cells show characteristic gene expression profiles with increased levels of selected HOX genes (1). We have shown that acute lymphoblastic leukemia (ALL) cell lines with and without MLL rearrangements could likewise be recognized by analysis of HOX gene expression (2). In contrast, MLL wild-type (MLLwt) and mutant (MLLmu) acute myeloid leukemia (AML) cell lines could not be distinguished by analysis of HOX genes, because even wild-type cell lines had a high expression background (2). It was our aim to find out whether MLLwt and MLLmu AML cell lines could be discriminated on the basis of gene expression - other than HOX genes. We performed gene expression analysis with pooled RNAs of MLLmu (n=8) and MLLwt (n=8) cell lines applying high density oligonucleotide Genechips from Affymetrix (HG-U133A). Defensin alpha4 (83x), defensin beta1 (32x), cathepsinG (9x) and FLT3 (7x) genes were overexpressed in MLLmu cell lines, stabilin1 (82x) and galectin10 (55x) in MLLwt cell lines. PCR analysis with individual (non-pooled) cDNAs of the 16 cell lines showed that none of the above genes was exclusively expressed by MLLmu or MLLwt cells. Thus, pooling RNAs has a major disadvantage: many PCRs have to be performed to establish faithful expression profiles for individual samples. BEX1 finally proved to be a gene that was exclusively overexpressed in one group of cell lines. It had been an interesting candidate already after oligonucleotide chip analysis, being both overexpressed (18x) in MLLmu cell lines, and - in contrast to the genes listed above - not a marker of myeloid differentiation. BEX1 is reportedly expressed in brain, testis and ovary, but not in peripheral blood leukocytes, lymph node and bone marrow (3). By RT-PCR analysis we showed that 7/8 MLLmu and 0/8 MLLwt cell lines expressed BEX1. Screening a panel of 54 hematopoetic cell lines gave the same result: BEX1 expression was restricted to MLLmu AML cell lines: 8/11 (73%) MLLmu AML cell lines expressed BEX1, but 43 other hematopoetic cell lines (including Hodgkin′s disease, anaplastic large cell lymphoma, ALL and MLLwt AML cell lines) tested negative. BEX1 expression may depend on the type of MLL rearrangement or the histological background of the cells, as MLLmu ALL cell lines (0/5) also tested negative. It has been shown in the mouse system, that BEX family members may be involved in cell signalling processes. Thus it will be interesting to elucidate whether BEX1 also participates in proliferative/antiapoptotic signalling processes in MLLmu AML cell lines.

Description: The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.

Description: Wild-type nucleophosmin (NPM) is a multifunctional protein shuttling between the nucleus and the cytoplasm. Chromosomal rearrangements leading to NPM fusion proteins occur in leukemias and lymphomas (e.g. with partners RARA, ALK). Recently, Falini et al. reported that 60% of acute myeloid leukemia (AML) patients with normal karyotype carry mutations at exon-12 of the NPM gene. This results in frame shifts that lead to alterations of the C-terminus of NPM resulting in the aberrant cytoplasmic localization of the mutated protein (NPMc+) (1). The effects of a mutationally altered protein on cellular functions like proliferation, differentiation or apoptosis, have often been revealed using immortalized cell lines that carry the mutation in question. Therefore, we screened a panel of 79 myeloid leukemia cell lines for presence of mutations - 4 bp insertions - at the exon-12 of the NPM gene. We performed polymerase chain reaction (PCR) analysis with fluorescent dye-labeled primers. For fragment size determination, the PCR products were mixed with dye-labeled size standards and separated by capillary electrophoresis. OCI-AML3 was the only cell line that expressed a signal in addition to and 4 bp larger than the wild-type NPM signal. Sequencing of the cloned NPM-mutated PCR product showed TCTG duplication at positions 956–959 of exon-12. This mutation was heterozygous and corresponded to the type that occurs in 77% of primary NPMc+ AMLs. OCI-AML3 cells have a hyperdiploid karyotype with 48(45–50)X/XY, +1, +5, +8, der(1)t(1;18)(p11;q11), i(5p),del(13)(q13q21), dup(17)(q21q25); sideline with r(Y)x1-2 and show the following immunoprofile: CD3−, CD4+, CD7−, CD8−, CD10−, CD13+, CD14−, CD15+, CD19−, CD30−, CD33−, CD34−, CD41+, CD42b−, CD68+, CD235a+, HLA-DR-. Especially the presence of myeloid markers and absence of CD34 is typical for NPMc+ cells (1). Furthermore, immunostaining with anti-NPM antibodies confirmed that the OCI-AML3 cells, like primary NPMc+ AML and in contrast to NPM wild-type cells, show cytoplasmic expression of NPM. Functional studies showed that the altered nucleo-cytoplasmic transport of NPM was nuclear export signalling-dependent and could be blocked by using the specific CRM1/exportin-1 inhibitor leptomycin B. In conclusion, cell line OCI-AML3 promises to be an important tool for studying the biological properties and response to new drugs of NPMc+ AML.