Journal: bioRxiv

Article Title: Targeting the SUMO pathway primes all- trans -retinoic acid-induced differentiation of non promyelocytic Acute Myeloid Leukemias

doi: 10.1101/254946

Figure Lengend Snippet: (A) Reduction of cell protein SUMOylation in the presence of SUMO pathway inhibitors . Cell extracts from U937 cells treated for 48 h with either 2-D08 (50 pM) or Anacardic Acid (AA) (25 pM) were used in immunoblotting experiments using antibodies directed to SUMO-1 and SUMO-2/3 (SUMO 2 and −3 being immunologically indistinguishable). SUMO-conjugated proteins appear as a smear, which is lighter in the presence of 2-D08 or AA. Immunoblotting against GAPDH was used as an electrophoresis loading control. (B) Inhibitors of SUMOylation increase the differentiation of U937 cells . U937 cells were treated for 5 days with ATRA (1 pM), 2-D08 (50 pM), AA (10 pM) or the combinations and the expression of CD15 was flow cytometry-assayed. Median fluorescent intensities (MFIs) normalized to mock-treated cells are indicated (mean +/−SEM n = 10). (C) SUMOylation inhibitors increase the differentiation of HL60 cells . HL60 cells were treated with ATRA (1 pM), 2-D08 (25 pM) or AA (25pM) alone or in combination for 5 days. CD11b expression was assayed by flow cytometry. MFIs, normalized to mock-treated cells are indicated on the graph (n = 11, mean +/−SEM) (D-F) Differentiation of THP1, MOLM14 and Ara-C-resistant U937 cells . Cells were treated for 9- (THP1), 2- (MOLM14) or 5 days (Ara-C-resistant U937) with ATRA (0.5 pM, 0.1 pM and 1 pM respectively), 2-D08 (50 pM) or ATRA+2-D08 and CD14, CD11b or CD15 expressions were assayed. MFIs, normalized to mock-treated cells are indicated on the graph (n = 4 for THP1, n = 7 for MOLM14, n = 3 for Ara-C resistant U937, mean +/−SEM).

Article Snippet: Anacardic acid was from Santa Cruz Biotechnologies and 2-D08 from Merck-Millipore.

Techniques: Western Blot, Electrophoresis, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Targeting the SUMO pathway primes all- trans -retinoic acid-induced differentiation of non promyelocytic Acute Myeloid Leukemias

doi: 10.1101/254946

Figure Lengend Snippet: (A) Morphological changes of U937 cells . U937 cells were treated for 5 days with ATRA (1 pM), 2-D08 (50 pM), AA (10 pM) or the combinations and May-Grumwald-Giemsa (MGG)-stained before microscopic analysis. Scale bar = 10 pm (n = 3). Cells were distributed in 4 groups, depending on their nuclear morphology and 3 groups for the number of cytosolic granules. Quantification was done on at least 100 cells from 3 independent experiments. (B) Morphological changes of HL-60 cells . HL-60 cells were treated for 9 days with ATRA (1 pM), 2-D08 (25 pM) or AA (25pM) alone or in combination. Scale bar = 10 pm (n=3). Cells were distributed in 4 groups, depending on their nuclear morphology as in A. Quantification was done on at least 100 cells from 3 independent experiments.

Article Snippet: Anacardic acid was from Santa Cruz Biotechnologies and 2-D08 from Merck-Millipore.

Techniques: Staining

Journal: bioRxiv

Article Title: Targeting the SUMO pathway primes all- trans -retinoic acid-induced differentiation of non promyelocytic Acute Myeloid Leukemias

doi: 10.1101/254946

Figure Lengend Snippet: (A) Expression of differentiation-associated genes . U937 cells were treated for 2 days with ATRA (1 pM), 2-D08 (50 pM) or ATRA+2-D08. mRNAs for the indicated genes were assayed by qRT-PCR and normalized to the TBP housekeeping mRNA. The results are presented as ratios to mock-treated cells (n = 6, mean +/− SEM). (B) Chromatin immunoprecipitation assays . U937 cells were treated as in (A) and used in ChIP experiments using control- and H3K4Me 3 antibodies. Genomic regions showing the highest enrichment of H3K4me3 in the ENCODE database on the RARA, ITGAX, CEBPA and TNFSF10 promoters in PBMCs were amplified from the immunoprecipitated DNA fragments (see primer list). The results are presented as percentage of input. The signal from the control IP was subtracted (n = 5, mean +/− SEM).

Article Snippet: Anacardic acid was from Santa Cruz Biotechnologies and 2-D08 from Merck-Millipore.

Techniques: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Amplification, Immunoprecipitation

Journal: bioRxiv

Article Title: Targeting the SUMO pathway primes all- trans -retinoic acid-induced differentiation of non promyelocytic Acute Myeloid Leukemias

doi: 10.1101/254946

Figure Lengend Snippet: (A-B) Cell proliferation . Parental or Ara-C-resistant U937 cells were treated with ATRA (1 pM), 2-D08 (50 pM) or the combinations for the indicated times. Viable cells were quantified using Trypan blue exclusion. The number of cells on Day 0 was set to 1. The p-values are between the mock-treated and treated cells (n = 3, mean +/− SEM). (C) Cell cycle distribution . U937 cells were treated as in A for 9 days and cell cycle distribution was monitored using propidium iodine (PI) staining (n=4, mean +/− SEM). (D) Increased expression of p21CIP1 . U937 cells were treated with ATRA (1 pM), 2-D08 (50 pM) or ATRA+2-D08 for 2 days. mRNAs for CDKN1A was assayed by qRT-PCR and normalized to the TBP housekeeping mRNA. The results are presented as ratios to mock-treated cells (n = 6, mean +/− SEM). (E-F) Xenografts growth . U937 cells were xenografted subcutaneously on each flank of NSG mice (2 independent experiments, 7 mice per group in each experiment). Four days after injection, mice were treated peritumorally with 2-D08 (10 mg/kg) and/or ATRA (2.5 mg/kg) every 2 days for 10 days. Tumor volumes were measured every 2 days and are presented for all mice at day 8 on (F). Tumors smaller than 30 mm 3 before the beginning of the treatment were excluded from the analysis. The p values indicated in (E) were calculated between the DMSO and the ATRA+2-D08 conditions.

Article Snippet: Anacardic acid was from Santa Cruz Biotechnologies and 2-D08 from Merck-Millipore.

Techniques: Staining, Expressing, Quantitative RT-PCR, Injection

Journal: bioRxiv

Article Title: Targeting the SUMO pathway primes all- trans -retinoic acid-induced differentiation of non promyelocytic Acute Myeloid Leukemias

doi: 10.1101/254946

Figure Lengend Snippet: (A) CD15 expression on primary AML samples . Primary AML cells from 16 patients were purified from bone marrow aspirate at diagnosis and treated in vitro with ATRA (1 pM), 2-D08 (50 pM), AA (25pM) or combinations of these drugs. Their differentiation was assessed 9 days later by flow cytometry assay of CD15. MFIs for each condition were corrected with isotypic controls and normalized to the mock condition (DMSO-treated samples). (B) Morphological changes . The bone marrow aspirate from one AML patient was treated as in (A) for 9 days and subjected to microscopic analysis after MGG staining. Scale bar = 10 pm. The arrows indicate cytosolic granules (also see Supplementary ) (C) Analysis of non-responsive or relapsed patient samples . Primary cells from 4 patients were treated and analyzed as in (A). AML-1 corresponds to a patient refractory to induction chemotherapy and AML-2 to −4 to patients at relapse. (D) Viability of patient cells . Primary AML cells taken at diagnosis or relapse (16 patients) were treated in vitro with ATRA (0.1 pM), 2-D08 (50 pM), AA (25 pM) or combinations of these drugs. The relative numbers of viable leukemic cells (CD45/SSC gating) were quantified 9 days later by flow cytometry using counting beads.

Article Snippet: Anacardic acid was from Santa Cruz Biotechnologies and 2-D08 from Merck-Millipore.

Techniques: Expressing, Purification, In Vitro, Flow Cytometry, Staining