Journal: Cell reports

Article Title: Negative feedback regulation of MAPK signaling is an important driver of chronic lymphocytic leukemia progression.

doi: 10.1016/j.celrep.2023.113017

Figure Lengend Snippet: Figure 2. DUSP1/6 inhibition is toxic specifically for CLL cells (A) Cytotoxic dose-response to increasing concentrations of the DUSP1/DUSP6 inhibitor BCI (0–5 mM) in primary CLL samples (n = 21). Viability was determined after 48 h treatment by flow cytometry via DAPI staining. The percentage of specific cell death was calculated as follows: 100 3 (% dead cells % baseline dead cells)/(100% % baseline dead cells). Data are presented as mean values ± SD. (B) Specific cell death was calculated after viability measurement upon 48 h treatment with 1.25 mM BCI in vitro in CD19+ healthy donor-derived B cells (n = 5) compared to primary CLL cells (n = 21). Data are presented as mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t test (p < 0.0001). (C) Time course of in vitro treatment of CLL-derived cell line MEC-1 compared to human diffuse large B cell lymphoma cell line (HBL-1) and T cell lymphoma cell lines (Jurkat and HUT78). Specific cell death was determined after 24- to 72-h treatment with 5 mM BCI (n = 2; independent experiments). Data are presented as mean values ± SD. (D–F) Analysis of in vivo treatment with the BCI derivate BCI-215. Splenocytes from TCL1-tansgenic (tg) mice were transplanted in WT mice and treated with 10 mg/kg BCI-215 (n = 5) or vehicle control (5% DMSO in PBS) (n = 5) for 10 days. Content of CLL cells in peripheral blood (PB), spleen (SP), or peritoneal cavity (PC) in percent was determined by flow cytometry in vehicle control group and BCI-215 treatment group. Representative result for 2 independent experiments is shown. Data are presented as mean values ± SD. (D) Evidence of CLL engraftment in non-irradiated C57Bl/6 WT mice prior treatment. (E) Content of total CLL counts (x108) in spleen. Statistical significance was assessed by a two-tailed unpaired Student’s t test (p = 0.0026). (F) Analysis of total CLL counts (x107) in the PC. Statistical significance was assessed by a two-tailed unpaired Student’s t test (p = 0.0029). (G–K) Evaluation of genetic knockout experiments by CRISPR-Cas9 system in MEC-1 cell line. (G) Representative immunoblot of DUSP1 protein expression in MEC-1 WT cells compared to KO clone. Beta actin served as a loading control. (H) Immunoblot of DUSP6 expression in MEC-1 WT cells compared to KO clones. Loading: WT MEC1, M = marker, ko1, ko2 clone. Beta Actin served as a control. (I and J) In vitro competitor growth assays of successfully generated gene knockouts in MEC-1 cell line: (I) DUSP1 knockout clones (n = 2) and GFP+ control clones (n = 2); (J) DUSP6 knockout clones (n = 2) and GFP+ control clones (n = 2). Fold change of percent GFP was determined after mixing GFP+ control cells and knockout cells. Data are presented as mean values (with ±SD for knockout clones) of the fold change of GFP expression over time. (K) Specific cell death of WT control cell line versus DUSP1 and DUSP6 knockout clones was calculated after viability measurement by flow cytometry via DAPI staining after 48-h treatment with 1.25 mM BCI or vehicle control. Pooled data from 4 independent ex- periments; data are presesented as mean values ± SD; statistical significance was assessed by a two-tailed unpaired Student’s t test (DUSP1 p = 0.0939; DUSP6 p = 0.0159).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies FACS: human anti-CD5 Pe-Cy7 (L17F12) BioLegend 364008 FACS: human anti-CD19 APC (HIB19) BioLegend 302212 FACS: human anti-CD19 PE (HIB19) BioLegend 302208 FACS: human anti-pERK Brilliant Violet 421 (6B8B69) BioLegend 369509 FACS: human anti-pH2AX PE (20E3) Cell Signaling Technology 5763S FACS: human anti-MKP3 (DUSP6) (SR39-09) Thermo Fisher Scientific MA5-31988 FACS: mouse anti-CD5 PE (53–7.3) BioLegend 115530 FACS: mouse anti-CD19 APC-Cy7 (6D5) BioLegend 100608 WB: anti-ATF2 (20F1) Cell Signaling Technology 9226 WB: anti-pATF2 (11G2) Cell Signaling Technology 5112 WB: anti-beta actin (8H10D10) Cell Signaling Technology 3700 WB: anti-pCHK1 (133D3) Cell Signaling Technology 2348 WB: anti-DUSP1 Abcam ab195261 WB: anti-DUSP6 Cell Signaling Technology 3058 WB: anti-pH2AX (20E3) Cell Signaling Technology 9718 WB: anti-Hsp60 BD Biosciences 611563 WB: anti-p44/42 MAPK (Erk1/2) Cell Signaling Technology 9102 WB: anti-pp44/42 MAPK (pErk1/2) Cell Signaling Technology 9101 WB: anti-pSAPK/JNK Cell Signaling Technology 9251 Biological samples Healthy human PBMCs Bavarian Red Cross, Munich N/A CLL patient PBMCs Munich Clinic Schwabing, Munich N/A CLL patient PBMCs MRI, Munich N/A CLL patient PBMCs National Center for Tumor Diseases, Heidelberg N/A Chemicals, peptides, and recombinant proteins AZD6672 Selleckchem S8843 BCI hydrochloride (DUSP1/6 inhibitor) Axon Medchem 2852 BCI-215 (DUSP1/6 inhibitor) MedChemExpress HY-121087 Emricasan (pan-caspase inhibitor) Selleckchem S7775 KU-55933 (ATM inhibitor) Selleckchem S1092 LY2603618 (CHK1/2 inhibitor) Cayman Chemical 20351 PD0325901(MEK1/2 inhibitor) Selleckchem S1036 PD98059 (MEK1/2 inhibitor) Hölzel Biotech HY-12028 QVD (pan-caspase inhibitor) D Biosciences 563828 SB 202190 (p38 inhibitor) MedChemExpress HY-10295 SP600125 (JNK inhibitor) MedChemExpress HY-12041 Trametinib (MEK1/2 inhibitor) Selleckchem S2673 N-acetylcysteine (NAC, antioxidant) Thermo Fisher Scientific C10491 cOmpleteTM, EDTA-free Protease Inhibitor Cocktail Roche 04693132001 Phosphatase Inhibitor Cocktail 1 Sigma Aldrich P2850 (Continued on next page) Cell Reports 42, 113017, October 31, 2023 15

Techniques: Inhibition, Cytometry, Staining, In Vitro, Derivative Assay, Two Tailed Test, In Vivo, Control, Irradiation, Knock-Out, CRISPR, Western Blot, Expressing, Clone Assay, Marker, Generated

Journal: Cell reports

Article Title: Gene regulatory network analysis predicts cooperating transcription factor regulons required for FLT3-ITD+ AML growth

doi: 10.1016/j.celrep.2023.113568

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: BCI , Selleckchem , S2837.

Techniques: Virus, Bacteria, Recombinant, Modification, Saline, Stripping Membranes, Protease Inhibitor, Gel Extraction, Plasmid Preparation, RNA Library Preparation, In Situ, Library Quantification, shRNA, Software