Journal: Nature Communications

Article Title: Systematic characterization of the HOXA9 downstream targets in MLL-r leukemia by noncoding CRISPR screens

doi: 10.1038/s41467-023-43264-5

Figure Lengend Snippet: A Competitive proliferation assay was conducted in Cas9-expressing SEM cells targeted with sgRNAs against HOXA9-bound sites close to the genes as PEBP4 , ZCCNC7 , AHI1 , RUNX1 , DCAF11 , CDK6 , NDUFS8 , and MAN1C1 . The sgRPS19 and sgFLT3-DE-1 sgRNA served as positive controls. Disruption of the loci targeted by these selected sgRNAs led to retarded cell growth of SEM cells in a time-dependent manner. The percentage of cell numbers was normalized to CFP + control cells infected with non-target sgRNA (sgNT). B Competitive proliferation assay was conducted in Cas9-expressing MOLM13 cells targeted with sgRNAs against HOXA9-bound sites close to the genes as PEBP4 , ZCCHC7 , AHI1 , RUNX1 , DCAF11 , CDK6 , NDUFS8 and MAN1C1 . The sgRPS19 and sgFLT3-DE-1 sgRNA served as positive controls. Disruption of the loci targeted by these sgRNAs led to retarded cell growth of MOLM13 cells in a time-dependent manner. The percentage of cell numbers was normalized to CFP + control cells infected with non-target sgRNA (sgNT). C Competitive proliferation assay was conducted in dCas9-KRAB-expressing SEM cells targeted with sgRNAs against HOXA9-bound sites close to the genes RUNX1 , DCAF11 , and CDK6 . The sgFLT3-DE-1 sgRNA served as positive control. D Competitive proliferation assay was conducted in dCas9-KRAB-expressing MOLM13 cells targeted with sgRNAs against HOXA9-bound sites close to the genes RUNX1 , DCAF11 , and CDK6 . The sgFLT3-DE-1 sgRNA served as positive control. E Characterization of the chromatin conformation change upon dCas9-KRAB targeting against the HOXA9-bound site in the intron of CDK6 . HiC (GEO: GSE138862), HOXA9 ChIP-seq, H3K27ac ChIP-seq and BRD4 ChIP-seq (GEO: GSE117864) tracks were shown to characterize the epigenetic status of the HOXA9-bound site. F Q-PCR was conducted to quantify the transcription decrease of CDK6 when CRISPRi targeted the HOXA9-bound site in the intron of CDK6 in SEM cells. G Q-PCR was conducted to quantify the transcription decrease of CDK6 when CRISPRi targeted the HOXA9-bound site in the intron of CDK6 in MOLM13 cells. H Total RNA-seq was performed using SEM cells targeted with sgCDK6 against the HOXA9-bound site in the CDK6 intron. Differential gene expression was defined by FDR < 0.01. The CDK6 expression is the top hit. Data are shown as mean values ± SEM of three biological replicates. P values were estimated using a two-tail un-paired t -test. Source data are provided as a Source Data File.

Article Snippet: MOLM13 was originally purchased from ACCEGEN.

Techniques: Proliferation Assay, Expressing, Disruption, Control, Infection, Positive Control, ChIP-sequencing, RNA Sequencing, Gene Expression

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) MOLM14 cells were added to a 384-well plate containing cytokines, chemokines, and growth factors with 10 nM AC220 (quizartinib). Viability was measured using MTS reagent, averaged over all concentrations (1, 10, and 100 ng/ml), and the results sorted according to highest average viability. Average viability >2 standard deviations above the mean are highlighted in red and by an *. B) MOLM14 cells were cultured in lower concentrations of recombinant proteins (1, and 0.1 ng/ml) in 96-well plates plus 10 nM AC220. Viability was assessed after 48 hours with MTS reagent and data plotted as percent of the respective untreated control. All wells were plated in triplicate with standard deviation indicated by error bars. C) MOLM14 cells were cultured continuously in 10 nM AC220 with FL, FGF2, or media alone as indicated (N=4 for each). Fresh media, AC220 and cytokine were replaced every 2–3 days over the indicated time period. Viable cell numbers were analyzed every 2–3 days with Guava ViaCount.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Cell Culture, Recombinant, Control, Standard Deviation

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) MOLM14 cells were cultured in media alone or media supplemented with 10 ng/ml FGF2, treated with 10 nM AC220, and exposed to 25, 50, and 100 nM of the FGFR inhibitor PD173074. Viability was measured by MTS assay after 48 hours and normalized to their respective untreated control (media alone, FGF2, 25 nM PD173074, etc.) B) MOLM14 cells were electroporated with siRNAs targeting FGFR1, FGFR2, FGFR3, FGFR4 and a non-specific (NS) control. After 48 hours the cells were pelleted and resuspended in media +/− FGF2 and +/− 10 nM AC220; and viability assessed by MTS after 48 hours. * indicates p<0.01 by t test. C) Western blot was used to evaluate FGFR1 protein expression after siRNA (see also supplemental Figure S1). D) BaF3 cells expressing FLT3-ITD (20) were retrovirally transfected with either empty pMX vector (pMX neg) or vector containing FGFR1, selected with puromycin, and analyzed by Western blot to show FGFR1 expression. The FLT3-ITD Baf3 cells with E) empty pMX vector or F) pMX with FGFR1 were then treated with a gradient of AC220 in either media alone or supplemented with 10 ng/ml FGF2. Viability was assessed after 48 hours with MTS reagent and data plotted as percent of the respective untreated control. All cell line experiments were performed in triplicate and error bars indicate standard deviation. Thawed frozen viable (see methods) and fresh primary FLT3-ITD AML samples were plated with media alone (no ligand) or with FGF2 at 1, 10 and 100 ng/ml concentration. The cells were then exposed to a gradient of AC220 and viability was measured after 72 hours by MTS. G) An example of ex vivo FGF2-mediated protection from AC220 with a primary FLT3-ITD AML sample. H) The area under the curve was calculated for 23 patient samples in the absence or presence of FGF2 ligand (see 2G) and graphed with mean and standard error of the mean shown. One-tailed t-tests were performed with p values indicated by *<0.05, **<0.005, and ***=0.0007.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Cell Culture, MTS Assay, Control, Western Blot, Expressing, Transfection, Plasmid Preparation, Standard Deviation, Concentration Assay, Ex Vivo, One-tailed Test

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) Naïve MOLM14 cells in media alone were exposed to a matrix of an AC220 gradient (0, 1.4, 4, 12, 37, 111, 333, 1000 nM) overlayed with the FGFR inhibitor PD173074 (0, 1.4, 4, 12, 37, 111, 333, 1000 nM) and compared to B) FGF2- and C) FL-dependent resistant MOLM14 cells (Figure 1C) in the same conditions. Viability was measured after 48 hours and average viability graphed as surface plots with gray scale denoting 20% increments. Graphs at right indicate viability with AC220 alone, PD173074 alone, and equimolar combination to highlight synergy. Calculation of the combination index is included in Supplemental Table 1 and synergy (CI<0.1) indicated by an *. All experiments were done in triplicate with average viability scaled to untreated condition (100%). Error bars represent standard deviation.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Standard Deviation

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) Naïve MOLM14 cells were treated for 24 hours −/+ 10 nM AC220 (first two lanes) and comparedto FGF2- and FL-dependent resistant cells (grown continuously in FGF2 or FL, lanes 3–10). The FGF2- and FL-dependent resistant cells were harvested 24 hours after addition of fresh media, recombinant protein and AC220. The cells were then lysed as described with Western blot analysis as per Materials and Methods. The FGF2-dependent resistant culture that developed a Y842C mutation is indicated.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Recombinant, Western Blot, Mutagenesis

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: The time to development of exponential growth (indicated by gray) was plotted over time for the extended cultures in Figure 1C. The timepoints at which Sanger or next-generation sequencing (at right, average read depth ~1500) was performed is indicated by arrows. If a mutation could not be detected it is indicated with “−”. A) MOLM14 cells cultured in media and AC220 alone (no ligand) developed exponential growth later than FGF2- and FL-dependent cultures (panels B and C) and developed early resistance mutations: D839V and D835H. B) FGF2-dependent and C) FL-dependent resistant cultures (cultured continuously in 10 ng/ml FGF2 or FL plus 10 nM AC220) were split at 4 months and 1×107 cells placed into fresh media without recombinant protein with continued 10 nM AC220 treatment. Fresh media and 10 nM AC220 were replaced every 2–3 days over the indicated time period and viable cell number was analyzed using Guava ViaCount. D) Naïve MOLM14 cells and resistant MOLM14 cells with FLT3 D839V mutation (R-1), FLT3 D842C mutation (FGF2 R-1), and non-mutated resistant cells (FL R1 after FL subtraction) were exposed to a gradient of FLT3 inhibitors: AC220 (quizartinib), crenolanib, sorafenib, and ponatinib. Viability was measured after 48 hours by MTS assay with average viability scaled relative to untreated cells (100%). The experiment was done in triplicate and error bars represent standard deviation.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Next-Generation Sequencing, Mutagenesis, Cell Culture, Recombinant, MTS Assay, Standard Deviation

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) The human stromal cell lines HS-5 and HS-27 were analyzed for FGF2 expression by Western blot. B) MOLM14 cells were cultured in media alone, 10 ng/ml FGF2, or in 1 μm transwells over HS-5 or HS-27 stromal cell lines. The cells were treated with 10 nM AC220 and/or 250 nM PD173074 as indicated and MOLM14 cells were analyzed for viable cell number after 4 days. Viable cells were plotted as percentage of untreated control. C) The corresponding stromal cells from the transwell co-culture experiments were also evaluated by MTS assay for viability. Results are shown relative to the untreated condition. All experiments were done in triplicate, error bars represent standard deviation, and * indicates p<0.05.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Expressing, Western Blot, Cell Culture, Control, Co-Culture Assay, MTS Assay, Standard Deviation