murine leukemia cell line  (ATCC)

Journal: Clinical Cancer Research

Article Title: Synergistic Activity of Combined FLT3-ITD and MDM2 Inhibition with Quizartinib and Milademetan in FLT3 -ITD Mutant/ TP53 Wild-type Acute Myeloid Leukemias

doi: 10.1158/1078-0432.CCR-24-2764

Figure Lengend Snippet: Quizartinib plus milademetan exerts highly synergistic proapoptotic activity through suppression of FLT3 downstream signaling and induction of apoptotic signaling and PUMA in FLT3 -ITD mutant/ TP53 WT AML cell lines. A, MOLM-13, MOLM-14, and MV-4-11 cell lines (all of these cell lines bear FLT3 -ITD mutations and p53 WT) and ( B ) OCI/AML3 ( FLT3 WT/ TP53 WT) and Tohoku Hospital Pediatrics 1 (THP-1; FLT3 WT/ TP53 mutation) cell lines were treated with indicated concentrations of milademetan and/or quizartinib for 48 hours. Apoptosis induction was measured by FCM with annexin V staining and CI. All experiments were repeated ≥3 times, and data are presented as mean ± SD. CIs were calculated using CalcuSyn software (version 2.0, PREMIER Biosoft). C, MOLM-13, MOLM-14, and MV-4-11 cell lines were treated with milademetan (30 nmol/L) or quizartinib (2 nmol/L) or Q/M combination for 24 hours. Phospho (p)-FLT3 and its downstream signaling proteins and Bcl-2 family proteins were measured by immunoblotting. Bim, Bcl-2–interacting mediator; CI, confidence interval; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The murine leukemia cell line Ba/F3 harboring FLT3 -ITD mutations (Ba/F3- FLT3 -ITD) was kindly provided by Dr. Donald Small (Department of Pediatric Oncology, Johns Hopkins University, Baltimore, MD).

Techniques: Activity Assay, Mutagenesis, Staining, Software, Western Blot

Journal: Clinical Cancer Research

Article Title: Synergistic Activity of Combined FLT3-ITD and MDM2 Inhibition with Quizartinib and Milademetan in FLT3 -ITD Mutant/ TP53 Wild-type Acute Myeloid Leukemias

doi: 10.1158/1078-0432.CCR-24-2764

Figure Lengend Snippet: Combination of Q/M-induced proapoptotic activity is p53-dependent, and the combination treatment impairs clonogenicity in FLT3 mutant/ TP53 WT leukemia blasts. A, MOLM-13-p53-Vec or MOLM-13-p53-KD cells were treated with increasing concentrations of single-agent milademetan or quizartinib or Q/M combination for 48 hours. Apoptosis was measured by FCM with annexin V staining. CIs were calculated using CalcuSyn software (version 2.0, PREMIER Biosoft). B, MOLM-13-p53-Vec or MOLM-13-p53-KD cells were treated with MOLM-13 and milademetan (60 nmol/L) or quizartinib (3 nmol/L) or the combination for 24 hours. Correlated signaling pathway proteins were measured with immunoblotting. C, Primary AML samples (three cases; harboring FLT3 mutant/ TP53 WT) and two normal BM samples from normal donors were treated with indicated concentrations of quizartinib and/or milademetan for 2 weeks in methylcellulose medium. CFU-GM colonies were counted, and the percentage of colony forming against the control group was calculated. The data were obtained from triplicated wells, and error bars are presented as mean ± SD. Asterisks indicate the level of statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001 as determined using a two-tailed unpaired t test. BAX, Bcl-2–associated X; CFU-GM, colony-forming unit granulocyte–macrophage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, no statistical significance.

Article Snippet: The murine leukemia cell line Ba/F3 harboring FLT3 -ITD mutations (Ba/F3- FLT3 -ITD) was kindly provided by Dr. Donald Small (Department of Pediatric Oncology, Johns Hopkins University, Baltimore, MD).

Techniques: Activity Assay, Mutagenesis, Staining, Software, Western Blot, Control, Two Tailed Test

Journal: Clinical Cancer Research

Article Title: Synergistic Activity of Combined FLT3-ITD and MDM2 Inhibition with Quizartinib and Milademetan in FLT3 -ITD Mutant/ TP53 Wild-type Acute Myeloid Leukemias

doi: 10.1158/1078-0432.CCR-24-2764

Figure Lengend Snippet: Co-targeting FLT3 and MDM2 synergistically induces apoptosis in quizartinib-sensitive and -resistant FLT3 - ITD and TKD double-mutant AML cells and in venetoclax-resistant cells. A, Murine FLT3 -ITD/ TP53 WT cells Ba/F3- FLT3 -ITD, Ba/F3- FLT3 -ITD + D835Y, and Ba/F3- FLT3 -ITD + F691L were treated with increasing concentrations of quizartinib and/or DS-5272 (murine MDM2i) for 48 hours. Apoptosis induction was measured by FCM with annexin V staining. CIs were calculated using CalcuSyn software (version 2.0, PREMIER Biosoft). B, MOLM-13 parental and venetoclax-resistant (MOLM-13-VEN-Resis) cells were treated with venetoclax (Bcl-2i) for 48 hours. Viable cells were measured using the CellTiter-Glo luminescent cell viability assay (Promega). C, MOLM-13-VEN-Resis cells were treated with quizartinib and/or milademetan for 48 hours. Apoptosis was determined by FCM with Annexin V staining. D, MOLM-13-VEN-Resis cells were treated with indicated concentrations of milademetan and/or quizartinib for 24 hours. Correlated proteins were determined using immunoblotting. Asterisks indicate the level of statistical significance. **, P < 0.01; ***, P < 0.001. BAX, Bcl-2–associated X; Bim, Bcl-2–interacting mediator; CI, confidence interval; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The murine leukemia cell line Ba/F3 harboring FLT3 -ITD mutations (Ba/F3- FLT3 -ITD) was kindly provided by Dr. Donald Small (Department of Pediatric Oncology, Johns Hopkins University, Baltimore, MD).

Techniques: Mutagenesis, Staining, Software, Cell Viability Assay, Western Blot

Journal: Clinical Cancer Research

Article Title: Synergistic Activity of Combined FLT3-ITD and MDM2 Inhibition with Quizartinib and Milademetan in FLT3 -ITD Mutant/ TP53 Wild-type Acute Myeloid Leukemias

doi: 10.1158/1078-0432.CCR-24-2764

Figure Lengend Snippet: Quizartinib plus milademetan significantly extended survival in a PDX model of treatment-resistant AML ( FLT3 -ITD positive + D835 mutant/ TP53 WT). Prevalence of xenografted cells in ( A ) PB and ( B ) BM was measured using FCM by gating with the mCD45 – /hCD45 + population. C, Spleen mass was determined using a digital balance after dissection from three mice per group. D, The survival curve for xenografted mice was analyzed using the Kaplan–Meier method, and log-rank statistics were used to assess survival differences between the groups. Asterisks indicate the level of statistical significance. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. NS, no statistical significance.

Article Snippet: The murine leukemia cell line Ba/F3 harboring FLT3 -ITD mutations (Ba/F3- FLT3 -ITD) was kindly provided by Dr. Donald Small (Department of Pediatric Oncology, Johns Hopkins University, Baltimore, MD).

Techniques: Mutagenesis, Dissection