human aml cell line u937  (ATCC)

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: FLT3-ITD AML cells are resistant to FLT3 inhibitors in hypoxia, associated with post-translational FLT3-ITD downregulation. A. Primary FLT3-ITD AML blasts from two patients were cultured for 48 hours under normoxia (21%O 2 ) or hypoxia (<1% O 2 ) with the FLT3 inhibitors gilteritinib or quizartinib at increasing concentrations and cytotoxicity was measured using the WST-1 assay. Cytotoxicity of both gilteritinib and quizartinib was reduced in hypoxia (red lines) compared with normoxia (black lines). Data represent means of three replicate wells. B. Blasts from three FLT3-ITD AML patients cultured in normoxia and hypoxia were harvested at 0, 48 and 96 hours and immunoblotted for expression of FLT3, p-STAT5, STAT5 and vinculin loading control. Bands were quantified by densitometry and FLT3, p-STAT5 and STAT5 were normalized to vinculin and to Time 0. Immunoblots and graphs are shown. FLT3-ITD and p-STAT5 were downregulated in hypoxia, but not in normoxia, while total STAT5 remained stable in both. C. FLT3 and GAPDH control mRNA was measured by RT-qPCR in blasts from three FLT3-ITD AML patients cultured for 0, 48 and 96 hours in hypoxia and normoxia. Graphs of FLT3 mRNA, normalized to GAPDH mRNA, show no decrease in FLT3 mRNA. D. FLT3-ITD AML patient blasts were treated with cycloheximide (CHX, 100 µg/mL) to block new protein translation, with or without addition of the proteasome inhibitor MG-132 (20 µmol/L) after 30 minutes to block proteasomal degradation, then harvested at 0, 1, 2, and 4 hours, starting 1 hour after addition CHX, and immunoblotted for FLT3 and vinculin control. Bands were quantified by densitometry and FLT3 protein half-lives were calculated by linear regression analysis. FLT3-ITD protein turnover was accelerated in hypoxia (1.0 vs. 2.5 hours), in the absence, but not presence, of MG-132, consistent with increased proteasomal degradation in hypoxia.

Article Snippet: The human FLT3-ITD AML cell lines MV4-11 and MOLM-14, with homozygous and heterozygous FLT3-ITD, respectively (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM L-glutamine, unless otherwise indicated, and were tested for Mycoplasma every six months ( ).

Techniques: Cell Culture, WST-1 Assay, Expressing, Control, Western Blot, Quantitative RT-PCR, Blocking Assay

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: A. Blasts from three FLT3-ITD AML patients cultured in normoxia and hypoxia were harvested at 0, 48 and 96 hours and immunoblotted for expression of c-CBL, p-c-CBL (Y371) and vinculin loading control, and bands were quantified by densitometry. Expression of c-CBL and p-c-CBL was upregulated in hypoxia, but not in normoxia. B. c-CBL and GAPDH control mRNA was measured at 0, 48 and 96 hours by RT-qPCR, and graphs of c-CBL mRNA normalized to GAPDH control mRNA are shown. c-CBL mRNA expression did not decrease in cells cultured in hypoxia. C . MOLM-14 cells were treated with cycloheximide (CHX, 100 µg/mL), with or without addition of the proteasome inhibitor MG-132 (20 µmol/L) after 30 minutes, then harvested at 0, 1, 2, and 4 hours, starting 1 hour after addition CHX, and immunoblotted for FLT3 and vinculin control. Bands were quantified by densitometry and FLT3 protein half-lives were calculated by linear regression analysis. FLT3-ITD protein turnover was similar in hypoxia and normoxia and the effect of MG-132 was also similar.

Article Snippet: The human FLT3-ITD AML cell lines MV4-11 and MOLM-14, with homozygous and heterozygous FLT3-ITD, respectively (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM L-glutamine, unless otherwise indicated, and were tested for Mycoplasma every six months ( ).

Techniques: Cell Culture, Expressing, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: A. Whole-cell lysates of MOLM-14 and MV4-11 FLT3-ITD AML cells and primary FLT3-ITD AML blasts (Patient 1) transfected with c-CBL siRNA or control (CTR) siRNA were immunoblotted and probed for c-CBL and vinculin loading control, and bands were quantified by densitometry. Immunoblots demonstrate c-CBL knockdown in cells transfected with c-CBL siRNA. B. MOLM-14 and MV4-11 FLT3-ITD AML cell lines and primary FLT3-ITD AML patient blasts transfected with c-CBL siRNA or CTR siRNA cultured in hypoxia were harvested at 0, 48, or 96 hours, and whole-cell lysates were probed for FLT3, c-CBL and vinculin loading control. Immunoblots and densitometric analysis are shown, demonstrating that c-CBL knockdown abrogates FLT3-ITD downregulation in hypoxia. C. c-CBL, FLT3 and GAPDH control mRNA expression was measured by RT-qPCR in MOLM-14, MV4-11 and primary FLT3-ITD AML patient blasts treated with c-CBL siRNA or CTR siRNA for 96 hours. Data are shown graphically, with bars showing mean fold expression (2^-ΔCt) ± SD in cells transfected with c-CBL siRNA, relative to CTR siRNA. c-CBL silencing decreases c-CBL, but not FLT3, mRNA, showing that, as expected, FLT3 downregulation in cells transfected with CTR siRNA is not transcriptional . D. MOLM-14 cells transfected with c-CBL siRNA or CTR siRNA were treated with CHX to inhibit new protein translation, with or without MG-132 to inhibit proteasomal degradation. FLT3 protein turnover was accelerated in cells transfected with control, but not c-CBL, siRNA, in the absence, but not presence, of MG-132, consistent with c-CBL silencing rescuing FLT3-ITD AML cells from increased FLT3-ITD proteasomal degradation in hypoxia.

Article Snippet: The human FLT3-ITD AML cell lines MV4-11 and MOLM-14, with homozygous and heterozygous FLT3-ITD, respectively (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM L-glutamine, unless otherwise indicated, and were tested for Mycoplasma every six months ( ).

Techniques: Transfection, Control, Western Blot, Knockdown, Cell Culture, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: Primary blasts from three patients with AML with WT FLT3 (Patients 7–9) cultured in hypoxia for 96 hours were immnunoblotted for FLT3, c-CBL and vinculin loading control. The findings suggest that, in contrast to FLT3-ITD AML, hypoxia does not uniformly induce FLT3 downregulation in FLT3-WT primary blasts, and that additional patient-specific factors may influence FLT3 stability under low-oxygen conditions.

Article Snippet: The human FLT3-ITD AML cell lines MV4-11 and MOLM-14, with homozygous and heterozygous FLT3-ITD, respectively (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM L-glutamine, unless otherwise indicated, and were tested for Mycoplasma every six months ( ).

Techniques: Cell Culture, Control

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: A. Primary FLT3-ITD AML cells from four patients were cultured for 48 hours with the FLT3 inhibitors gilteritinib or quizartinib in increasing concentrations in normoxia or hypoxia in the presence or absence of 2 mM glutamine, and cytotoxicity was measured using the WST-1 assay. FLT3-ITD AML cells remained sensitive to FLT3 inhibitors in hypoxia in the absence of glutamine. B. Primary FLT3-ITD AML cells cultured in hypoxia in the presence or absence of 2 mM glutamine were harvested for immunoblotting at 0, 48 and 96 hours. FLT3-ITD and p-STAT5 expression decreased progressively in the presence, but not absence, of 2 mM glutamine, while total STAT5 expression did not change, demonstrating that FLT3-ITD and p-STAT5 downregulation in hypoxia is abrogated in the absence of glutamine. C. FLT3-ITD AML cells were treated with CHX to block new protein synthesis, with or without addition of the proteasome inhibitor MG-132, and cultured in hypoxia in the presence or absence of 2 mM glutamine. Increased FLT3-ITD proteasomal degradation of occurred in hypoxia in the presence, but not absence, of glutamine.

Article Snippet: The human FLT3-ITD AML cell lines MV4-11 and MOLM-14, with homozygous and heterozygous FLT3-ITD, respectively (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM L-glutamine, unless otherwise indicated, and were tested for Mycoplasma every six months ( ).

Techniques: Cell Culture, WST-1 Assay, Western Blot, Expressing, Blocking Assay

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: FLT3-ITD AML blasts were cultured in hypoxia for 96 hours in medium with or without 2 mM L-glutamine and immunoblotted for expression of c-CBL and vinculin loading control. Immunoblot and graphic representation are shown. c-CBL protein expression was increased in cells cultured with, but not without, glutamine supplementation.

Article Snippet: The human FLT3-ITD AML cell lines MV4-11 and MOLM-14, with homozygous and heterozygous FLT3-ITD, respectively (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM L-glutamine, unless otherwise indicated, and were tested for Mycoplasma every six months ( ).

Techniques: Cell Culture, Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: A. Primary FLT3-ITD AML cells cultured in hypoxia with and without glutamine (2 mM), with telaglenastat (100 nM) and with glutamine and telaglenastat and harvested at 0, 48 and 96 hours were immunoblotted for c-CBL, FLT3, p-STAT5, STAT5 and vinculin expression, and bands were quantified by densitometry. Immunoblots and densitometric analysis are shown. B. MOLM-14 cells and primary FLT3-ITD AML cells from two patients were cultured in hypoxia in the presence of glutamine with telaglenastat and the FLT3 inhibitors gilteritinib or quizartinib at diverse concentrations. Drug combination effects were analyzed using the Chou-Talalay method. Combination index <1, =1 or >1 indicated synergy, additivity and antagonism, respectively. Synergistic effects were seen.

Article Snippet: The human FLT3-ITD AML cell lines MV4-11 and MOLM-14, with homozygous and heterozygous FLT3-ITD, respectively (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM L-glutamine, unless otherwise indicated, and were tested for Mycoplasma every six months ( ).

Techniques: Cell Culture, Expressing, Western Blot