Research ArticleSIGNAL TRANSDUCTION

Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia

See allHide authors and affiliations

Science Advances  18 Sep 2015:
Vol. 1, no. 8, e1500221
DOI: 10.1126/sciadv.1500221
  • Fig. 1 Increased Pim kinase expression is found in sorafenib-resistant primary AML samples and confers resistance to FLT3 inhibition in vivo.

    (A) Left: Western blotting with Pim-1 and Pim-2 antibodies was performed on seven paired primary AML samples from patients before TKI treatment [naïve (N)] and after relapse (R) following sorafenib therapy. Right: FLT3-TKD sequencing (−, absence of mutation; +, detection of a D835 mutation) in naïve or resistant/relapsed (Res) AML samples. (B to F) Six- to 8-week-old B6 mice were transplanted with bone marrow (BM)–derived hematopoietic precursors transduced with FLT3-ITD (black), FLT3-ITD and human Pim-2 (FLT3-ITD/PIM2; gray), or control (white) constructs. Eight weeks after adoptive transfer, body weight (B), spleen weight (C), spleen cell counts (D), and peripheral blood counts (total leukocytes, monocytes, and platelets) (E) were determined, and immunohistochemical analysis of FLT3 and Pim-2 protein expression was performed on spleen sections (F) (n = 4 for each). (G and H) Peripheral blood counts (total leukocytes, monocytes, and platelets) from mice adoptively transferred with bone marrow precursors expressing FLT3-ITD (black bars) or FLT3-ITD/PIM2 (gray bars), treated or not with AC220 for 1 week (n = 4 for each). (I) Spleen weight relative to body weight in mice transduced with FLT3-ITD (white bars) or FLT3-ITD/PIM2 (black bars) and treated with vehicle or AC220. (J) Hemalun-Erythrosine-Safran (HES) and Ki-67 staining of spleen sections from (G) and (H). Results in the graphs are expressed as means ± SEM. *P < 0.05; **P < 0.01; ns, not significant. β-Actin was used as a loading control for all Western blots.

  • Fig. 2 Pim kinases are FLT3-ITD targets involved in resistance to FLT3 inhibition in AML.

    (A) AML cell lines (HL-60, OCI-AML3, MV4-11, and MOLM-14) were transduced via lentivirus with Dox-inducible anti-FLT3 shRNA vectors. shRNA induction was achieved with Dox (200 ng/ml). Western blots were performed using FLT3, Pim-1, and Pim-2 antibodies. WT, wild type. (B) MOLM-14 and OCI-AML3 cells were cultured with FLT3-L and/or 5 nM AC220. Tyrosine phosphorylation was evaluated in FLT3 immunoprecipitates. Pim-1, Pim-2, phospho-STAT5 (Y694), and STAT5 levels were detected in whole-cell lysates by immunoblotting. (C) MOLM-14 cells were treated for 24 hours with 5 nM AC220 (left), and MOLM-14 and MV4-11 cells were transduced with inducible shFLT3 and treated with Dox (200 ng/ml) for 48 hours (right). Pim-1 and Pim-2 mRNA levels were quantified by real-time polymerase chain reaction. Gene expression was normalized to the HPRT (hypoxanthine-guanine phosphoribosyltransferase) levels (n = 3). (D) STAT5A/B gain (right) or loss (left) of function in MOLM-14 cells transduced with lentivirus. STAT5A, STAT5B, Pim-1, Pim-2, and Bcl-xL protein levels were evaluated by immunoblotting. (E) MOLM-14 cells were separately transduced with a PIM1- or PIM2-expressing vector or an empty vector. Ectopic expression of Pim-1 and Pim-2 was verified by immunoblotting (right). Cells were treated with vehicle or 1 nM AC220 for 48 hours, and cell viability was assessed by an UptiBlue assay (left) (n = 3). (F) MOLM-14 cells expressing an FLT3 shRNA in a Dox-inducible manner were transduced with a Pim2 (murine Pim-2) allele or a control vector. After 48 hours of culture in the presence of Dox (200 ng/ml), FLT3 and murine Pim-2 expression was assessed by immunoblotting, and apoptosis was measured by annexin V staining. (G and H) MOLM-14 cells were transduced via lentivirus with a control vector (Dox-inducible anti–Pim-2 shRNA vector) or with Pim2 lentivirus and xenografted into nude mice. (E) Tumor growth was assessed in mice treated with AC220 (1 mg/kg) (initiated once the tumor size reached 100 mm3) with (black circle) or without (white circle) Pim2 transduction. Growth of untreated xenotransplanted MOLM-14 cells is also provided (white square box). The means of the individual tumor sizes are plotted (n = 8). (F) Kaplan-Meier survival curve analysis of MOLM-14 cells transduced (dashed line) or not (solid line) with Pim2, transplanted into nude mice, and treated with AC220 (n = 8). Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blots. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

  • Fig. 3 Pim kinases, with a predominant implication of Pim-2, regulate FLT3i-naïve and FLT3i-resistant AML cell survival.

    (A) AML cell lines (MOLM-14, MV4-11, HL-60, OCI-AML3, and THP-1) were transduced via lentivirus with a vector promoting the expression of an anti–Pim-2 shRNA after induction with Dox (200 ng/ml). Apoptosis was quantified by annexin V staining after 4 days of shRNA induction (n = 3). The extent of Pim-2 knockdown in each cell line was determined by Western blot (bottom). (B) Western blot for Pim-2 expression (top) and annexin V staining in normal CD34+ hematopoietic progenitor cells lentivirally transduced with scrambled (−) or Pim-2 (+) shRNA (n = 3). (C) Pim-2 shRNA–transduced MOLM-14 cells were cotransfected with Pim2 or Pim2KD as indicated. Cell viability after Dox treatment was assessed by staining with the UptiBlue fluorescent reagent (n = 6). (D to G) Tumor growth and survival (Kaplan-Meier curve) of MOLM-14 cells transfected with scrambled or Pim-2 shRNA and xenografted into nude mice. Animals were treated with vehicle [phosphate-buffered saline (PBS), black line, n = 8] or Dox (200 μg/ml) (Pim-2 shRNA in red, scrambled shRNA in blue; n = 8 for each). (H) Tumor sections stained by HES and TUNEL or labeled with Pim-1, Pim-2, and phospho-4E-BP1 (S65) antibodies. Representative images from three separate experiments are shown. (I) MOLM-14 cells were harvested from AC220-treated xenografted mice as depicted in Fig. 2, E and F. Pim-2 knockdown was induced with Dox (200 ng/ml) for 24 hours, and 2 nM AC220 was then added to the culture for an additional 24 hours. Apoptosis was quantified by annexin V staining. Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blots. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 Pim kinase inhibition directly facilitates FLT3-ITD receptor blockade by AC220.

    (A) Ba/F3 cells expressing FLT3-ITD, Pim2, and Pim2KD alleles as single or combined transfectants as indicated were stained with Fluo-4 AM and treated or not with FLT3-L (30 ng/ml). Variations in intracellular calcium concentrations ([Ca2+]i) were evaluated. Fluorescence (485-nm excitation/516-nm emission) was acquired over time to evaluate the kinetics of response. Variations are expressed as differences between the baseline and experimental [Ca2+]i elevations (ΔF/F0) (left), and [Ca2+]i elevations (ΔF/F0) are expressed as the mean area under the curves [change in relative fluorescence units (ΔRFU)] (right). Results are expressed as the mean of at least four independent samples. Thapsigargin (10 μM) was used as a control for calcium mobilization. (B) Protein extracts from MOLM-14 cells expressing a control vector, Pim2, or Pim2KD were assessed for FLT3 tyrosine phosphorylation (after FLT3 immunoprecipitation) as well as STAT5 (Y694) phosphorylation and Pim-2 and STAT5 expression by immunoblotting. (C) Parental Ba/F3 cells and Ba/F3 cells expressing FLT3-ITD or FLT3-ITD-D835Y alleles were treated with vehicle or 1 μM LGB321 for 1 hour, and calcium flux was measured as detailed in (A). (D) STAT5, Pim-2, and FLT3 recombinant proteins were mixed together in a kinase buffer without or with 1, 2, 5, 10, or 50 nM AC220 and without or with 1 μM LGB321 for 1 hour. Then, 200 μM ATP was added for 30 min, and proteins were solubilized in Laemmli buffer and analyzed by immunoblotting with a phospho-STAT5 Y694 antibody. A representative Western blot is provided (top). Signal intensity was quantified using MultiGauge software (Fujifilm), and results are presented with AC220 concentrations given in log scale and using the log(inhibitor) versus response variable slope (four parameters) function of GraphPad v6 software. Results of IC50 for STAT5 phosphorylation without (−) or with (+) LGB321 are provided (bottom) (n = 3). (E) Schematic representation of FLT3-ITD receptors and of Pim kinase consensus S935 site with either nonphosphomimetic or phosphomimetic amino acid substitutions. (F and G) Ba/F3 cells were transduced with FLT3-ITD receptors either unmodified (ITD) or harboring nonphosphomimetic (ITD-S935A) or phosphomimetic (ITD-S935D) amino acid substitutions. (F) Western blotting with phospho-FLT3 (Y591), phospho-ERK (T202/Y204), phospho-STAT5 (Y694), FLT3, STAT5, and ERK antibodies. (G) Ba/F3 cells were cultured for 48 hours with vehicle or 5 nM AC220. Apoptosis was measured by annexin V binding. Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blotting experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 Combined inhibition of Pim and FLT3 eradicates FLT3-mutated cells.

    (A) Schematic representation of FLT3i resistance-conferring mutations within the FLT3-ITD kinase domain. (B) Cell viability measured by a CellTiter-Glo assay in FLT3-ITD-D835Y–transduced Ba/F3 cells treated with log dilutions of AC220 without or with 3.2 nM LGB321. Relative luminescence (RLU) was normalized to vehicle-treated cells for each condition (normalized RLU). Results are presented using the log(inhibitor) versus response variable slope (four parameters) function of GraphPad v6 software. The IC50 values for this assay are provided (bottom) (n = 3). (C) Ba/F3 ITD-D835Y cells were cultured without or with 5 nM AC220 and/or 1 μM LGB321 for 4 hours. Western blots were done with phospho-STAT5 (Y694), phospho-ERK (T202/Y204), phospho-P70S6K (T389), STAT5, ERK, and P70S6K antibodies. (D) Ba/F3 cells expressing FLT3-ITD or FLT3-ITD-F691L alleles were cultured for 48 hours with 1 μM LGB321 and increasing doses of TKIs (AC220, sorafenib, PKC412, and crenolanib), as indicated. Apoptosis was determined by annexin V staining (n = 3 for each). (E and F) Ectopic expression of FLT3-ITD or FLT3-ITD-D835Y alleles with a lentiviral vector in MOLM-14 cells. (E) Western blotting with a Pim-2 antibody, using protein extracts from MOLM-14-ITD or MOLM-14-ITD-D835Y cells treated with vehicle or 5 nM AC220 for 24 hours. (F) Cells were treated with vehicle, 1 μM LGB321, 5 nM AC220, or the combination of LGB321 and AC220 for 48 hours, and apoptosis was quantified by annexin V staining. (G and H) AML cell lines (MOLM-14, MV4-11, OCI-AML2, OCI-AML3, and THP-1) (G) and primary FLT3-ITD+ (n = 7) or FLT3-WT (n = 3) AML samples (H) were cultured with vehicle, 1 μM LGB321, 5 nM AC220, or the combination of LGB321 and AC220 for 48 hours, and annexin V staining was measured. (I) Explanatory model. Baseline: FLT3-ITD receptors (ITD) constitutively activate prosurvival signaling pathways including Pim-1 (through STAT5 activation) and Pim-2 (through an unknown STAT5-independent mechanism). FLT3i inhibit FLT3 activity and induce cytotoxicity. FLT3i resistance: Increased Pim kinase expression is found in FLT3i-resistant AML cells. Pim kinases have intrinsic oncogenic properties in favor of AML cell survival. They also induce direct FLT3-ITD receptor modifications (including S935 phosphorylation) contributing to FLT3i resistance. Pim/FLT3 inhibition: Genetic or pharmacological Pim kinase inhibition restores FLT3i activity against FLT3-ITD receptors, leading to synergistic cytotoxicity. β-Actin was used as a loading control for all Western blotting experiments. Results are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Table 1 Clinicopathologic characteristics of seven patients with FLT3-ITD+ AML treated on sorafenib monotherapy trial.

    G, gender; A, age (in years); FAB, French-American-British classification; WCC, white cell count (109/liter); Blast, percentage of bone marrow blast cells; NPM1, nucleophosmin 1 mutation status; Karyo., karyotype; FLT3-TKD, FLT3 tyrosine kinase domain mutation status; Naïve, pre-sorafenib sample; Res., sample collected after disease evolution upon sorafenib treatment; NS, not specified; NA, not available; WT, wild type; Mut, mutated; 7 + 3, daunorubicin and cytarabine; HDAC, high-dose cytarabine; MTZ/VP16/AC, mitoxantrone, etoposide, and cytarabine; MACE-DEX, mitoxantrone, cytarabine, etoposide, and dexamethasone; ICE, idarubicin, cytarabine, etoposide; CLARA, clofarabine, cytarabine; MTZ/MDAC, mitoxantrone, medium-dose cytarabine; 5AZA, 5-azacytidine; Y and N, presence or absence of TKD mutations in sorafenib-naïve and sorafenib-resistant samples.

    PatientG/AFABWCCBlastNPM1Karyo.Previous treatmentsFLT3-TKD*
    NaïveRes.
    AML#1M/54NS16781NAComplex7 + 3, HDAC×2, MACE-DEXNN
    AML#2M/34M626 (75)WT46, XY7 + 3, ICE, CLARA, MTZ/MDAC, 5AZANY
    AML#3F/57M11954WT46, XX7 + 3, MTZ/MDAC×3NN
    AML#4§F/47NS2578Mut.46, XX7 + 3, ICE×2NY
    AML#5F/87M126094NA46, XXNot eligibleNY
    AML#6F/43NS1855WTComplex7 + 3, HDAC×2, MTZ/VP16/ACNN
    AML#7F/67M110295NA46, XX7 + 3, HDAC×2, 5AZANY

    *D835 mutation of FLT3-TKD was examined as described (18) on peripheral blood cells. †42~48,XY,del(5)(p13),+del(6)(q16),del(12)(p11.2),-14,-15,-17,t(17;19)(q21;p13),-20,+1~6mar[cp8]/46,XY[7]. ‡The percentage of blast cells of bone marrow was 75% among the nucleated cells. §The clinical information and the FLT3-TKD status of patient AML#7 have been reported (18). ¶46,XX,i(17)(q10)[6]/46,XX,del(5)(q34)[3]/46,XX,add(11)(p11.2)[2]/46,XX,del(11)(15.2)[2]/46,XX[20].

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/8/e1500221/DC1

      Fig. S1. Pim kinase expression in AML.

      Fig. S2. Pim-2 regulation by downstream FLT3-ITD receptors.

      Fig. S3. Pim-1 and Pim-2 regulation by FLT3-ITD receptors.

      Fig. S4. Direct phosphorylation of FLT3 receptors by Pim-2.

      Fig. S5. Dual inhibition of FLT3 and Pim kinases produces synergistic cytotoxicity in AML.

      Table S1. Genotyping of AML cell lines used in the current study.

      Table S2. References of the antibodies used in the current study.

      Materials and Methods

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Pim kinase expression in AML.
      • Fig. S2. Pim-2 regulation by downstream FLT3-ITD receptors.
      • Fig. S3. Pim-1 and Pim-2 regulation by FLT3-ITD receptors.
      • Fig. S4. Direct phosphorylation of FLT3 receptors by Pim-2.
      • Fig. S5. Dual inhibition of FLT3 and Pim kinases produces synergistic cytotoxicity in AML.
      • Table S1. Genotyping of AML cell lines used in the current study.
      • Table S2. References of the antibodies used in the current study.
      • Materials and Methods

      Download PDF

      Files in this Data Supplement:

    Stay Connected to Science Advances

    Navigate This Article