Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6

Abstract

Up to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD(+) acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration-approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors.

Figure 1

Focused chemical genetic screen reveals sensitivity of FLT3-mutant AML cell lines to several FDA-approved compounds. (A) Heat map shows treatment response of FLT3-ITD (MOLM-14 and MV4-11) or FLT3 wild-type (WT) (THP-1, ML-2, KU812, and K562) leukemic cells. Viability measurements were conducted by the CellTiterGlo (CTG) Viability Assay. For full data set, see supplemental Figure 1A. Blue, sensitivity; red, resistance. (B) Significance of viability difference between FLT3 WT and ITD⁺ cells upon drug exposure. (C) Dose-response curve of ITD⁺ (red) or control (black) leukemic cells with CDK4/6 inhibitor palbociclib. Cells were incubated with increasing concentrations for 72 hours. Cell viability and proliferation were assessed by using the CTG assay. IC₅₀ values were calculated by using GraphPad Prism software. Error bars indicate ± SEM.

Figure 2

Palbociclib selectively and potently induces apoptosis in FLT3-ITD leukemic cells. (A-B) Cells were incubated with palbociclib (1 µM) for 72 hours, stained with propidium iodide, and analyzed by flow cytometry. Treatment induces apoptotic sub-G₁ fraction in FLT3-mutant cells (MOLM-14, PL-21, and MV4-11) but not in control cells (THP-1 and NOMO-1). (A) Representative dot blots and (B, lower panel) 1 representative histogram are depicted. (B, upper panel) Bar graphs show distribution of indicated cells in sub-G₁ fraction. (C) Palbociclib (1 µM)-induced apoptosis was evaluated on day 4 by labeling indicated cells with annexin V/7-aminoactinomycin D (7-AAD) via fluorescence-activated cell sorting analysis. The percentage of cells in the upper left quadrant denotes cells that stained positive for annexin V only (early apoptosis). The cells in the upper right quadrant stained positive for annexin V and 7-AAD (late apoptosis). The percentage of cells in the lower right quadrant represents cells that stained positive for 7-AAD only (necrosis). Three independent experiments were carried out. Error bars indicate ± SEM. **P < .001. n.s., not significant. PI-A, propidium iodide area; PI-W, propidium iodide width.

Figure 3

CDK6 but not CDK4 binds the promoter of the FLT3 gene and regulates FLT3 transcription in a kinase-dependent manner. (A-B) Inhibition of FLT3 protein expression with CDK4/6 inhibitor palbociclib at indicated concentrations in a time-dependent manner is depicted. Cells were harvested (A) between 24 and 120 hours or (B) at 48 hours. Cell lysates were subjected to western blot analysis for total FLT3. β-actin was used as loading control. (C) Cells were incubated with increasing concentrations of palbociclib. A time- and dose-dependent decrease in FLT3 phosphorylation at residue Y591 and in STAT5 phosphorylation at residue Y694 was detected by immunoblotting. (D) Palbociclib inhibits FLT3-dependent signaling in a dose-dependent manner. MOLM-14 cells were incubated with palbociclib at indicated concentrations for 4 days. Total cell lysates were immunoblotted with the indicated antibodies: total FLT3, total STAT5, phospho-STAT5, and total MYC. (E) PIM1 gene expression was analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) in FLT3-mutant (MOLM-14, MV4-11, and PL-21) and FLT3-WT (THP-1 and NOMO-1) cell lines after palbociclib (1 µM) administration for 72 hours. Relative PIM1 expression was normalized to the housekeeping gene RPLP0. (F) Effects of individual CDK4 and CDK6 suppression on FLT3 protein levels. (G) FLT3 gene expression was analyzed by quantitative RT-PCR in indicated cell lines after palbociclib (1 µM) administration for 72 hours. Relative FLT3 expression levels were normalized to RPLP0 mRNA. (H-I) Chromatin immunoprecipitation (ChIP) experiments were performed in (H) a murine HPC7 hematopoietic progenitor cell line and in (I) indicated human AML cells. Protein-DNA complexes were immunoprecipitated by using (H) home-made sera against Cdk6 or (I) by using a commercial anti-CDK6 antibody and were analyzed by quantitative PCR (qPCR) for their presence on the FLT3 promoter region. EGR1, p16^INK4a, and VEGF-A promoter regions served as positive controls. Bar graphs depict fold enrichment over a negative region as described in the supplemental Data. *P < .05; **P < .01; ***P < .001; ****P < .0001. shRNA, short hairpin RNA.

Figure 4

Combined CDK6 and FLT3 kinase inhibition reveals synergistic effects. (A) Cells were sensitized to palbociclib administration by a single dose of FLT3 inhibitor TCS-359 for 3 days. Cell viability and proliferation were assessed by using the CTG assay. Analysis was carried out in triplicate. Error bars indicate ± SEM. A one-way analysis of variance was used for statistical comparison. (B-C) Combined effects of palbociclib with different FLT3 inhibitors tested (TCS-359, quizartinib, and tandutinib) exceeds Bliss prediction indicating synergy. Dose-response surfaces are centered on the half maximal effective concentration (EC₅₀) of each compound in the MOLM-14 cells (B, upper panel). Analysis was carried out in triplicate. Values depicted represent absolute deviations. Observed values were divided through standard deviations (SDs) plus 15th percentile. Needle graphs indicated deviation from Bliss predicted additivity in AML cells carrying mutant FLT3 kinase (MOLM-14) (B, lower panel and C). (D) Potential synergistic drug combination was evaluated in MOLM-14 cells by isobologram analysis using CompuSyn software. The obtained combination index values (<1) indicated synergy. Analysis was performed in triplicate. (E) Dose-response curve with FLT3 inhibitor TCS-359 alone or in the presence of 30 nM palbociclib (based on the isobologram analysis) in the MOLM-14 cell line. Three independent experiments were carried out. Error bars indicate ± SEM. ****P < .0001.

Figure 5

Pharmacologic CDK6 blockade reduces the clonogenicity of primary ITD⁺ AML patient biopsies. (A) Fold change in FLT3 gene expression upon pharmacologic CDK6 inhibition (#1, 1 µM; #2, 0.3 µM) relative to vehicle computed from qPCR experiments in primary patient CD34⁺ cells. (B) FLT3-ITD AML patient material was subjected to palbociclib (#1, 3 µM; #4, 1 µM; #2, 0.3 µM), stained with FLT3 phycoerythrin antibody, and analyzed by flow cytometry for FLT3 mean fluorescence intensity. (C) Viability measurements upon CDK6 kinase inhibition (1 µM) were conducted by using the CTG Assay. Analysis was carried out in triplicate. Two-tailed unpaired Student t test was used for statistical comparison. (D) Patient AML samples (n = 6) were embedded in methylcellulose with recombinant cytokines and erythropoietin (MethoCult H4434) in the presence of CDK6 inhibitor (palbociclib) or FLT3 kinase inhibitor (TCS-359). Colonies were counted 10 days after seeding. Representative data are depicted (magnification: ×4). *P < .05; **P < .01; ***P < .001; ****P < .0001. n.s., not significant.

Figure 6

CDK6 directly regulates PIM1 kinase. (A-B) ChIP assays were performed in (A) murine HPC7 hematopoietic progenitor lines and in (B) indicated human AML cell lines as described in Figure 3H-I. (C) PIM1 gene expression was analyzed by quantitative RT-PCR in primary CD34⁺ cells bearing FLT3-ITD after palbociclib (#1, 1 µM; #2, 0.3 µM) administration. Relative PIM1 expression levels were normalized to RPLP0 mRNA. (D) Combined effects of palbociclib with PIM1 inhibitor SGI-1776 free base exceeds Bliss prediction indicating synergy. Dose-response surfaces are centered on the EC₅₀ of each compound in the MOLM-14 cells (upper panel). Analysis was carried out in triplicate. Values depicted represent absolute deviations. Observed values were divided through SDs plus 15th percentile. Needle graphs indicate deviation from Bliss-predicted additivity in FLT3-ITD–expressing AML cells (MOLM-14) (lower panel). (E) Dose-response curve with PIM1 inhibitor SGI-1776 free base alone or in the presence of 10 nM palbociclib (based on the Bliss prediction) in the MOLM-14 cell line. *P < .05; **P < .01.

Figure 7

CDK6 is required for FLT3-ITD–driven tumor formation and leukemogenesis in vivo. (A) Kaplan-Meier plot depicting disease onset of immune-compromised Rag2^−/−γc^−/− recipients injected with FLT3-ITD⁺ cells (MOLM-14). On day 5 after engraftment, mice were randomly divided into 2 groups and dosed once per day with vehicle (n = 5) or palbociclib (n = 6). Mean survival with vehicle, 43 days; with palbociclib, 73.5 days. Log-rank test was used for statistical comparison. (B) Bone marrow–infiltrating MOLM-14 cells isolated from diseased mice (n = 3 for each group) shown in (A) were analyzed for human PIM1 and FLT3 gene expression upon treatment with either vehicle or palbociclib. (C-D) FLT3-ITD⁺ (MOLM-14) cells were subcutaneously (s.c.) injected into both flanks of immune-compromised Rag2^−/−γc^−/− recipients. Mice were treated once per day with vehicle or palbociclib on (C) day 0 (n = 4 mice for each group) or on (D) day 5 (vehicle, n = 3 mice; palbociclib, n = 2 mice) until terminal workup at day 12. (E) Human PIM1 and FLT3 gene expression was analyzed by quantitative RT-PCR in subcutaneously grown tumors shown in (D) after treatment with either vehicle or palbociclib. (F) Scheme of the mechanism of action of palbociclib in FLT3-ITD leukemic cells: blockade of CDK6 kinase activity upon palbociclib exposure impairs cell cycle progression from G₁ phase to S phase and inhibits transcription of FLT3 and PIM1 leading to survival inhibition. *P < .05; **P < .01.