Molecular Interaction Between the Microenvironment and FLT3/ITD+ AML Cells Leading to the Refractory Phenotype

Internal tandem duplication mutations in the FLT3 gene (FLT3/ITD) are detected in 10–15% of children and 30% of adult patients with AML and are associated with an extremely poor prognosis. Although several antagonists against FLT3/ITD have been developed, few of them are effective for the treatment of FLT3/ITD+ AML because of the emergence of drug-resistant cells. The mechanisms responsible for drug resistance include the acquisition of additional mutations in the FLT3 gene and/or activation of other prosurvival pathways such as microenvironment-mediated resistance. Recent studies have strongly suggested that the reciprocal interaction between the microenvironment and AML cells generates specific machinery that leads to chemoresistance. This chapter describes the molecular mechanism responsible for the refractory phenotype of FLT3/ITD+ AML cells resulting from the communication between the microenvironment and FLT3/ITD+ leukemia cells. Understanding this mechanism enables the discovery of novel and innovative therapeutic interventions for resistant FLT3/ITD+ AML

Internal Tandem Duplication in FLT3 Attenuates Proliferation and Regulates Resistance to the FLT3 Inhibitor AC220 by Modulating p21Cdkn1a and Pbx1 in Hematopoietic Cells

Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression

Internal tandem duplication mutations in FLT3 gene augment chemotaxis to Cxcl12 protein by blocking the down-regulation of the Rho-associated kinase via the Cxcl12/Cxcr4 signaling axis

Internal tandem duplication mutations in the Flt3 gene (ITD-FLT3) enhance cell migration toward the chemokine Cxcl12, which is highly expressed in the therapy-protective bone marrow niche, providing a potential mechanism underlying the poor prognosis of ITD-FLT3(+) acute myeloid leukemia. We aimed to investigate the mechanisms linking ITD-FLT3 to increased cell migration toward Cxcl12. Classification of the expression of Cxcl12-regulated genes in ITD-FLT3(+) cells demonstrated that the enhanced migration of ITD-FLT3(+) cells toward Cxcl12 was associated with the differential expression of genes downstream of Cxcl12/Cxcr4, which are functionally distinct from those expressed in ITD-FLT3(-) cells but are independent of the Cxcr4 expression levels. Among these differentially regulated genes, the expression of Rock1 in the ITD-FLT3(+) cells that migrated toward Cxcl12 was significantly higher than in ITD-FLT3(-) cells that migrated toward Cxcl12. In ITD-FLT3(-) cells, Rock1 expression and Mypt1 phosphorylation were transiently up-regulated but were subsequently down-regulated by Cxcl12. In contrast, the presence of ITD-FLT3 blocked the Cxcl12-induced down-regulation of Rock1 and early Mypt1 dephosphorylation. Likewise, the FLT3 ligand counteracted the Cxcl12-induced down-regulation of Rock1 in ITD-FLT3(-) cells, which coincided with enhanced cell migration toward Cxcl12. Rock1 antagonists or Rock1 shRNA abolished the enhanced migration of ITD-FLT3(+) cells toward Cxcl12. Our findings demonstrate that ITD-FLT3 increases cell migration toward Cxcl12 by antagonizing the down-regulation of Rock1 expression. These findings suggest that the aberrant modulation of Rock1 expression and activity induced by ITD-FLT3 may enhance acute myeloid leukemia cell chemotaxis to the therapy-protective bone marrow niche, where Cxcl12 is abundantly expressed

FLT3-ITD Attenuates the Growth Factor-independent Proliferation and Cell Cycle Progression of Hematopoietic Progenitor Cells by Up-regulating p21^Cdkn1a Expression.

<p>(A) Growth factor-independent proliferation of CFCs in mouse bone marrow cells lacking p21 or Survivin and transduced with <i>FLT3</i>-ITD. Bone marrow cells from Survivin^fx/fx, Cre-ER Survivin^fx/fx, p21^+/+ or p21^-/- mice were retrovirally transduced with wild-type <i>FLT3</i> or N51-<i>FLT3</i>-ITD. A total of 500,000 GFP⁺ cells were plated on either 0.3% agar or methylcellulose medium containing 30% FBS in the absence of hematopoietic growth factors. To delete the <i>Survivin</i> gene, cells from Cre-ER Survivin^fx/fx or Survivin^fx/fx mice were cultured in the presence of 1 μM 4-hydroxy tamoxifen at the time of plating. The colonies were enumerated after 14 days. The presented data are the averages of 9 experiments for p21 deletion and 2 experiments for Survivin deletion (P < 0.05). The numbers of CFCs generated by p21^+/- cells transduced with <i>FLT3</i>-ITD compared to those generated by p21^+/+ and p21^-/- cells are shown in the lower panel. The data shown were obtained from one of two experiments that yielded identical results. (B) The proliferation of bone marrow c-kit⁺, Sca-1⁺, lineage^- (KSL), c-kit⁺, lineage^- (KL), lineage^- (Lin^-) or lineage⁺ (Lin⁺) cells transduced with <i>FLT3</i>-ITD derived from p21^+/+ and p21^-/- mice. Following transduction, the GFP⁺ cells were sorted and cultured in 10% FBS/IMDM in the absence of hematopoietic growth factors. The cells were enumerated 7 days after culture. The fold change in the number of FLT3-ITD⁺ p21^-/- cells compared to the FLT3-ITD⁺ p21^+/+ cells is shown. The presented data are the averages of three experiments (P < 0.05). The cell number from one representative experiment is shown in the lower panel. (C) The effect of ectopic p21 expression on FLT3-ITD⁺ KSL cell proliferation. Bone marrow cells from control Survivin^fx/fx mice or Cre-ER Survivin^fx/fx mice were transduced with N51-<i>FLT3</i>-ITD and <i>p21</i> using a MIEG3 vector or a negative MIEG3 vector control. Following transduction, the GFP⁺ cells were cultured for 2 weeks in 10% FBS/IMDM containing 1μM 4-hydroxy tamoxifen at a density of 5 x 10⁵ cells/ml. The cells were stained for c-kit, Sca-1 and lineage markers to quantify the number of KSL cells. The data shown were obtained from one of two experiments that were analyzed in triplicate. (D) Ba/F3 cells expressing FLT3-ITD (N51) were transduced with either a <i>p21</i> shRNA or a control shRNA and were cultured in 1% FBS/RPMI without any growth factors. The viable cells were enumerated using the trypan blue exclusion assay. The data shown were obtained from one of two experiments that were performed in quadruplicate with identical results (*: P < 0.01). The expression levels of the p21 protein in both cell lines are shown in the inset. The lower panel shows a cell cycle histogram of FLT3-ITD⁺ Ba/F3 cells transduced with a control shRNA (left plot) and the p21 shRNA (right plot). The histogram represents one of three experiments with identical results. (E) Dose-dependent growth inhibition by Ara-C in N51-FLT3-ITD-Ba/F3 cells transduced with the <i>p21</i> shRNA and control cells. The cells were cultured in the presence of DMSO alone (control) or increasing doses of Ara-C in 1% FBS/RPMI-1640 without any growth factors for 24 hours. The y-axis represents the % inhibition of viable cells incubated with Ara-C compared to DMSO. The data shown represent one of three experiments with identical results.</p

Blocking FLT3-ITD Using AC220 Decreases p21 Expression Coincident with Emergence of FLT3-ITD⁺ Cells Refractory to AC220.

<p>(A) The panel shows FLT3 phosphorylation in N51-FLT3-ITD-Ba/F3 cells incubated with 1, 2 or 5 nM AC220 for 24 hours. The cells were stained for phospho-FLT3 using a rabbit monoclonal antibody raised against Tyr591 of human FLT3, followed by staining with a PE-conjugated anti-rabbit IgG secondary antibody and flow cytometry analyses. (B) The left panel indicates the numbers of viable N51-FLT3-ITD-Ba/F3 cells incubated with 1 or 2 nM AC220 compared to the DMSO control (0 nM). Cells plated at a density of 1 x 10⁵ cells/ml were incubated with 1 or 2 nM AC220 or control DMSO for 12 days. The right panel represents the number of viable cells that were incubated with 5 nM AC220 for 22 days. The medium was replaced every 5 days and contained 5 nM fresh AC220. The cells were enumerated using the trypan blue exclusion assay. The y-axis represents the cell number on the log scale. The data shown represent one of the three experiments that were analyzed in triplicate with identical results (*: P < 0.05 compared to time 0, †: P < 0.05 compared to 1 or 2 nM AC220). (C) The relative expression of p21 in N51-FLT3-ITD-Ba/F3 cells that were sensitive or refractory to AC220 and incubated with 2 nM AC220 compared to those incubated with the DMSO control. The refractory cells, which were established by incubation with 2 nM AC220, were washed and cultured for 48 hours in fresh medium without AC220. The cells were subsequently incubated with 2 nM AC220 for 48 hours. Similarly, parental cells that were sensitive to AC220 were incubated with 2 nM AC220 for 48 hours. The <i>p21</i> mRNA levels were quantitated before and after incubation with AC220 (*: P < 0.05, N = 3).</p

FLT3 Signaling Up-regulates p21^Cdkn1a Expression in Ba/F3 Cells.

<p>(A) Expression of p21 and p27 in Ba/F3-FLT3 cells before and after IL-3 withdrawal. The blots are from triplicate cultures. The total number of viable cells was enumerated at the time of harvest using the trypan blue exclusion assay, and lysates from 5x10⁵ viable cells were loaded into each lane. The p21/p27 ratio was determined by densitometry and is shown beneath the blot. (B) The number of viable Ba/F3-FLT3 cells stimulated with or without FL. Following IL-3 withdrawal for 16 h, the cells were resuspended in 10% FBS/RPMI-1640 at a concentration of 5x10⁵ cells/ml with FL concentrations ranging from 0 to 200 ng/ml. The number of viable cells was counted 24 and 48 h after incubation. P21 protein expression was analyzed by Western blotting at 48 h (top). The data are representative of two experiments with identical results. (C) Proliferation of Ba/F3 cells expressing wild-type or FLT3-ITD. The cells were washed three times and cultured for 72 h in 1% FBS/RPMI-1640, and the numbers of viable Ba/F3 cells expressing wild-type or FLT3-ITD derived from 3 different patients (N51, N73 and N78) were counted every 24 h (*: P < 0.05). The data shown are from one of three experiments that were performed in triplicate with identical results. (D) The upper panel shows the expression of the p21, Survivin and p27 proteins, as analyzed by Western blotting. The level of p21 relative to actin is shown beneath the blot. The middle panel shows the relative <i>p21</i> mRNA expression levels in Ba/F3 cells expressing wild-type or N51-FLT3-ITD, which were determined using quantitative RT-PCR. The presented data are the averages of two independent experiments (*: P < 0.05). The lower panel shows the <i>p21</i> mRNA expression levels in FLT3-ITD (N51) Ba/F3 cells incubated with 1 or 2 nM AC220 for 48 h compared to the control cells, which were incubated with dimethyl sulfoxide (DMSO) alone (N = 3, *: P < 0.05). (E) The blot shows p21 protein expression in RS4;11 cells and MV4-11 cells, as determined by Western blot analysis. (F) The <i>p21</i> mRNA expression levels in FLT3-ITD⁺ KSL cells compared to freshly isolated bone marrow KSL cells. C57BL/6 mouse bone marrow cells were retrovirally transduced with N51-<i>FLT3</i>-ITD using the MSCV-IRES-EGFP vector and were cultured for 2 weeks without hematopoietic growth factors. The GFP⁺ KSL cells were sorted by FACS, and <i>p21</i> mRNA expression was compared with freshly isolated KSL cells from the same donor. The presented data are the averages of three independent experiments (*: P < 0.005).</p

Suggested Roles of P21-mediated Inhibitory Signaling Pathways that Contribute to the Development of FLT3-ITD⁺ Cells that are Refractory to AC220.

<p>(A) FLT3-ITD stimulates various signaling molecules that promote cell proliferation or survival, such as MAP kinase, Akt, Stat5 and Survivin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158290#pone.0158290.ref020" target="_blank">20</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158290#pone.0158290.ref021" target="_blank">21</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158290#pone.0158290.ref030" target="_blank">30</a>]. The present study shows that FLT3-ITD also generates a growth-inhibitory signal through p21, which is partially mediated by Pbx1. Because the signaling activity that stimulates cell proliferation or survival dominates the p21-mediated inhibitory signal, the FLT3-ITD⁺ cells proliferate in the absence of a FLT3-ITD antagonist. (B) Antagonizing FLT3-ITD with AC220 can not only eradicate the growth-stimulatory signals but also disrupt the growth-inhibitory signals by inhibiting p21 expression. FLT3-ITD⁺ cells do not proliferate immediately after incubation with AC220. (C) Prolonged incubation of FLT3-ITD⁺ cells with AC220 allowed the cells to re-proliferate, indicating that the cells acquire resistance to AC220, which is most likely mediated by the activation of stimulatory signals and/or disrupting the growth-inhibitory signals. Our data show that antagonizing FLT3-ITD with AC220 can decrease p21 expression and functions as a growth-inhibitory signal. Although p21 inactivation is not sufficient to initiate proliferation and other pro-survival pathways are required, the lack of p21 facilitates the development and proliferation of AC220-refractory cells. The mechanism responsible for the activation of the pro-survival pathways includes additional mutations of the FLT3-ITD gene, microenvironment-mediated resistance, or autocrine or paracrine stimulation of FLT3-ITD by the FLT3-ligand [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158290#pone.0158290.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158290#pone.0158290.ref023" target="_blank">23</a>]. These data strongly suggest that the disruption of p21 expression by FLT3-ITD inhibition facilitates the development and proliferation of AC220-resistant FLT3-ITD⁺ cells. Treatments targeting FLT3-ITD can potentially contribute to the refractory phenotype of FLT3-ITD⁺ AML cells toward FLT3-ITD inhibitors by eradicating the growth-inhibitory signals.</p

Pbx1 Knockdown Abrogates the Enhanced Proliferation of FLT3-ITD⁺ HPCs Induced by p21 Deletion.

<p>(A) Bone marrow cells from p21^+/+ and p21^-/- mice were transduced with <i>FLT3</i>-ITD. Following the sorting of FLT3-ITD⁺ cells (using GFP as a marker) by FACS, the cells were transduced with a control shRNA or <i>Pbx1</i> shRNA (shRNA-1 or shRNA-2) using a pSINsi-mU6 plasmid, and the number of growth factor-independent CFCs grown in methylcellulose was quantified 14 days later. The data are shown as the fold changes in the number of CFCs compared to those of FLT3-ITD⁺ p21^+/+ cells transduced with the control shRNA (N = 3). Identical results were obtained after 1 week of culture. Quantitative RT-PCR was performed to compare <i>Pbx1</i> expression relative to <i>Hprt</i> in the <i>Pbx1</i> shRNA-transduced samples and p21^+/+ cells transduced with the control shRNA (inset). (B) FLT3-ITD⁺ Ba/F3 cells (N51) harboring a <i>p21</i> shRNA or control shRNA were transduced with a <i>Pbx1-</i>targeted shRNA or control shRNA. The cells were transfected with a p21 shRNA or a control shRNA and selected in the presence of 3 μg/ml puromycin for 2 weeks. The reduction in p21 expression was validated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158290#pone.0158290.g003" target="_blank">Fig 3D</a>. The cells were then transfected with the control shRNA or two different shRNAs for Pbx1 (Pbx1 shRNA-1 and Pbx1 shRNA-2) and selected with 1 mg/ml G418 for 2 weeks. The reduction in <i>Pbx1</i> mRNA expression was validated by Q-RT-PCR and shown in the inset (right). The cells were cultured in 1% FBS/RPMI without any growth factors for 10 days and enumerated using the trypan blue exclusion assay. The data shown represent one of three experiments that were performed in quadruplicate with identical results (*: P < 0.01 compared to the control shRNA paired with the <i>Pbx1</i> shRNA). The top inset indicates the fold change in the cell number on day 10 compared to the FLT3-ITD⁺ Ba/F3 cells containing two control shRNAs. The inset on the right demonstrates the fold change in the <i>Pbx1</i> mRNA expression in the FLT3-ITD⁺ Ba/F3 cells transduced with the <i>Pbx1</i> shRNA compared to those transduced with the control shRNA paired with the <i>Pbx1</i> shRNA (N = 3, P < 0.05).</p

Silencing p21 Accelerates the Emergence and Proliferation of FLT3-ITD⁺ Cells Refractory to AC220.

<p>(A) The numbers of viable N51-FLT3-ITD-Ba/F3 cells transfected with the <i>p21</i> shRNA or control shRNA were quantitated in the presence or absence of 2 nM AC220. The cells were plated at a density of 1x10⁵ cells/ml and incubated with 2 nM AC220 or control DMSO for 10 days. The viable cells were enumerated using the trypan blue exclusion assay. The data shown represent one of three experiments that were analyzed in triplicate with identical results (*: P < 0.05 compared to the control shRNA). The expression levels of the p21 protein in both cell populations are shown in the inset (the same blot is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158290#pone.0158290.g003" target="_blank">Fig 3D</a>). The right panel indicates the AC220 dose-dependent inhibition of the control FLT3-ITD⁺ cells containing empty vector and those with shRNA for p21. The cells were incubated with different concentrations of AC220 for 10 days, and the percent inhibition of viable cells was calculated compared to the control cells incubated with DMSO. (B) The panel indicates the numbers of viable FLT3-ITD⁺ cells containing the empty vector or p21 shRNA that were cultured in the presence of 2 nM AC220 for 60 days. The cells were plated at a density of 1x10⁵ cells/ml and were incubated with 2 nM AC220 or the DMSO control for 60 days. The medium was replaced every 5 days and contained 2 nM fresh AC220.</p