Effective targeting of quiescent chronic myelogenous leukemia stem cells by histone deacetylase inhibitors in combination with imatinib mesylate

Abstract

Imatinib mesylate (IM) induces remission in chronic myelogenous leukemia (CML) patients but does not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. Here we investigated the ability of HDAC inhibitors (HDACis) to target CML stem cells. Treatment with HDACis combined with IM effectively induced apoptosis in quiescent CML progenitors resistant to elimination by IM alone, and eliminated CML stem cells capable of engrafting immunodeficient mice. In vivo administration of HDACis with IM markedly diminished LSCs in a transgenic mouse model of CML. The interaction of IM and HDACis inhibited genes regulating hematopoietic stem cell maintenance and survival. HDACi treatment represents an effective strategy to target LSCs in CML patients receiving tyrosine kinase inhibitors.

Figure 1. Effect of IM and LBH treatment on HDAC activity and BCR-ABL expression and activity in CML CD34+ cells

CD34+ cells from newly-diagnosed CML patients were cultured in the presence of LBH and IM for 24 hours. (A) Histone proteins were extracted and analyzed by Western blotting for levels of acetylated and total Histone H3 and H4. (B) Total proteins were extracted and analyzed by Western blotting for BCR-ABL, P-CrkL, phospho-tyrosine, p27 and p21 and actin. Results for 3 CML samples are shown. See also Figure S1.

Figure 2. Apoptosis and proliferation of CML and CB CD34+CD38− primitive progenitors and CD34+CD38+ committed progenitors following IM and LBH treatment

CD34+ cells from newly diagnosed CML patients (n=3) and CB (n=3) were labeled with CFSE and CD34+CD38− and CD34+CD38+ populations selected by flow cytometry sorting. Cells were exposed to LBH (25nM or 50nM), with or without IM (1μM) for 96 hours. Apoptosis was analyzed by flow cytometry as the percentage of cells labeled by Annexin V-PE. Results for CML (A) and normal (B) cells are shown. Apoptosis of undivided cells for samples co-treated with LBH and 1μM imatinib are shown for CML CD34+CD38− and CD34+CD38+ cells in (C) and for normal cells in (D). Cell divisions were analyzed by flow cytometry based on reduction in CFSE intensity, and a proliferation index was determined using ModFit software. Proliferation indices were normalized to untreated controls. Results for CML (E) and normal (F) cells are shown. Results shown represent Mean±SEM. P values: * p<0.05, ** p<0.01, ***p<0.001, compared with untreated cells. See also Figure S2.

Figure 3. The combination of LBH and IM eliminates CML cells capable of long-term multilineage engraftment in immunodeficient mice

CD34+ cells from CML patients (1×10⁶ – 2×10⁶ cells per mouse) and normal BM (1×10⁵– 8×10⁵ cells per mouse) were injected into sublethally irradiated (300cGy) NSG mice. Human cell engraftment measured in peripheral blood samples taken from mice (n=9 for CML; n=7 for normal) at 4 week intervals by flow cytometric assessment of human CD45+ cell engraftment is shown in (A). BM cells were obtained from femurs of mice 16 weeks after transplantation (n=6 for CML; n=7 for normal) and specific human cell subsets were detected by staining with antibodies to human CD34, CD33, CD11b, and CD19 as shown in (B). CML CD34+ cells (2×10⁶ cells per mouse, 8 mice per condition) and normal BM CD34+ cells (1×10⁵ cells per mouse, 7 mice per condition) were cultured for 96 hours in the absence of drug (control), with IM (1μM) alone, LBH (50nM) alone, or IM in combination with LBH and then transplanted into NSG mice. Mice were euthanized after 16 weeks and PB, BM and spleen cells were analyzed by flow cytometry. Representative results of engraftment in BM for CML are shown in (C) and for CB in (G). Combined results for engraftment of cells in BM for CML are shown in (D) and for CB in (H). Results shown represent the mean ± SEM for multiple samples. Human CD45+ cells were enriched from BM of mice engrafted with CML CD34+ cells using immunomagnetic column selection. The percentage of human CD45+ cells present in column-selected cells were as follows: untreated 43.9±2.6%, IM 20.9±6.3%, LBH 5.5±1.5% and IM+LBH 0. (E) 1×10⁵ CD45+ selected cells were plated in CFC assay using human specific growth factors. (F) BCR-ABL mRNA levels in CD45+ selected cells were measured by Q-PCR. Results shown represent Mean±SEM. P values: * p<0.05, ** p<0.01, ***p<0.001, compared with untreated cells. See also Figure S3.

Figure 4. In vivo administration of LBH and IM inhibits myeloproliferation and induces apoptosis in LSC in transgenic BCR-ABL expressing mice

BCR-ABL expression was induced in Scl-tTa-BCR-ABL/GFP mice by tetracycline withdrawal. BM cells were obtained 4 weeks after induction and GFP expressing cells selected using flow cytometry were transplanted into wild-type FVB/N recipient mice irradiated at 900cGy (10⁶ cells/mouse). Treatment with IM (200mg/kg daily by gavage), LBH (30 mg/kg body weight intraperitoneally 3 days per week on Monday, Wednesday and Friday), or LBH in combination with IM or vehicle alone (controls) was initiated 4 weeks after transplantation and continued for 4 weeks. Apoptosis in BM LSK cells was evaluated 5 days after start of treatment by Annexin-V and DAPI labeling. Representative results are shown in (A) and compiled results from 3 experiments are shown in (B). To evaluate stem cell cycling, mice were injected intraperitoneally with EdU and sacrificed 2 hours later. The percentage of stem cells in S-phase was determined based on EdU incorporation in BM LSK cells measured by flow cytometry as described in the methods. Representative results from one of two such experiments are shown in (C). PB total WBC counts (D), neutrophil percentage and absolute neutrophil counts (E, F), and GFP+ WBC percentage and absolute counts (G, H) were measured 4 weeks after start of treatment. Results represent the mean ± SEM for 5–6 mice per treatment. P values: *p<0.05, **p<0.01, ***p<0.001, compared with no treatment.

Figure 5. LBH and IM administration profoundly depletes leukemia stem and progenitor cells in the BM and spleen of transgenic BCR-ABL expressing mice

Mice from the cohort described in Figure 4 were euthanized after 4 weeks of treatment with IM, LBH, IM combined with LBH or vehicle alone and BM and spleen cells obtained (A). GFP-expressing hematopoietic populations were analyzed by flow cytometry. Results for the following GFP+ populations in the BM (n=6 mice per treatment) are shown: total GFP+ WBC (B), immature myeloid cells (Gr-1+Mac-1+) (C), GMP (D), CMP (E), and LSC (LSK cells) (F). Representative flow cytometry plot for BM LSK cells is shown in (G). Results for GFP+ populations in the spleens (n=5 mice per treatment) are as follows: total GFP+ WBC (H), Gr-1+Mac-1+ cells (I), GMP (J), CMP (K), and LSK cells (L). A representative flow cytometry plot for splenic LSK cells is shown in (M). Results represent the mean ± SEM for multiple samples. Significance values: *p<0.05, **p<0.01, ***p<0.001, compared with no treatment. See also Figure S4.

Figure 6. Transplantation of BM from IM and LBH treated mice into secondary recipients

(A) BCR-ABL expression was induced in GFP+/SCLtTA/BCR-ABL mice and BM cells obtained from mice 4 weeks after induction. GFP expressing cells were selected and transplanted into wild-type FVB/N recipient mice (10⁶ cells/mouse). Treatment with IM, LBH, LBH in combination with IM or vehicle alone, was initiated 4 weeks after transplantation and continued for 4 weeks (8 mice per condition) as described for Figures 4 and 5. (B) Mice were followed after discontinuation of treatment and survival was monitored for 90 days. The total WBC count (C) and GFP+ WBC count (D) in PB of mice 8 weeks after discontinuation of treatment are shown. (E) BCR-ABL expression was induced in GFP+/SCLtTA/BCR-ABL mice and BM cells were obtained 4 weeks after induction. GFP expressing cells were transplanted into wild-type FVB/N recipient mice irradiated at 900cGy (10⁶ cells/mouse). Treatment with IM, LBH, and LBH in combination with IM or vehicle alone was initiated 4 weeks after transplantation and continued for 4 weeks (5 mice per condition). Mice were then euthanized and BM cells were obtained. The BM cells were pooled from 3–5 mice and 2×10⁶, 1×10⁶, 5×10⁵ cells/mouse (8 mice/dose/condition) mixed with 2×10⁵ BM cells/mouse from wild-type FVBN mice were transplanted into wild-type FVB/N recipient mice irradiated at 900cGy. Engraftment was monitored by drawing PB every 4 weeks. The percentage of GFP+ cells in PB was analyzed by flow cytometry. The mean ± SEM of GFP+ WBC at 8 and 16 weeks after transplantation are shown in (F). A percentage of GFP⁺ cells in PB ≥ 0.5% was considered positive for engraftment. The frequency of LSC after treatment is shown in (G). The fraction of mice showing evidence of engraftment at 16 weeks after secondary transplantation was shown in (H).

Figure 7. Gene expression changes induced by combined IM and LBH treatment

CD34+CD38− cells from 3 CML patients were selected using flow cytometry sorting and cultured with IM (1μM), LBH (50nM), the combination of IM and LBH, or without exposure to drugs (controls) for 24 hours. RNA was extracted, amplified, labeled and hybridized to Affymetrix HG U133 plus 2.0 Arrays. Microarray data analyses were performed and differentially expressed genes were identified as described in the methods. (A) The log₂ fold changes in gene expression after IM, LBH and IM+LBH treatment compared to controls are shown. The column labeled “interaction” shows the fold elevation or reduction of gene expression with the IM+LBH combination compared to the sum of the effects of IM given alone and LBH given alone. Genes shown are those whose expression is significantly altered as a result of the interaction between IM and LBH (p<0.01, fold change≥3). (B) GSEA was performed to detect enrichment of predetermined gene sets following IM, LBH and combination treatment. The normalized enrichment scores (NES) for these gene sets are shown. The column labeled “interaction” shows gene sets that are enriched for genes whose expression is significantly altered as a result of the interaction between IM and LBH. The highest ranked gene sets within the most common functional categories are displayed (FDR<0.1 with up to 10 gene sets per category). (C) A summary of regulatory mechanisms that are significantly affected by the interaction of LBH and IM is shown.