Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity

Abstract

Imatinib therapy, which targets the oncogene product BCR-ABL, has transformed chronic myeloid leukemia (CML) from a life-threatening disease into a chronic condition. Most patients, however, harbor residual leukemia cells, and disease recurrence usually occurs when imatinib is discontinued. Although various mechanisms to explain leukemia cell persistence have been proposed, the critical question from a therapeutic standpoint--whether disease persistence is BCR-ABL dependent or independent--has not been answered. Here, we report that human CML stem cells do not depend on BCR-ABL activity for survival and are thus not eliminated by imatinib therapy. Imatinib inhibited BCR-ABL activity to the same degree in all stem (CD34+CD38-, CD133+) and progenitor (CD34+CD38+) cells and in quiescent and cycling progenitors from newly diagnosed CML patients. Although short-term in vitro imatinib treatment reduced the expansion of CML stem/progenitors, cytokine support permitted growth and survival in the absence of BCR-ABL activity that was comparable to that of normal stem/progenitor counterparts. Our findings suggest that primitive CML cells are not oncogene addicted and that therapies that biochemically target BCR-ABL will not eliminate CML stem cells.

Figure 1. Inhibition of phosphotyrosine by imatinib in CML stem and progenitor cells.

(A) Intracellular phosphotyrosine levels were evaluated by FACS in Lin^– CML cells costained for CD34, CD38, and CD133. Representative phosphotyrosine histograms are shown in CML committed progenitor (CD34⁺CD38⁺) and stem cell (CD34⁺CD38^– and CD133⁺) populations treated for 4 hours with or without 5 μM imatinib (IM). FACS data are displayed on a logarithmic scale. Background signal was established in the same populations by staining with a matched isotype control. MFI ± SEM for phosphotyrosine signal of treated and untreated cells relative to isotype are shown (n = 10 [CML]; 3 [normal]). (B) Suppression of phosphotyrosine FACS signal by imatinib in primary CML stem and progenitor cell populations was compared with 3 BCR-ABL–expressing cell lines. Signal over isotype is shown as percent of untreated. A representative phosphotyrosine immunoblot is shown for the leukemic cell line Kcl22 treated or not with 5 μM imatinib. (C) Dose-dependent inhibition of BCR-ABL activity by imatinib in CD34⁺CD38⁺ and CD34⁺CD38^– cells was evaluated by phosphotyrosine FACS in 3 independent CML samples. Signals are graphed as percent of untreated.

Figure 2. Inhibition of phospho-CRKL by imatinib in CML stem and progenitor cells.

(A) Phospho-CRKL levels in normal CD34⁺ cells were compared with CML CD34⁺ cells treated or not with imatinib. Total CRKL and actin are shown as loading controls. (B) Phospho-CRKL and (C) phospho-STAT5 immunoblots with lysate from sorted CD34⁺CD38⁺ and CD34⁺CD38^– CML cells treated 4 hours with or without 5 μM imatinib demonstrated inhibition of BCR-ABL by imatinib in both populations. Total CRKL and total STAT5 are shown as respective loading controls. (D) Phospho-CRKL, phospho-STAT5, and phospho-AKT immunocytochemical stains of sorted CD34⁺CD38^– and CD34⁺CD38⁺ CML cells treated 4 hours with or without 5 μM imatinib demonstrated BCR-ABL inhibition in all cells within the population. Representative cells are shown. Identical data were obtained for a second sample (not shown).

Figure 3. Inhibition of phospho-CRKL by imatinib in quiescent and cycling CML cells.

(A) Lin^–Ki-67^– and Lin^–Ki-67⁺ CML cells were FACS sorted into quiescent and cycling fractions, respectively. Representative histograms are shown. (B) Immunoblots of Ki-67 and PCNA in sorted Lin^–Ki-67^– and Lin^–Ki-67⁺ CML cells were used to confirm sort purity, as indicated by exclusive expression of these proteins in the cycling fraction. Representative blots are shown. Actin is included as a loading control. (C) Ki-67 expression was evaluated by FACS prior to and following 4-hour culture with or without 5 μM imatinib. (D) Lin^– cells stained with CFSE and cultured for 72 hours with 5 μM imatinib were analyzed for Ki-67 expression. Ki-67 expression is shown in undivided (CFSE^hi) cells and in cells having undergone 1 or more divisions (CFSE^lo/mod). (E) Phospho-CRKL immunoblots of quiescent versus cycling cells treated 4 hours with or without imatinib are shown for 2 newly diagnosed CML samples. Total CRKL is included as a loading control.

Figure 4. Proliferation and survival of CML progenitor cells in short-term imatinib culture.

(A) Cell numbers of newly diagnosed CML or normal Lin^– cells with or without imatinib were monitored daily during a 4-day culture in the absence of cytokines or in the presence of a cytokine cocktail that included SCF, FLT3 ligand, GCSF, IL-3, and IL-6. Normalized cell counts (mean ± SEM) are plotted for CML (n = 10) and normal (n = 6) cells treated or not with 5 μM imatinib. (B) Apoptosis in untreated versus imatinib cultures was evaluated by annexin-V staining (n = 3, CML and normal). Mean ± SEM on day 4 is shown for cultures with no cytokines and the 5-cytokine cocktail. Moreover, imatinib-treated CML cells costained with annexin-V, Lin cocktail, CD34, and CD38 on day 4 (n = 3) showed differing apoptosis frequencies in different cell types. (C) Percent BCR-ABL⁺ cells were determined by FISH following culture with or without 5 μM imatinib. (D) Fold expansion of Lin^– cells (n = 3, CML and normal) with or without imatinib was evaluated following a 4-day culture in a cytokine cocktail that was serially diluted relative to the conditions in A. Error bars represent SEM.

Figure 5. Inhibition of BCR-ABL activity in cultured CML Lin^– cells.

(A) Phospho-CRKL and total phosphotyrosine levels in bulk cultured Lin^– cells were analyzed by immunoblot. Untreated cells, 5 μM imatinib–treated cells in which imatinib was washed out then cultured without imatinib prior to immunoblots (washout), and cells treated continuously with 5 μM imatinib are shown from a representative sample (n = 3). Lanes were run on the same gel but were noncontiguous (white lines). (B) Phosphotyrosine levels were analyzed by intracellular FACS analysis in bulk cultured Lin^– cells. Matched isotype was used to establish background staining in each population. Graphs represent mean ± SEM fluorescence signal relative to isotype control (n = 3).

Figure 6. Survival and expansion of different CML stem and progenitor cell immunophenotypic and functional subtypes following culture with imatinib.

(A) Relative frequencies of Lin^–, CD34⁺, CD34⁺CD38^–, and quiescent cells prior to and following 4-day CML Lin^– cell culture. Values are mean ± SEM percent of total population (n = 6 [CML]; 3 [normal]). (B) Absolute cell numbers (normalized relative to starting cell density) of Lin^–, CD34⁺, and CD34⁺CD38^–, and quiescent cells prior to and following culture with or without imatinib. Values are mean ± SEM (n = 6 [CML]; 2 [normal]). (C) Expansion of CD34⁺ and CD34⁺CD38^– CML cell numbers in the presence of imatinib shown for individual patient samples. (D) Phosphotyrosine levels were analyzed by intracellular FACS analysis in CD34⁺CD38^– and CD34⁺CD38⁺ cells following culture. Matched isotype was used to establish background staining in each population. Values are mean ± SEM fluorescence signal relative to isotype control (n = 3). (E) Colony-forming and LTC-IC assays of postculture CML progenitors (n = 3) were performed. CFCs were evaluated at 0, 3, and 6 weeks of culture on murine stromal cells. Mean ± SEM CFC numbers are shown as percent of untreated.

Figure 7. Sensitivity of CML stem and progenitor cells to imatinib, dasatinib, and nilotinib.

A) Lin^– CML cells were cultured for 4 days with SCF, FLT3 ligand, GCSF, IL-3, and IL-6 in the presence of imatinib, dasatinib, and nilotinib. Daily cell counts were used to compare cell proliferation for each inhibitor. A representative sample is shown. (B) BCR-ABL activity following culture with imatinib, dasatinib (das), and nilotinib (nil) was determined by phosphotyrosine FACS in CD34⁺ and CD34⁺CD38^– cells. Matched isotype was used to determine background levels. Mean fluorescence signal relative to background shown for a representative sample includes untreated cells, inhibitor-treated cells in which inhibitor was washed out prior to analysis (washout), and inhibitor-treated cells.

Figure 8. Model of CML disease persistence with imatinib treatment.

Orange cells represent CML cells in a BCR-ABL–active state. Gray cells, which indicate lack of BCR-ABL activity, are either normal (nonleukemic) cells or CML cells in which BCR-ABL is suppressed by imatinib treatment. Brown cells represent stroma, and small dark brown circles are secreted cytokines. (A) BCR-ABL activity drives expansion of primitive CML cells. (B) Imatinib treatment inhibits BCR-ABL activity in CML stem cells, restoring normal homeostasis and eliminating the proliferative advantage of leukemic cells. Normal hematopoiesis is reestablished; however, the bone marrow microenvironment supports survival of CML stem cells that are not oncogene addicted.