Targeting multiple kinase pathways in leukemic progenitors and stem cells is essential for improved treatment of Ph+ leukemia in mice

Abstract

It is generally believed that shutting down the kinase activity of BCR-ABL by imatinib will completely inhibit its functions, leading to inactivation of its downstream signaling pathways and cure of the disease. Imatinib is highly effective at treating human Philadelphia chromosome-positive (Ph(+)) chronic myeloid leukemia (CML) in chronic phase but not Ph(+) B cell acute lymphoblastic leukemia (B-ALL) and CML blast crisis. We find that SRC kinases activated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells, suggesting that imatinib does not inactivate all BCR-ABL-activated signaling pathways. This SRC pathway is essential for leukemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Inhibition of both SRC and BCR-ABL kinase activities by dasatinib affords complete B-ALL remission. However, curing B-ALL and CML mice requires killing leukemic stem cells insensitive to both imatinib and dasatinib. Besides BCR-ABL and SRC kinases, stem cell pathways must be targeted for curative therapy of Ph(+) leukemia.

Fig. 1.

SRC kinases remain active after inhibition of BCR-ABL kinase activity by imatinib. (A) Activation of SRC kinases by BCR-ABL does not require BCR-ABL kinase activity. P190- or P210-expressing ENU cells were cultured in the presence or absence of imatinib for 12 h, and ENU cells bearing empty vector were used as controls. Protein lysates were analyzed by Western blotting with antibodies against phosphotyrosine (p-Tyr), ABL, activated SRC kinases (p-SRC-Tyr-416) (29), and LYN. (B) BCR-ABL-transduced BM cells were cultured under Whitlock–Witte conditions for 5 days. The cells were treated with imatinib at the concentrations indicated for 2 days. Protein lysates were analyzed by Western blotting with the antibodies indicated.

Fig. 2.

SRC kinases play a critical role in maintaining survival and promoting proliferation of pre-B leukemic cells. (A) BM cells from B6 mice were transduced with the empty vector or v-SRC retrovirus and cultured under Whitlock–Witte conditions for 14 days. (B) Dasatinib inhibits activity of both BCR-ABL and SRC kinases. BCR-ABL-transduced BM cells were cultured under Whitlock–Witte conditions for 5 days. Different concentrations of dasatinib were added to the culture for 48 h, and protein lysates were analyzed by Western blotting. (C) Inhibition of SRC kinases reduces survival of BCR-ABL-T315I-expressing B-lymphoid cells. BCR-ABL-T315I-transduced BM cells were cultured at 1 × 10⁵ cells per well in 24-well plates, and different concentrations of dasatinib were added to the culture for 5 or 7 days. Viable cells were counted. (D) Therapeutic effect of imatinib and dasatinib on BCR-ABL-T315I-induced B-ALL. BMT, BM transplantation. (E) In vivo inhibition of SRC kinase activity with dasatinib. Mice with BCR-ABL-T315I-induced B-ALL were treated with a placebo or dasatinib for 3 days. After the last dose, leukemic cells from peripheral blood of the mice were analyzed by Western blotting. Each lane represents a mouse from the indicated treatment group. (F) BCR-ABL-transduced wild-type or Lyn/Hck/Fgr triple knockout BM cells were transplanted into wild-type recipient mice to induce B-ALL. GFP⁺ cell counts (percentage of GFP⁺ cells × white blood cell count) were measured at different time points after the induction of leukemia. (G) Percentages of GFP⁺ B-leukemic cells in peripheral blood were determined by FACS analysis as described in F.

Fig. 3.

Simultaneous targeting of kinase activity of both BCR-ABL and SRC kinases results in long-term survival of mice with B-ALL. (A) Mice with BCR-ABL-induced B-ALL were treated with a placebo, imatinib, or dasatinib. BMT, BM transplantation. (B) Reduction of GFP⁺ leukemic cells in peripheral blood of the treated B-ALL mice. (C) In vivo inhibition of BCR-ABL autophosphorylation by imatinib and dasatinib. B-ALL mice were treated with placebo, imatinib, or dasatinib for 3 days. After the last dose, leukemic cells from the pleural effusion were analyzed by Western blotting. Each lane represents a mouse from the indicated treatment group. (D) The SRC-selective kinase inhibitor PP2 alone or with imatinib has an inhibitory effect on proliferation of BCR-ABL-transduced BM cells in Whitlock–Witte culture. The transduced cells were cultured at 1 × 10⁵ per well in 24-well plates for 5 days, and the two drugs were added to the culture for the last 2 days. Viable cells were counted. (E) Lack of LYN, HCK, and FGR prevents CML transition to lymphoid blast crisis. Wild-type and Lyn/Hck/Fgr triple knockout BM cells from CML mice were transferred into wild-type recipient mice to assay CML transition to B-ALL by FACS analysis of GFP⁺ B-leukemic cells in peripheral blood. (F) Dasatinib, but not imatinib, is effective at suppressing p53-deficient leukemic cells in B-ALL mice.

Fig. 4.

Dasatinib efficiently kills highly proliferating B-leukemic cells, but not stem cells, in B-ALL mice. (A) B-ALL reappeared in most of the mice after dasatinib treatment stopped (−); the relapsed mice remained sensitive to dasatinib therapy (+). (B) A low level of GFP⁺ pro- or pre-B cells (<1%) persisted in dasatinib-treated mice. (C) B220⁺/CD43⁺ pro-B leukemic cells function as leukemic stem cells in B-ALL. The sorted GFP⁺/B220⁺/CD19⁺ cells from BM of B-ALL mice transfer B-ALL to secondary recipients after 2 months, and leukemic cells in peripheral blood are B220⁺/CD43⁺ pro-B cells.

Fig. 5.

Imatinib and dasatinib fail to eradicate BCR-ABL-expressing HSCs completely. (A) CML mice treated with imatinib and dasatinib. BMT, BM transplantation. (B) GFP⁺ leukemic cell counts in peripheral blood (PB) of CML mice treated with imatinib and dasatinib. (C) Comparison of the percentages of BCR-ABL-expressing HSCs (GFP⁺CD34⁻c-kit⁺Hoe⁻) in side populations (SP) of BM cells from placebo-, imatinib-, and dasatinib-treated CML mice. (D) Dasatinib inhibits BCR-ABL kinase activity in CML stem cells. BM cells from CML mice were treated with a placebo or dasatinib (100 nM) in culture for 24 h, and BCR-ABL-expressing HSCs (GFP⁺CD34⁻c-kit⁺Hoe⁻) were identified by FACS. Intracellular levels of BCR-ABL phosphorylation were determined by FACS with anti-Abl-Y412 antibody, which detects the active form of BCR-ABL. (E) A representative CML mouse treated with dasatinib for 16 weeks still contains large numbers of BCR-ABL-expressing HSCs.

Fig. 6.

Identification of BM cell populations that function as CML stem cells. (A) BCR-ABL-transduced BM cells from B6 mice were sorted by Sca-1 MACS columns (Miltenyi Biotec, Gladbach, Germany), followed by transferring a Sca-1⁻ or Sca-1⁺ population into B6 mice (1 × 10⁵ cells per mouse; four mice per cell population group) to induce CML. GFP⁺ myeloid cells (Gr-1⁺) in peripheral blood (PB) of the mice were examined at days 9 and 19 after the induction of leukemia. All mice receiving the Sca-1⁺ population died of CML by day 42. (B) BCR-ABL-expressing HSCs function as CML stem cells. BM cells from CML mice in B6 background were sorted by FACS for BCR-ABL-expressing HSCs (GFP⁺Lin⁻c-kit⁺Sca-1⁺), followed by transfer into lethally irradiated B6 mice (2 × 10⁴ cells per mouse). GFP⁺ myeloid cells (Gr-1⁺) were detected in peripheral blood. In contrast to the normal control mice, CML mice showed complete infiltration of the lungs with myeloid leukemic cells and complete disruption of follicular architecture of the spleen by infiltrating leukemic cells.