Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia

Abstract

Targeting of cancer stem cells is believed to be essential for curative therapy of cancers, but supporting evidence is limited. Few selective target genes in cancer stem cells have been identified. Here we identify the arachidonate 5-lipoxygenase (5-LO) gene (Alox5) as a critical regulator for leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML). In the absence of Alox5, BCR-ABL failed to induce CML in mice. This Alox5 deficiency caused impairment of the function of LSCs but not normal hematopoietic stem cells (HSCs) through affecting differentiation, cell division and survival of long-term LSCs (LT-LSCs), consequently causing a depletion of LSCs and a failure of CML development. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly by affecting LT-LSCs, and prolonged survival. These results demonstrate that a specific target gene can be found in cancer stem cells and its inhibition can completely inhibit the function of these stem cells.

Figures

Figure 1. Alox5 is essential for the induction of CML induced by BCR-ABL

a, Bone marrow cells from C57BL/6 mice (B6) were transduced with retrovirus containing BCR-ABL/GFP or GFP alone (BCR-ABL-IRES-GFP-pMSCV or IRES-GFP-pMSCV), and then transferred into B6 recipient mice to induce CML. One group of CML mice was treated with imatinib (150 mg/kg body weight/ per dose, once every 4 hours) for 5 doses beginning at day 13 post bone marrow transplantation (BMT). Bone marrow cells were isolated from CML mice, and were sorted by FACS for GFP+Lin−c-Kit+Sca-1+ cells (normal or CML stem cells). Total RNA was isolated from these sorted cells for DNA micorarray analysis. Expression of the Alox5 gene was up-regulated by BCR-ABL in CML stem cells as compared to the sorted GFP+Lin−c-Kit+Sca-1+ cells that did not express BCR-ABL, and this up-regulation was not prevented by imatinib treatment. b, Bone marrow cells from C57BL/6 mice (B6) were transduced with retrovirus containing BCR-ABL/GFP or GFP alone to induce CML as described in a. Bone marrow cells were isolated from the mice were sorted by FACS for GFP+Lin−c-Kit+Sca-1+ cells, and then total RNA was isolated from these sorted cells for comparing Alox5 expression between GFP vector-transduced normal stem cells and BCR-ABL-transduced LSCs by RT-PCR. Expression of the Alox5 gene was significantly up-regulated by BCR-ABL in LSCs as compared to the sorted GFP vector-transduced normal stem cells (p< 0.001). c, The plasma level of LTB4 in recipients of BCR-ABL-transduced bone marrow cells was significantly higher than that in recipients of bone marrow cells transduced with GFP-containing retrovirus (p< 0.05), and this increased level of LTB4 was not observed in recipients of Alox5−/− bone marrow cells transduced by BCR-ABL. These results indicated that BCR-ABL up-regulates LTB4 through Alox5. d, Kaplan-Meier survival curves for recipients of BCR-ABL-transduced bone marrow cells from wild type or Alox5−/− donor mice (10 mice per group). All recipients of BCR-ABL-transduced bone marrow cells from wild type donor mice developed CML and died within 4 weeks after bone marrow transplantation (days post BMT), whereas recipients of BCR-ABL-transduced bone marrow cells from Alox5−/− donor mice survived. e, Gross appearance of the lungs and spleens showed severe lung hemorrhages and splenomegaly of recipients of BCR-ABL-transduced bone marrow cells from wild type but not Alox5−/− donor mice. f, Photomicrographs of haematoxylin and eosin-stained lung and spleen sections from recipients of BCR-ABL-transduced bone marrow cells from wild type or Alox5−/− donor mice. g, FACS analysis showed gradual disappearance of GFP+Gr-1+ cells in peripheral blood (PB) and bone marrow (BM) of recipients of BCR-ABL-transduced bone marrow cells from Alox5−/− but not wild type donor mice. h, In recipients of BCR-ABL-transduced bone marrow cells from Alox5−/− donor mice, GFP+Gr-1+ cells in PB gradually decreased with time, whereas the GFPGr-1+ cells that did not express BCR-ABL gradually increased, showing that Alox5 deficiency significantly inhibited engraftment of BCR-ABL-expressing but not normal BM cells in the same animals. Mean percentage for each group (n=5) was shown.

Figure 2. Alox5 transgene rescues defective CML phenotype

a, The rescue construct (BCR-ABL-IRES-Alox5-pMSCV) was used to transfect 293T cells to test for expression of 5-LO with mock- or BCR-ABL-IRES-GFP-pMSCV-transfected cells as controls. BCR-ABL and 5-LO were detected by Western blotting using antibodies against Abl and 5-LO. 5-LO protein was detected in cells transfected with BCR-ABL-IRES-Alox5-pMSCV. b, Bone marrow cells from B6 mice were transduced with retrovirus containing IRES-GFP-pMSCV (empty vector) or BCR-ABL-IRES-Alox5-pMSCV, and then transferred into B6 recipient mice to induce CML. The plasma level of LTB4 in recipients of BCR-ABL-IRES-Alox5-pMSCV-transduced bone marrow cells was significantly higher than that in recipients of bone marrow cells transduced with empty vector-containing retrovirus (p< 0.01), confirming that the BCR-ABL-IRES-Alox5-pMSCV construct induced LTB4 production in mice. c, Kaplan-Meier survival curves for recipients of BCR-ABL-IRES-Alox5-pMSCV-transduced bone marrow cells from wild type (n=10) or Alox5−/− (n=9) donor mice. All recipient mice died. d, FACS analysis showed appearance of GFP+Gr-1+ cells in peripheral blood of recipients of BCR-ABL-IRES-Alox5-pMSCV-transduced bone marrow cells from both wild type and Alox5−/− donor mice. e, Peripheral blood smears showed accumulation of neutrophils, indicating high white blood cell counts in these CML mice. f, Photomicrographs of haematoxylin and eosin-stained lung and spleen sections from recipients of BCR-ABL-IRES-Alox5-pMSCV-transduced bone marrow cells from wild type or Alox5−/− donor mice.

Figure 3. Loss of Alox5 impairs the function of CML stem cell

a, BCR-ABL-expressing (GFP+) and non-BCR-ABL-expressing (GFP) Lin−c-Kit+Sca-1+ cells (CML stem cells) in BM and the spleens (SPL) were analyzed by FACS in recipients of BCR-ABL-transduced BM cells from wild type or Alox5−/− donor mice (n=4 for each group at two time points after BMT). Total number of GFP+Lin−c-Kit+Sca-1+ cells for each mouse was calculated as percentage of GFP+Lin−c-Kit+Sca-1+ cells x total cell count for the cells from femurs and tabias. Loss of Alox5 caused significant reduction of CML stem cells in BM (p< 0.05). b, Equal numbers of the sorted GFP+Lin−c-Kit+Sca-1+ cells from recipients of BCR-ABL-transduced bone marrow cells (5000 cells each) from wild type (CD45.1) or Alox5−/−(CD45.2) donor mice were mixed, followed by transplantation into lethally irradiated wild type mice. At days 14 and 25 after BMT, FACS analysis showed that the percentages of CD45.1+ cells were much higher than those of CD45.2+ cells. All these mice died of CML, presumably due to the development of CML from CD45.1+ cells. c, GFP+Lin−c-kit+Sca-1+ cells sorted by FACS from BCR-ABL-transduced BM cells from wild type or Alox5−/− mice were injected into lethally irradiated wild type recipient mice (15000 GFP+Lin−c-kit+Sca-1+ cells per recipient mouse). At day 14 after BMT, GFP+ Gr-1+ cells in peripheral blood of the mice were analyzed by FACS. Alox5-deficient GFP+Lin−c-kit+Sca-1+ poorly engrafted. The mice receiving the Alox5-deficient GFP+Lin−c-kit+Sca-1+ cells survived (n=4), whereas the mice receiving the wild type GFP+Lin−c-kit+Sca-1+ cells died of CML (n=2). d, FACS analysis indicated the percentages of BCR-ABL-expressing (GFP+) and non-BCR-ABL-expressing (GFP) CMP, GMP, and MEP cells in BM of recipients of BCR-ABL-transduced wild type or Alox5−/− donor BM cells (n=4). The results showed that loss of Alox5 caused depletion of BCR-ABL-expressing but not non-BCR-ABL-expressing CMP, GMP, and MEP cells in BM of the mice.

Figure 4. Loss of Alox5 function blocks differentiation of LT-LSCs

a, Bone marrow cells were isolated from recipients of BCR-ABL-transduced bone marrow cells from wild type or Alox5−/− donor mice, and FACS analysis showed that the percentages and total numbers of LT-LSCs (GFP+Lin−c-Kit+Sca-1+CD34-) and ST-LSCs /BCR-ABL-expressing MPP cells (Lin−c-Kit+Sca-1+CD34+) in recipients of BCR-ABL-transduced Alox5−/− (middle and right panels) donor BM cells were much lower than those in recipients of BCR-ABL-transduced wild type (left panel) donor BM cells (n=4). In addition, LT-LSCs in recipients of BCR-ABL-transduced Alox5−/− donor BM cells relatively accumulated due to the blockade of differentiation, resulting in depletion of ST-LSCs /BCR-ABL-expressing MPP cells as assayed at day 90 post BMT. b, Recipients of BCR-ABL-transduced bone marrow cells from wild type donor mice were treated with Zileuton (300 mg/kg, twice a day) beginning at 8 days after BMT. At days 20 and 90 post BMT, FACS analysis showed the depletion of ST-LSCs /BCR-ABL-expressing MPP cells in Zileuton-treated CML mice, indicating a blockade of differentiation of LT-LSCs. In contrast, in the same animals the percentages and total numbers of ST-HSCs /MPP cells were higher than those of LT-LSCs, indicating that Zileuton treatment did not lead to a blockade of differentiation of normal LT-HSCs in Zileuton-treated CML mice. c, Mice receiving wild type donor BM cells transduced with MSCV-IRES-GFP retrovirus were treated with a placebo or Zileuton as described in b. At day 14 after BMT, the numbers of GFP+Lin−c-Kit+Sca-1+ cells in BM and the spleens (SPL) of placebo- and Zileuton-treated mice were compared. Zileuton treatment did not result in a reduction of normal HSCs in the mice. d, At day 14 after BMT, bone marrow cells were isolated from recipients of BCR-ABL-transduced bone marrow cells from wild type or Alox5−/− donor mice. The cells were stained with Hoechst Blue, and DNA contents, represented by the percentages of three LSC populations (total LSCs, LT-LSCs, and ST-LSCs+MPPs) in the S+G2M phase of the cell cycle, was examined by FACS. Mean percentage for each cell population (n=5) was shown. e, At day 14 after BMT, bone marrow cells were isolated from recipients of BCR-ABL-transduced bone marrow cells from wild type or Alox5−/− donor mice. The cells were stained with PI and Hoechst Blue, and the percentages of LSCs positive for PI and Hoechst Blue, representing apoptotic cells, were determined by F ACS. f, Bone marrow GFP+Lin−c-Kit+Sca-1+ cells were sorted by FACS from recipients of GFP vector- or BCR-ABL-transduced bone marrow cells from wild type or Alox5−/− donor mice for isolation of total RNA, and expression of β-catenin, GATA-1 and FOG were detected by RT-PCR. Each bar represents the mean value of three samples. BMCs: bone marrow cells.

Figure 5. Inhibition of Alox5 prolongs survival of CML mice

a, Zileuton treatment resulted in a reduction of the plasma LTB4 level in CML mice. b, Kaplan-Meier survival curves for CML mice treated with a placebo, Zileuton alone, imatinib alone, or both Zileuton and imatinib in combination. Zileuton-treated CML mice were much healthier than placebo-treated CML mice, and inhibition of Alox5 by Zileuton significantly prolonged survival of CML mice. c, Photomicrographs of haematoxylin and eosin-stained lung and spleen sections from CML mice treated with a placebo or Zileuton. d, FACS analysis showed gradual disappearance of GFP+Gr-1+ cells in PB of CML mice treated with Zileuton. e, In CML mice treated with Zileuton, GFP+Gr-1+ cells in PB gradually decreased with time, whereas the GFP−Gr-1+ cells that did not express BCR-ABL gradually increased, showing that inhibition of Alox5 with Zileuton significantly inhibited engraftment of BCR-ABL-expressing but not normal BM cells in the same animals. f, FACS analysis showed gradual disappearance of GFP+Gr-1+ cells in BM of CML mice treated with Zileuton.

Figure 6. Alox5 deficiency does not significantly affect normal HSCs

a, Bone marrow cells from wild type and Alox5−/− mice were analyzed by FACS for the percentages of total HSCs (Lin−c-Kit+Sca-1+), LT-HSCs (Lin−c-Kit+Sca-1+CD34−), ST-HSCs (Lin−c-Kit+Sca-1+CD34+Flt3), and MPPs (Lin−c-Kit+Sca-1+CD34+Flt3+). b, Cells from bone marrow and peripheral blood of wild type and Alox5−/− mice were analyzed by FACS for the percentages of Gr-1+, Mac-1+, B220+, and CD3E+ cells. c, Three doses (1×105, 5×104, and 2.5×104) of wild type or Alox5−/− BM cells were injected into lethally irradiated recipient mice. Survival curves showed that there was only a minor engraftment defect of BM in Alox5−/− mice. d, Alox5−/−(CD45.2) and wild type (CD45.1) BM cells were 1:1 mixed and then transferred into recipient mice (n=20). 12 weeks after BMT, FACS analysis was carried out to compare the percentages of wild type and Alox5−/−Gr-1+, Mac-1+, B220+, and CD3E+ cells in PB of recipient mice. e, BM cells from BM of wild type and Alox5−/− mice were analyzed by FACS for the numbers of CMPs, GMPs, MEP, and CLPs. f, Bone marrow cells were isolated from wild type or Alox5−/− mice, and stained with Hoechst Blue. DNA contents in total HSCs, LT-HSCs, and ST-HSCs+MPPs in the S+G2M phase of the cell cycle were examined by FACS. g, Bone marrow HSCs were sorted by FACS from wild type or Alox5−/− mice for isolation of total RNA, and expression of β-catenin, GATA-1 and FOG were detected by RT-PCR. Each bar represents the mean value of three samples.

Figure 7. Alox5 is not required for the induction of lymphoid leukemia by BCR-ABL

a, Kaplan-Meier survival curves for recipients of BCR-ABL-transduced bone marrow cells from wild type (n=6) or Alox5−/− (n=8) donor mice. Both groups of mice developed and died of ALL. b, At day 14 post BMT, FACS analysis showed no difference in the percentages of GFP+B220+ cells in PB of recipients of BCR-ABL-transduced bone marrow cells from wild type or Alox5−/− donor mice. c, FACS analysis showed similar numbers of GFP+B220+ cells in PB of recipients of BCR-ABL-transduced bone marrow cells from wild type or Alox5−/− donor mice. d, The plasma levels of LTB4 in recipients of BCR-ABL-transduced bone marrow cells from wild type or Alox5−/− donor mice were compared. Loss of Alox5 did not result in a reduction of the plasma LTB4 level in ALL mice.