Differential regulation of myeloid leukemias by the bone marrow microenvironment

Abstract

Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM) and may be the cause of relapse following chemotherapy. Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. CD44 (refs. 3,4) and interleukin-6 (ref. 5) have been implicated previously in the LSC niche. Transforming growth factor-β1 (TGF-β1) is released during bone remodeling and plays a part in maintenance of CML LSCs, but a role for TGF-β1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell-specific activation of the parathyroid hormone (PTH) receptor attenuates BCR-ABL1 oncogene-induced CML-like myeloproliferative neoplasia (MPN) but enhances MLL-AF9 oncogene-induced AML in mouse transplantation models, possibly through opposing effects of increased TGF-β1 on the respective LSCs. PTH treatment caused a 15-fold decrease in LSCs in wild-type mice with CML-like MPN and reduced engraftment of immune-deficient mice with primary human CML cells. These results demonstrate that LSC niches in CML and AML are distinct and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSCs, a prerequisite for the cure of CML.

Figure 1

BCR-ABL1-induced CML-like MPN is attenuated in Col1-caPPR recipients. (a and b) Cumulative mortality from CML-like MPN (a) and overall survival (b) for WT (n=20, solid) or Col1-caPPR (n=26, dashed) recipients of BCR-ABL1-transduced WT BM (P< 0.001 and P = 0.002, respectively; logrank tests). (c) Disease clonality in spleens of WT (lanes 1–9) and Col1-caPPR (lanes 10–22) recipients (WT, 6.6±2.1 clones; Col1-caPPR, 7.2±2.1 clones; P = 0.52, t-test). (d) Absolute number of GFP⁺ (BCR-ABL1⁺) KLS cells in the BM of Col1-caPPR mice (P = 0.03, t-test, gray bar, n=20) versus WT mice (black bar, n=20). (e) Hoechst⁺ BCR-ABL1⁺ cycling c-Kit⁺Lin⁻ cells in the BM of Col1-caPPR (gray) mice compared to WT mice (black) (P < 0.001, t-test). (f) Absolute number of viable BM cells from Col1-caPPR (gray) versus WT (black) recipients of BCR-ABL1-transduced BM (P < 0.001, t-test). (g) Number of colonies in methylcellulose from BM cells (squares) from Col1-caPPR (open squares) or WT (solid squares) mice with CML-like MPN (P = 0.02, t-test) and from spleen cells (circles) from Col1-caPPR (open circles) or WT (solid circles) mice with CML-like MPN (P = 0.02, t-test). (h) Overall survival for WT secondary recipients of BCR-ABL1⁺ BM grafts from a WT (solid, n=30), compared to a Col1-caPPR (dashed, n=18) microenvironment (P = 0.04). (i) Survival curves for WT (n=18, solid) or Col1-caPPR (n=21, dashed) recipients of MLL-AF9-transduced WT donor BM (P < 0.0001, logrank test). (j) Clonality of MLL-AF9⁺ AML-like disease in spleen of WT recipients (lanes 2–7) compared to spleen of Col1-caPPR recipients (lanes 8–14) (P = 0.01, t-test) and in BM of WT (lane 15) or Col1-caPPR recipients of MLL-AF9-transduced WT BM (lanes 16–17).

Figure 2

TGF-β1 signaling is increased in the bones of Col1-caPPR mice and suppresses the growth of BCR-ABL1⁺ cells in vitro. (a) Incidence of CML-like MPN in WT (left) and Col1-caPPR (right) mice treated with osteoprotegerin (WT: light gray, Col1-caPPR: white, n=10) or saline (wt: black, Col1-caPPR: dark gray, n=10) (P = 0.02, Chi-square). (b) Immunoblotting of lysates from crushed bone from WT or Col1-caPPR mice probed with antibodies to TGF-β1 (molecular mass 25 kDa) or actin (42 kDa) (mean intensity of TGF-β1 bands normalized to actin control: WT, 0.6±0.2; Col1-caPPR, 2.5±0.3; P < 0.001, t-test) (c) Bioactive TGF-β1 in Col1-caPPR bones (white bar, n=8) compared to WT bones (dark gray bar, n=9) (P = 0.02, t-test). (d) Immunohistochemistry (top) for TGF-β1 and hematoxylin & eosin stains (bottom) (Scale bar indicates 100 μm) on bones of Col1-caPPR mice with CML-like MPN treated with saline (left) or OPG-Fc (right). The arrows depict TGF-β1⁺ osteoblastic cells (bold arrow) and osteoclasts (thin arrow). (e) Immunohistochemistry (Scale bar indicates 100 μm) for pSMAD2/3 (detected by immunoperoxidase using yellow/brown horseradish-peroxidase chromogen) on bones of representative Col1-caPPR mice (bottom) with CML-like MPN treated with saline (left) or OPG-Fc (right). (f) Growth of BCR-ABL1⁺ K562 (circles) and MLL-AF9⁺ THP-1 (triangles) cells in the presence of vehicle (solid line) or TGF-β1 (dashed line); the differences in cell number at day 3 and 4 were significant (P < 0.001, t-tests). (g) Hematoxylin-eosin-stained sections of BM of a representative (n=3) WT mouse with CML-like MPN treated by intrafemoral injections with PBS (left panel) or TGF-β1 (right panel) (Scale bar indicates 100 μm). (h) Immunoblotting of lysates from crushed bone or spleen from WT or Col1-caPPR mice with CML-like MPN probed with an antibody to TGF-β1 as in b. (i) Overall survival of WT secondary recipients of BCR-ABL1⁺ BM or spleen grafts from a WT (BM: solid, n=30; spleen: long dashes, n=24), compared to a Col1-caPPR (BM: dots, n=18; spleen: short dashes, n=20) microenvironment. Significant differences in survival (logrank tests) are indicated.

Figure 3

Modulation of TGFβRI on leukemic cells differentially affects BCR-ABL1⁺ (chronic) and MLL-AF9⁺ (acute) myeloid neoplasms in a Col1-caPPR microenvironment. (a) Survival curves for WT or Col1-caPPR recipients of BCR-ABL1-transduced WT BM that was co-transduced with lentivirus expressing either scrambled shRNA (WT: solid line; Col1-caPPR: dotted line) or Tgfbr1 shRNA (WT: long dashes; Col1-caPPR: short dashes). Survival of Col1-caPPR recipients of BCR-ABL1⁺ Tgfbr1 shRNA⁺ BM was significantly shortened (P = 0.007, logrank test) compared to Col1-caPPR recipients of BCR-ABL1⁺ scrambled shRNA⁺ BM. All Col1-caPPR recipients of BCR-ABL1⁺ Tgfbr1 shRNA⁺ BM succumbed to CML-like MPN. (b) Survival curves for WT or Col1-caPPR recipients of WT BM transduced with MLL-AF9 retrovirus alone (WT: solid line; Col1-caPPR: dotted line) or co-transduced with a retrovirus expressing TGFβRI (WT: long dashes; Col1-caPPR: short dashes). Survival of Col1-caPPR recipients of MLL-AF9⁺ TGFβRI⁺ BM (n=10) was significantly prolonged (P = 0.02, logrank test) compared to Col1-caPPR recipients of BM transduced with MLL-AF9 only (n=8). (c) pSMAD2/3 immunofluorescence staining of Lin⁻ MLL-AF9⁺ (left) or BCR-ABL1⁺ KLS cells treated in vitro with 5 ng/ml TGF-β1 (bottom) or saline (top). (d) Quantitation of individual cells from c by confocal microscopy. pSMAD2/3 staining was significantly increased in CML-initiating cells treated with TGF-β1 compared to saline-treated cells (P = 0.01, t-test), but not in AML-initiating cells.

Figure 4

PTH treatment reduces leukemia burden and LSC abundance in wt mice and leads to more longterm survivors in combination with imatinib. (a) Frequency of leukemia-free untreated secondary recipients of BCR-ABL1⁺ BM from saline- (black circles) or PTH- (open squares) treated donors in relation to the dose of transplanted bone marrow. The lines fitted by regression analysis (L-Calc) allow estimation of leukemia-initiating cell frequency, which was significantly lower in recipients of BM from PTH-treated donors (P = 0.0006). (b) Overall survival of WT secondary recipients of BCR-ABL1⁺ BM grafts from WT mice treated with PTH (dotted line, n=27) compared to WT mice treated with saline (solid line, n=29) (P = 0.03, logrank test). (c) Overall survival of WT recipients of BCR-ABL1-transduced BM treated with daily subcutaneous injections of saline (solid line, n=19) or PTH (dotted line, n=19), imatinib (long dashes, n=18) or a combination of PTH and imatinib (short dashes, n=18), randomized prior to transplantation. (d) Percentage of cycling GFP⁺c-Kit⁺Lin⁻ cells is significantly decreased in animals treated with PTH with or without imatinib (P = 0.04, t-test) compared to imatinib alone. (e) Levels (mean and s.d.) of human hematopoietic engraftment (measured or estimated percentage of hCD45⁺ leukocytes, left panel) or BCR-ABL1⁺ engraftment (estimated by levels of BCR-ABL1 transcripts, right panel) in BM aspirates from individual NSG recipients of primary human CML subject samples (n=7) that were treated with either saline or PTH (P = 0.049, t-test). Recipients of BM grafts from the same CML subject sample are indicated by the individual symbols. The two samples in the PTH-treated cohort at the bottom of the plot had BCR-ABL1 transcript levels that were below the level of detection (estimated <0.001%) despite detectable levels of human cell engraftment.