Spred1 deficit promotes treatment resistance and transformation of chronic phase CML

Abstract

Spred1 is highly expressed in normal hematopoietic stem cells (HSCs). Lack of Spred1 function has been associated with aberrant hematopoiesis and acute leukemias. In chronic myelogenous leukemia (CML), Spred1 is reduced in patients with accelerated phase (AP) or blast crisis (BC) CML, thereby suggesting that deficit of this protein may contribute to disease transformation. In fact, Spred1 knockout (KO) in SCLtTA/BCR-ABL CML mice either globally, or restricted to hematopoietic cells (i.e., HSCs) or to endothelial cells (ECs), led to transformation of chronic phase (CP) CML into AP/BC CML. Upon BCR-ABL induction, all three Spred1 KO CML models showed AP/BC features. However, compared with global Spred1 KO, the AP/BC phenotypes of HSC-Spred1 KO and EC-Spred1 KO CML models were attenuated, suggesting a concurrent contribution of Spred1 deficit in multiple compartments of the leukemic bone marrow niche to the CML transformation. Spred1 KO, regardless if occurred in HSCs or in ECs, increased miR-126 in LSKs (Lin^-Sca-1⁺c-Kit⁺), a population enriched in leukemic stem cells (LSCs), resulting in expansion of LSCs, likely through hyperactivation of the MAPK/ERK pathway that augmented Bcl-2 expression and stability. This ultimately led to enhancement of Bcl-2-dependent oxidative phosphorylation that supported homeostasis, survival and activity of LSCs and drove AP/BC transformation.

Figure 1.. Downregulation of Spred1 was observed in BM and CD34⁺ cells from patients with BC CML and associated with an increased “stemness” phenotype.

A Expression of SPRED1 in BM CD34⁺ cells from patients with BC CML and CP CML by Q-RT-PCR (n=8 samples for BC CML and n=12 samples for CP CML) and western blot and in BM by immunohistochemistry staining (one of three independent experiments with similar results was shown) (left), and expression of miR-126 in CD34⁺ and CD34⁺CD38⁻ cells from BC CML (n=6 samples) and CP CML (n=10 samples) patients by Q-RT-PCR (right). B SPRED1 mRNA expression by Q-RT-PCR and protein expression by western blot, miR-126 levels by Q-RT-PCR, cell cycling by Ki-67 and DAPi staining (top) or by cell trace violet staining (bottom) followed by flow cytometry analysis in CML CD34⁺ cells transduced with SPRED1 siRNA to knock-down (KD) SPRED1 or with a non-targeting control siRNA (Ctrl). UND: undivided cells, G0; DIV: division. C Representative colonies and quantification of colony forming cells (CFC) in CML CD34⁺ (left) and CD34⁺CD38⁻ (right) cells transduced with Spred1 siRNA to KD SPRED1 or with ctrl siRNA (n=3). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 2.. Spred1 deficit promotes CML transformation.

A Schematic design of the mouse crossings. B Schematic design and results of the experiments. After tetracycline withdrawal to induce BCR-ABL expression, Spred1 wt SCLtTA/BCR-ABL and Spred1 KO SCLtTA/BCR-ABL mice (n=15 mice per group) were monitored for white blood cell (WBC) counts in peripheral blood (PB) every two weeks. Four weeks post BCR-ABL induction, blood and BM leukemic blasts by microscopy and LSKs and GMPs by flow cytometry, spleen size and weight, and splenic LSKs and GMPs by flow cytometry, in the Spred1 KO CML mice were compared with Spred1 wt CML mice. C Survival of Spred1 wt SCLtTA/BCR-ABL and Spred1 KO SCLtTA/BCR-ABL mice after BCR-ABL induction (n=10 mice per group). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Figure 3.. BM or LSK cells from Spred1 KO CML mice recapitulated AP/BC phenotype in recipient mice.

A Schematic design of the experiments. Three weeks after tetracycline withdrawal to induce BCR-ABL expression, BM MNCs from Spred1 wt SCLtTA/BCR-ABL and Spred1 KO SCLtTA/BCR-ABL mice (B6-Ly5.2, n=3 mice per group) were selected and transplanted into congenic normal wt recipient mice (B6-Ly5.1, n=10 mice per group), followed by measurement of WBC counts, engraftment and survival. B WBC counts, blood CML engraftment rates analyzed by flow cytometry (left), BM leukemic blasts by microscopy (middle), and survival (right) of recipient mice transplanted with BM MNCs from Spred1 wt CML or from Spred1 KO CML mice. C Schematic design and results of the experiments. After three weeks of BCR-ABL induction by tetracycline withdrawal, BM LSKs from Spred1 wt SCLtTA/BCR-ABL and Spred1 KO SCLtTA/BCR-ABL mice (B6-Ly5.2, n=3 mice per group) were selected and transplanted into congenic normal wt recipient mice (B6-Ly5.1, n=10 mice per group) and survival of the recipient mice was shown. Results shown represent mean ± SEM. Significance values: *, p<0.05; ****, p<0.0001.

Figure 4.. Spred1 insufficiency in HSCs caused an attenuated CML transformation phenotype.

A Schematic design of the mouse crossing. Spred1^flox(f)/f (B6-Ly5.2) mouse was crossed with Mx1-Cre+ (B6-Ly5.2; Jax lab, #2527) mouse to generate Spred1^f/fMx1-Cre+ (Spred1 KO in HSCs, hereafter called Spred1^HSCΔ/Δ) mice. Spred1^f/fMx1-Cre+ mice were then bred with SCLtTA/BCR-ABL mice to obtain SCLtTA/BCR-ABL/Spred1^f/fMx1-Cre+ (Spred1^HSCΔ/Δ SCLtTA/BCR-ABL) or Cre- (Spred1 wt SCLtTA/BCR-ABL) mice. B Schematic design and results of the experiments. Spred1 wt SCLtTA/BCR-ABL and Spred1^HSCΔ/ΔSCLtTA/BCR-ABL mice were treated with 7 doses of poly(I:C) to activate Cre activity, followed by tetracycline withdrawal to induce BCR-ABL expression (n=12 mice per group). WBC counts, blood and BM leukemic blasts by microscopy and LSKs and GMPs by flow cytometry, spleen size and weight and LSK and GMP numbers by flow cytometry in Spred1 wt CML and Spred1^HSCΔ/Δ CML mice measured at four weeks after poly(I:C) administration and BCR-ABL induction. C Survival of Spred1 wt SCLtTA/BCR-ABL and Spred1^HSCΔ/ΔSCLtTA/BCR-ABL mice after poly(I:C) administration and BCR-ABL induction (left) and survival of Spred1^HSCΔ/ΔSCLtTA/BCR-ABL (HSC-Spred1 KO) and Spred1^−/−SCLtTA/BCR-ABL (global KO) after BCR-ABL induction (n=13 mice per group). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Figure 5.. SPRED1 KO in ECs increases arterioles in the BM niche.

A Schematic design of the mouse crossings. Spred1^f/f (B6-Ly5.2) mouse was crossed with Tie2-Cre+ (B6-Ly5.2; Jax lab, #8863) mouse to generate Spred1^f/fTie2-Cre+ (Spred1 KO in ECs, hereafter called Spred1^ECΔ/Δ) mice. Spred1^f/fTie2-Cre+ mice were then bred with SCLtTA/BCR–ABL mice to obtain SCLtTA/BCR-ABL/ Spred1^f/fTie2-Cre+ (i.e., Spred1^ECΔ/ΔSCLtTA/BCR-ABL, or Spred1^ECΔ/Δ CML, or EC-Spred1 KO CML) and Cre- (i.e., Spred1 wt SCLtTA/BCR-ABL, or Spred1 wt CML) mice. B Representative plots (left) and aggregate results (right) of BM EC Sca-1^high and Sca-1^low subfractions from Spred1 wt SCLtTA/BCR-ABL and Spred1^ECΔ/ΔSCLtTA/BCR-ABL mice after four weeks of BCR-ABL induction by tet withdrawal, analyzed by flow cytometry (n=4 mice per group). C CD31 (FITC) and Sca-1 (PE) immunofluorescence (IF) staining (left, top, tibia; left, bottom, enlarged representative region) and quantification (right, top, arbitrary units representing arterioles; right, bottom, aggregate results of arbitrary units from three representative regions) of CD31⁺Sca-1^high EC-lined vessels (i.e., arterioles, indicated by yellow arrows, see supplementary methods for the details how the arterioles were quantified using arbitrary units) in the tibias from Spred1 wt CML and EC-Spred1 KO CML mice (n=3 mice per group). Results shown represent mean ± SEM. Significance values: *, p<0.05.

Figure 6.. Spred1 loss in the BM vascular niche independently contributes to CML transformation.

A Schematic design of the experiments. After BCR-ABL induction by tetracycline withdrawal, Spred1 wt SCLtTA/BCR-ABL and Spred1^ECΔ/ΔSCLtTA/BCR-ABL mice were monitored for WBC counts, leukemic blasts and survival. B WBC counts, blood and BM leukemic blasts by microscopy and LSK and GMP numbers by flow cytometry, spleen size and weight and LSK and GMP numbers by flow cytometry, in Spred1 wt SCLtTA/BCR-ABL and Spred1^ECΔ/ΔSCLtTA/BCR-ABL mice measured at four weeks after BCR-ABL induction (n=8 mice per group). C Survival of Spred1 wt SCLtTA/BCR-ABL and Spred1^ECΔ/ΔSCLtTA/BCR-ABL mice after BCR-ABL induction (n=13 mice per group, left) and survival of Spred1^−/−SCLtTA/BCR-ABL (global KO, n=14 mice), Spred1^HSCΔ/ΔSCLtTA/BCR-ABL (HSC KO, n=16 mice) and Spred1^ECΔ/ΔSCLtTA/BCR-ABL (EC KO, n=13 mice) mice after BCR-ABL induction (right). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Figure 7.. Spred1 loss in the BM vascular niche independently contributes to CML transformation.

A Schematic design and results of the experiments. LSKs (5000 cells/mouse) from SCLtTA/BCR-ABL CML mice (B6-Ly5.1/5.2, BCR-ABL was induced for three weeks by tet withdrawal) were selected and transplanted into congenic Spred1 wt (B6-Ly5.2, n=14) and Spred1^ECΔ/Δ (B6-Ly5.2, n=10) recipient mice respectively (top). WBC counts, CD31 (FITC) and Sca-1 (PE) IF staining of CD31⁺Sca-1^high EC-lined vessels (i.e., arterioles, indicated by yellow arrows) in the tibias, and survival (bottom) of the Spred1 wt (n=14) and Spred1^ECΔ/Δ (n=10) recipient mice receiving CML LSKs. B Schematic design and results of the experiments. LSKs (5000 cells/mouse) from Spred1 wt SCLtTA/BCR-ABL and Spred1^ECΔ/ΔSCLtTA/BCR-ABL mice (both are B6-Ly5.2, BCR-ABL was induced for three weeks by tet withdrawal) were transplanted into congenic normal wt recipient mice (B6-Ly5.1, n=10 mice per group) (top). WBC counts and CML cell engraftment rate in PB by flow cytometry, leukemia blasts in BM by microscopy, and survival of recipient mice (n=10 mice per group) transplanted with LSKs from Spred1 wt CML or Spred1^ECΔ/Δ CML mice (bottom). Results shown represent mean ± SEM. Significance values: **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.

Figure 8.. Spred1 depletion promotes TKI resistance in CML.

A Schematic design and results of the experiments. BM cells (1×10⁶/mouse) from Spred1 wt SCLtTA/BCR-ABL and Spred1^HSCΔ/ΔSCLtTA/BCR-ABL leukemic mice (B6-Ly5.2, BCR-ABL was induced for three weeks by tet withdrawal) were transplanted into congenic normal wt mice (B6-Ly5.1, n=20 mice per group). Two weeks after transplantation, these mice were divided into four groups and treated with vehicle or nilotinib (NIL, 50mg/kg, oral gavage, daily) for three weeks (wt+vehicle, wt+NIL, HSC KO+vehicle, HSC KO+NIL; top). WBC counts and CML cell engraftment rates in PB measured at four weeks after transplantation by flow cytometry, and survival of the four groups of mice were shown (bottom). B Schematic design and results of the experiments. After two weeks of BCR-ABL induction by tet withdrawal, Spred1 wt SCLtTA/BCR-ABL and Spred1^ECΔ/ΔSCLtTA/BCR-ABL mice (B6-Ly5.2, n=8 mice per group) were treated with NIL (50mg/kg, oral gavage, daily) or vehicle for three weeks. BM cells (1×10⁶/mouse) from the treated primary mice were pooled and transplanted into secondary recipient mice (B6-Ly5.1, n=10 mice per group). WBC counts and survival of primary treated mice (bottom, left), and WBC counts, CML cell engraftment rates in PB, and survival of secondary recipient mice (bottom, right) were shown. Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.