Overcoming Wnt–β-catenin dependent anticancer therapy resistance in leukaemia stem cells

Abstract

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt–β-catenin and PI3K–Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate β-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt–β-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated β-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, β-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated β-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.

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Fig. 1: Cooperative activation of the Wnt–β-catenin and PI3K–Akt pathways successively expands HSPCs, LSCs and T-ALL blast cells.
Fig. 2: DXR inhibits β-catenin activation by Akt.
Fig. 3: Differential response of LSCs, HSPCs and blast cells to treatment.
Fig. 4: LSCs uniquely express immune checkpoints that can be inhibited with low-dose DXR.
Fig. 5: Chemotherapy induction combined with maintenance targeted/low-dose DXR treatment increases survival.
Fig. 6: Low-dose DXR treatment reduces the number of persistent pS552-β-cat+ cells from MRD+ human leukaemia.

Data availability

ChIP–seq, RNA-seq and ATAC-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE105049. Original data underlying this manuscript can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/LIBPB-791. Source data for Figs. 1–6 and Extended Data Figs. 1, 3 and 5–7 are presented with the paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank H. Marshall, K. Zapien, J. McCann, S. Billinger, J. Haug, A. Box, C. Semerad, J. Park, L. Blunk, S. Chen, T. Parmely, A. Peak, K. Hall, B. Slaughter and J. Unruh for technical support; K. Tannen for proofreading and editing; H. Wu for providing Pten conditional-mutant mice; J. Goethert for providing HSC-SCL-Cre-ERT mice; M. Taketo for providing the conditional β-catAct mutants; and A. McMahon for providing FLAG:β-catenin ES cells. D. Xu is supported by NIH (R01 GM100701). The synthesis and characterization of polymers for NanoDXR was partly supported by a grant from the National Science Foundation NSF CAREER Award to R.M.K. (DMR-0748398) and ACS PRF 5247200 to R.M.K., managed by the American Chemical Society. The NanoDXR preparation was supported by the Academic Plan Grant from the University of Connecticut. We acknowledge support from the University of Kansas Cancer Center’s Biospecimen Repository Core Facility staff for helping obtain human specimens and the University of Kansas Cancer Center’s Cancer Center Support Grant (P30 CA168524). Research reported in this publication was supported by Stowers Institute for Medical Research, Children’s Mercy Kansas City, Braden’s Hope for Childhood Cancer, the Leukemia and Lymphoma Society (LLS), Kansas Bioscience Authority, Hall Family Foundation and the University of Kansas Cancer Center (KUCC) National Cancer Institute Cancer Center Support Grant (NCI-CCSG) P30 CA168524 and used the KUCC Lead Development and Optimization Shared Resource.

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Contributions

J.M.P. designed and conducted the primary experiments and wrote the manuscript. F.T. conducted ChIP–seq and ATAC-seq experiments. A.R. and G.S.S. conducted high-throughput screening. T.L. conducted the clinical trial. X.C.H., A.M. and D.D. conducted transplantation and drug treatments. X.L., R.M.K., T.-H.T., P.D. and C.T.N. designed and synthesized nanoDXR. S.J.W., E.G., K.A., A.S.G., R.R., and M.B. provided insights into clinical treatment. A.G. oversaw patient biospecimen acquisition. Z.H. and D.X. conducted computational simulation. S.D. provided β-catenin inhibitor. J.N., L.R., X.Y., J.P., K.S., M.Z., A.V., P.Q., Z.L. and M.H. helped in scientific discussion and facilitated some experiments. S.C. and A.P. conducted bioinformatics analysis. L.L. provided overall supervision of the project. All authors reviewed and approved the manuscript.

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Correspondence to Linheng Li.

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Extended data

Extended Data Fig. 1 HTS screening and in vitro analysis shows DXR preferentially inhibits LSC expansion.

a, Flow chart showing HTS design. b, Vector designs for cells expressing Akt and β-catenin and TCF reporter activity. c, FRET verification between Akt and β-catenin. While FRET was observed in mCherry-β-catenin + EGFP-AKT transfected cells and could be inhibited by DXR, essentially no discernible FRET occurred when mCherry-β-catenin was transfected with EGFP alone (see also Fig. 2h-i). Two biologically independent experiments with n=62, 19, 27, and 36 independent cells for vehicle, [Low]DXR, and vehicle (negative), [Low]DXR (negative), respectively; mean ± SD is indicated. d-e, BM isolated from leukemic Pten:β-catAct mice at 8 wpi was cultured in HSC expansion media (see Methods). Doxorubicin (white), 103506 (light grey), and thioguanosine (dark grey) were added to 11, 33 or 100 nM and cultured for 72 hours and analyzed by flow cytometry for LSCs (d) and HSPCs (e) as in Fig. 1. Fold change before and after culture for each population is indicated relative to equivalent vehicle control concentrations. n=3 biologically independent replicates for each condition; mean ± SEM is indicated. Statistics determined by unpaired two-tailed t-test. Source data

Extended Data Fig. 2 Schematic representation of experimental setup and treatment scheme for leukemic mice.

See Methods for additional detail.

Extended Data Fig. 3 Low-dose DXR inhibits downstream Wnt signaling.

a, Heatmap of Hallmark MYC target genes (V1) up and downregulated in HSPCs and LSCs treated with vehicle and LSCs treated with low-dose DXR. Data from two biological replicates of each, differing by <0.3 standard deviations, are shown. b, c, Gene ontology enrichment analysis using -log10 of the uncorrected p value as x axis. The upregulated or downregulated enriched terms are shown in red or blue, respectively. Numbers correspond to the same term upregulated in b but downregulated in c. b, Upregulated terms in LSCs vs. HSPCs in leukemic mice (treated only with vehicle control). c, Downregulated terms in LSCs from low-dose DXR vs. vehicle control treated leukemic mice. n=2 biologically independent replicates for each (see Methods). d, TCF/Lef:H2B-GFP mice were stained with CD3 and c-Kit and analyzed by FACS. Percent of TCF/Lef:H2B-GFP+ cells for each population are indicated. Mean of n=2 biologically independent replicates for each is indicated. e, TCF/Lef:H2B-GFP mice were treated with vehicle or low-dose nanoDXR and analyzed by FACS at 3 hours post-injection for TCF/Lef:H2B-GFP+ cells. n=5 biologically independent replicates for each condition; mean ± SD is indicated. Statistics determined by unpaired two-tailed t-test. f, Representative FACS plots for data quantified in (e) and including untreated, TCF/Lef:H2B-GFP negative littermate control analysis. n=4,5, and 5 biologically independent replicates as indicated; mean ± SD is indicated. Statistics determined by unpaired two-tailed t-test. Source data

Extended Data Fig. 4 β-catenin binds multiple immune checkpoint gene loci; low-dose DXR has differential effects on IC genes in LSCs and blast cells.

a, CHIP-seq was performed on 2×107 cells from the β-catenin-3xFlag mouse ES cell line. Genome browser view of β-catenin binding density at the Pdcd1 (Pd-1), Havcr2 (Tim-3), Cd24a, and Ctla4 gene loci promoter regions and/or intergenic regions. b, ATAC-seq was used to show chromatin accessibility profiles of Wnt target genes observed in blast cells (~30k cells per replicate) and leukemic stem cells (~15k cells per replicate). c, Accessibility profiles of immuno-checkpoint genes observed by ATAC-seq in blast cells (~30k cells per replicate) and LSCs (~15k cells per replicate). Cells were sorted from BM pooled from 20 leukemia mice treated with low-dose DXR and 8 leukemia mice treated with vehicle control (b-c). Experiment was repeated 1 time with similar results.

Extended Data Fig. 5 Single-dose DXR-loaded nanoparticles substitute for multiple doses of free DXR in reducing LSCs. [Low]NanoDXR treatment reduces functional LSCs in vivo.

a, b, Leukemic mice established as described in Extended Data. 2 were treated with 5 daily injections of free DXR at 0.5 or 0.15 µg/g with and without chemotherapy. Alternatively, a single injection on day 1 of 0.8 or 2.5 µg/g of DXR-loaded nanoparticles (NanoDXR) was given with and without chemotherapy. At 10 days post-treatment, BM was analyzed by flow cytometry to determine frequency of LSCs (a) and HSPCs (b). n=6,10,3,6,10,6,3 (a) and n=6,10,8,6,10,6,3 (b) (left to right, respectively) biologically independent replicates for each condition; mean ± SEM is indicated. Note that 5 doses of 0.15 µg/g DXR is ineffective; however, a single NanoDXR injection with a similar cumulative dose (0.8 µg/g) is most effective at reducing LSCs while allowing for HSPC recovery. c-d, Cohorts of leukemic mice were prepared and treated as in Figure S2 but with low-dose NanoDXR. At 12 days post-treatment, BM was harvested from treated mice and transplanted into sub-lethally irradiated NSG recipients. c, Treatment schematic and Kaplan-Meier curves of recipient mice. The free low-dose DXR treatment group (solid line) from Fig. 5f is shown for comparison (n = 30 per group). d, Recipients of BM from low-dose DXR and low-dose NanoDXR treated leukemic mice were analyzed by flow cytometry at 6 months post-transplant for Blast cells, HSPCs and LSCs. n=27 (free_ and 24 (nano) biologically independent replicates; mean ± SD is indicated. Statistics determined by unpaired two-tailed t-test. Source data

Extended Data Fig. 6 Low-dose DXR treatment reduces leukemia-initiating activity only in human leukemia exhibiting chemoresistant pS552-β-cat+ LSCs.

a, b, Summary of pediatric leukemia patients analyzed by FACS for LSCs and pS552-β-cat+ LSCs. B- and T-lymphoid LSCs were identified as enriched in CD45+ CD34+ CD19+ and CD45+ c-Kit+ CD3+ cells, respectively. BM samples at diagnosis and at day 29 post-chemotherapy treatment. Pt samples 019 and 034, B and T-lymphoid leukemias exhibiting chemoresistant pS552-β-cat+ LSCs, were subjected to further in vivo treatment and analysis. Pt 024 and 031, B and T-lymphoid acute leukemias lacking chemoresistant pS552-β-cat+ LSCs, were also tested. c, Experimental schematic of establishment and treatment of patient-derived xenografts (PDX) (see Methods). d, e, Kaplan-Meier survival curve of 1° PDX recipients of diagnostic and post-chemotherapy BM from Pt 019 (d) and Pt 034 (e). n=3 biologically independent replicates each. f, g, Kaplan-Meier survival curve for 2° PDX recipients treated with vehicle or low-dose nanoDXR. BM from 1° recipients of diagnostic (d) or post-chemotherapy (e) was transplanted into 2° PDX recipients and treated with low-dose NanoDXR 14 days post-transplant. h, i, FACs analysis of 2° PDX recipient BM from (f-g). Human CD45+ and human LSC engraftment was determined after succumbing to leukemia or at experimental endpoint. n=10 (each, h) and n=10 and 9 (vehicle and low-dose NanoDXR, respectively, i) biologically independent replicates; mean ± SEM is indicated. Statistics determined by unpaired two-tailed t-test. jo, Similar analysis to D-I but of patient-derived xenografts (PDX) from Pt 024 and Pt 031, which lack chemoresistant pS552-β-cat+ LSCs. n=3 biologically independent replicates each (j-k). n = biologically independent replicates as indicated (f, g, l, m). n=9,10 (vehicle and low-dose NanoDXR, respectively, n) and n=10,9 (vehicle and low-dose NanoDXR, respectively, o) biologically independent replicates; mean ± SEM is indicated. Statistics determined by unpaired two-tailed t-test. (N.S. = not significant). Statistics determined by Log-rank (Mantel-Cox) test for survival curves. Source data

Extended Data Fig. 7 Low-dose Daunorubicin performs similarly to Low-dose Doxorubicin.

a, b, Daunorubicin (DNR) dose-response and FACs analysis of LSC, HSPC and Blast Cell frequency in leukemic mice treated with chemotherapy with either low-dose DXR or low-dose DNR. Experiment was repeated 1 time with similar results (a). n=5 biologically independent replicates for each; mean ± SD is indicated. Statistics determined by unpaired two-tailed t-test. Source data

Extended Data Fig. 8 Targeting Akt-activated β-catenin dependent immune escape in LSCs.

The cooperative role of the Wnt/β-catenin and PI3K/Akt pathway in resistance to anti-cancer therapies, including immune escape, made the Pten:β-catAct double mutant mice served as an ideal model to study cancer therapy resistance. We found that cooperative Akt:β-catenin signaling is particularly critical for therapy-resistant LSCs. a, Investigating the mechanism underlying this resistance, we unexpectedly found that Akt-activated β-catenin binds to multiple IC genes, which are expressed on LSCs. b, In identifying DXR as an inhibitor of Akt:β-catenin interaction at low doses, we found that DXR could be repurposed as a targeted therapy for resistant LSCs, in part by inhibiting multiple ICs, particularly PD-1/PD-L1. Notably, LSCs but not blast cells exhibit unique properties of immune resistance, which can be reduced with low-dose DXR.

Supplementary information

Supplementary Information

Supplementary ATAC-seq QC analysis.

Reporting Summary

Supplementary Tables

Supplementary Table 1, related to Fig. 3. Limiting Dilution Assays. Recipients of sorted blast cells or LSCs (see Figure 3) from chemotherapy or [Low]DXR treated mice were analyzed at 10-12 weeks post-transplant. Recipient mice exhibiting ≥ 1% CD45Hi blast cells in bone marrow were considered engrafted. Upper and lower confidence intervals for the estimated CRU frequency were obtained using ELDA software (http://bioinf.wehi.edu.au/software/elda/index.html). Supplementary Table 2, related to Extended Data 6. Pediatric ALL Sample Characteristics and Analysis. Characteristics of pediatric acute leukemia samples examined. Supplementary Table 3, related to Figure 6l-n. Relapsed/Refactory AML Patient Characteristics. Characteristics of adult relapsed/refractory AML patients taking part in the study (Fig. 6l-n).

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Perry, J.M., Tao, F., Roy, A. et al. Overcoming Wnt–β-catenin dependent anticancer therapy resistance in leukaemia stem cells. Nat Cell Biol (2020). https://doi.org/10.1038/s41556-020-0507-y

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