PTPσ inhibitors promote hematopoietic stem cell regeneration

Abstract

Receptor type protein tyrosine phosphatase-sigma (PTPσ) is primarily expressed by adult neurons and regulates neural regeneration. We recently discovered that PTPσ is also expressed by hematopoietic stem cells (HSCs). Here, we describe small molecule inhibitors of PTPσ that promote HSC regeneration in vivo. Systemic administration of the PTPσ inhibitor, DJ001, or its analog, to irradiated mice promotes HSC regeneration, accelerates hematologic recovery, and improves survival. Similarly, DJ001 administration accelerates hematologic recovery in mice treated with 5-fluorouracil chemotherapy. DJ001 displays high specificity for PTPσ and antagonizes PTPσ via unique non-competitive, allosteric binding. Mechanistically, DJ001 suppresses radiation-induced HSC apoptosis via activation of the RhoGTPase, RAC1, and induction of BCL-X_L. Furthermore, treatment of irradiated human HSCs with DJ001 promotes the regeneration of human HSCs capable of multilineage in vivo repopulation. These studies demonstrate the therapeutic potential of selective, small-molecule PTPσ inhibitors for human hematopoietic regeneration.

Fig. 1

DJ001 is a non-competitive, allosteric inhibitor of PTPσ. a Chemical structures of compounds 3071, 5205, and 5075. b Concentration-inhibition curves and IC₅₀ values for 3071, 5205, 5075, and control (DMSO) following incubation with PTPσ (n = 3). c Concentration-inhibition curves and IC₅₀ values for DJ001 and control following incubation with PTPσ (n = 3). d In silico three-dimensional docking of DJ001 (Z isomer) (represented as ball and stick in yellow color) to the PTPσ allosteric binding site located between domain 1 (green) and domain 2 (blue) of PTPσ. e At left, substrate titration reveals DJ001 as a non-competitive inhibitor that inhibits substrate catalysis (V_max), but not substrate binding of PTPσ (constant K_m). At right, plots of K_m and V_max as a function of compound concentration delivered from nonlinear regression in substrate titration. Error bars represent SEM. Source data are provided as Source Data File

Fig. 2

PTPσ inhibition promotes hematopoietic regeneration. a Mean numbers of CFCs from BM KSL cells following 300 cGy irradiation and culture for 3 days in TSF media ± DJ001 (n = 12). b Mean numbers of BM CFCs in Ptprs^+/+ and Ptprs^−/− mice at day + 10 following 600 cGy TBI (n = 12). c Mean numbers of PB WBCs, neutrophils (NEU), and lymphocytes (LYMPH) in mice at day + 10 following 750 cGy and treatment with DJ001 or 10% DMSO (vehicle) (n = 10). d Representative flow cytometric analysis of percentages of KSL cells and c-kit⁺sca-1⁻ progenitor cells at day + 10 post 750 cGy in the treatment groups shown. e Mean percentages and numbers of BM KSL cells and c-kit⁺sca-1⁻ cells at day + 10 post 750 cGy (n = 9). f Percent survival of mice after 750 cGy and treatment with DJ001 (27/29 alive) or vehicle (12/29 alive); Log-rank test. g Mean percentages of donor CD45.2⁺ cells in the PB of CD45.1⁺ recipient mice over time following transplantation of 5 × 10⁵ BM cells from CD45.2⁺ mice at day + 10 following 750 cGy TBI and treatment with DJ001 or vehicle, along with 1 × 10⁵ CD45.1⁺ BM competitor cells (n = 18–21/group). h Mean percentages of total donor CD45.2⁺ cells and CD45.2⁺ Mac1⁺Gr1⁺ myeloid cells, CD45.2⁺B220⁺ B cells, and CD45.2⁺CD3⁺ T cells in the PB of recipient mice at 20 weeks post transplantation (n = 18/group). i Mean numbers of PB WBCs and NEU counts in mice at day + 0 (pre-5FU), + 8, + 11, and + 14 following 5FU treatment (n = 4–7/group). Error bars represent SEM. Source data are provided as Source Data File. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

DJ001 promotes HSC regeneration via RAC1 activation and induction of BCL-X_L. a %p250GAP phospho-tyrosine (pTyr) in BM lin⁻ cells cultured ± DJ001 (media, n = 10; DJ001, n = 7). b At left, representative images of PLA in BM KSL cells treated ± DJ001. Red = DsRed⁺, PTPσ-p250GAP complex; blue = DAPI, original magnification ×63, scale bars = 10 µm). At right, numbers of DsRed⁺ KSL cells in each condition (n = 48, control; n = 36, DJ001). c %RAC1-GTP⁺ KSL cells from Ptprs^+/+ and Ptprs^−/− mice treated ± DJ001 (n = 5). Two-way ANOVA with Sidak’s multiple comparison test. d Percentages of p-PAK1⁺ KSL cells following treatment with media alone, 1 μg/mL DJ001, or DJ001 + 6 μg/mL EHT1864 (n = 5). One-way ANOVA with Tukey’s multiple comparison test. e At left, Annexin V/7AAD staining of BM KSL cells from mice at 24 h post 500 cGy and treatment with vehicle, 5 mg/kg DJ001, DJ001 + 40 mg/kg EHT1864, or EHT1864 alone. Numbers represent percentages in each gate. At right, %Annexin⁺7AAD⁻ BM KSL cells (media and DJ001, n = 9; DJ001 + EHT1864, n = 7; EHT1864, n = 4). f Percent survival of mice following 750 cGy and 10-day treatment with DJ001 (11/15 alive), vehicle (4/15 alive), or DJ001 + EHT1864 (2/15 alive; Log-rank test). g Fold changes (2^−ΔΔCt) of gene expression in BM KSL cells at 12 h post 300 cGy and treatment with or without DJ001 (n = 3–6/group). Two-way ANOVA with Sidak’s multiple comparison test. h At left, BCL-X_L protein levels in non-irradiated BM KSL cells (red), 300 cGy-irradiated KSL cells in media alone (black) or treated with DJ001 (blue) or DJ001 + EHT1864 (gray). At right, %BCL-X_L⁺ KSL cells (n = 6). One-way ANOVA with Tukey’s multiple comparison test. i CFCs from BM KSL cells at 48 h following 300 cGy and culture with and without DJ001, and with and without shRac1 or shBcl2l1 (n = 3–9/group). Two-way ANOVA with Sidak’s multiple comparison test. Error bars represent SEM. Source data are provided as Source Data File. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

DJ001 promotes HSC proliferation via induction of CDK2. a At left, representative cell cycle analysis of BM KSL cells at 36 h following 300 cGy and culture with media ± 1 μg/mL DJ001. At right, mean percentages of KSL cells in G₀ (Ki67⁻7AAD⁻), G₁ (Ki67⁺7AAD⁻) and G₂/S/M phase (Ki67⁺7AAD⁺) are shown (n = 7). b Fold changes (2^−ΔΔCt) of cell cycle regulatory gene expression in KSL cells at 36 h following 300 cGy and culture with media ± DJ001 (n = 3). Gene transcript changes are normalized to Gapdh and media treatment. c Fold changes (2^−ΔΔCt) of Cdk2 and Cyclin E expression in BM lin⁻ cells in response to DJ001 at 48 h after 300 cGy, with and without Rac1-shRNA treatment (n = 3–5). d Top panel shows representative cell cycle analysis of KSL cells at 36 h following 300 cGy and culture with media ± 1 µg/mL DJ001, DJ001 ± 6 µg/mL EHT1864, and DJ001 ± 240 ng/mL CDK2 inhibitor SU9516. Bottom panel shows the percentages of BM cells in G₀, G₁, and G₂/S/M phase for each condition (media, n = 6; DJ001 and DJ001 + SU9516 groups, n = 5; DJ001 + EHT1864, n = 8). Two-way ANOVA with Sidak’s multiple comparison test for a–c. One-way ANOVA with Tukey’s multiple comparison test for d. Source data are provided as Source Data File. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

DJ001 promotes human HSC regeneration following irradiation. a At left, representative flow cytometric analysis of CD34⁺CD38^- cells at 36 h after 300 cGy irradiation of BM CD34⁺ cells and culture with media ± 1 µg/mL DJ001. At right, mean percentages and numbers of CD34⁺CD38⁻ cells in each group (Day 0, n = 6; media, DJ001, n = 10). One-way ANOVA with Tukey’s multiple comparison test. b Numbers of CFU-GEMMs within cultures of CD34⁺ cells at 72 h after 300 cGy and culture with media ± DJ001 (n = 6). c At left, representative flow cytometric analysis of Annexin V/7AAD staining of CD34⁺ cells at 24 h after 300 cGy and culture with media ± DJ001 ± EHT1864. At right, percentages of live and apoptotic CD34⁺CD38⁻ cells in each condition (media and DJ001, n = 10; DJ001 + EHT1864, n = 5). One-way ANOVA with Tukey’s multiple comparison test. d Fold changes (2^−ΔΔCt) in BCL2L1 and MCL-1 gene expression in CD34⁺CD38⁻ cells at 12 h after 300 cGy in media ± DJ001 (n = 3). Gene transcript changes were normalized to GAPDH and media treatment. Two-way ANOVA with Sidak’s multiple comparison test. e Schematic representation of NSG mice transplantation assay using the progeny of human BM CD34⁺ cells irradiated with 300 cGy and treated with or without DJ001 × 36 h. f Representative flow cytometric analysis of human CD45⁺ cells, human CD34⁺ cells, human CD19⁺ B cells, human CD33⁺ myeloid cells, and human CD3⁺ T cells engraftment in the BM of NSG mice at 12 weeks post transplantation. g Percent engraftment of human hematopoietic cell subsets in the BM of NSG mice at 12 weeks post transplantation (n = 11–12/group). Source data are provided as Source Data File. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001