SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

Abstract

Activating mutations of Src homology-2 domain-containing protein tyrosine phosphatase-2 (Shp2) cause multiple childhood conditions for which there is an unmet therapeutic need, including juvenile myelomonocytic leukemia (JMML) and Noonan syndrome. SFX-01, an α-cyclodextrin-stabilized sulforaphane complex currently in clinical development, covalently adducts cysteine residues. Using unbiased proteomics, its protein targets were identified, including Shp2. SFX-01 induced an inhibitory dithiolethione modification at the Shp2 active site cysteine. Importantly, in a transgenic mouse model of human Noonan syndrome with hyperactive D61G Shp2, SFX-01 concomitantly normalized their phosphatase activity and myeloid cell count. Furthermore, SFX-01 also attenuated JMML human patient-derived hematopoietic stem cell proliferation that was linked to STAT1 signaling and decreased cyclin D1 expression, resulting in cell-cycle arrest. We conclude that SFX-01 is an activating mutant Shp2 inhibitor and may offer beneficial effects in patients with JMML or Noonan syndrome.

Keywords: Myeloproliferative Disorders; Noonan Syndrome; SFX-01; Shp2; Sulforaphane.

Figure 1. Detection and identification of sulforaphane-modified proteins.

(A, B) Immunoblot and immunofluorescence analysis showing multiple proteins in HEK 293 cells modified in a concentration-dependent manner by SFN upon treatment with R-, S- or R, S-isomers. (C) Immunoblot analysis showing multiple proteins modified by SFN in widespread tissues 3-h after oral gavage of WT mice with SFN or SFX-01. (D) A silver-stained polyacrylamide gel showing immunoprecipitation of SFN-adducted proteins from the cardiac tissue of WT mice using the anti-SFN antibody following oral gavage of the electrophile. (E) Volcano plot showing cardiac proteins in the upper right quadrant that were significantly and reproducibly modified by SFN in mice (n = 4 biological replicates). Source data are available online for this figure.

Figure 2. Sulforaphane adducts Shp2 to induce an inhibitory dithiolethione modification in vitro.

(A) Recombinant human Shp2 activity after 30-min incubation with SFX-01. Data represent mean activity ( ± SEM; n = 3 biological replicates) and were fitted to a one-phase exponential decay curve (gray line; r² = 0.987). (B) Shp2 activity after incubation with SFX-01 for the indicated times and concentrations. Data shown are mean activity ( ± SEM; n = 3 biological replicates). (C) Shp2 activity after 30-minute incubation with or without bisphosphorylated IRS1 and SFX-01. Bar represents mean activity (± SEM; n = 4 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. (D) Representative immunoblots showing SFN-modification of recombinant Shp2 (1.6 nM) following incubation with 1.75 or 0.109 µM SFX-01 for 30 min. (E) Precursor isotopic envelop spectrum of 0.1 µM recombinant human Shp2 protein incubated with equimolar SFN for 6 h at 37 °C corresponding to a dithiolethione modification adducted between Cys³³³ and Cys³⁶⁷. (F) Schematic representing the proposed mechanism of Shp2-dithiolethione formation by SFN. The isothiocyanate group reacts with a cysteine residue to form a dithiocarbamate intermediate, which further reacts with a second cysteine residue to yield the dithiolethione modification. (G) Immunoblot of immunoprecipitated WT or active site mutant Shp2 and SFN-modification from HEK cells treated with SFX-01. “E” represents non-transfected cells, and 0 h represents untreated cells. The graph represents densitometric analysis of Shp2-SFN adduct formation in WT or mutant Shp2 exposed to SFX-01 for 2 or 4 h. Bars represent mean values (± SEM; n = 3 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. Source data are available online for this figure.

Figure 3. SFX-01 inhibits wild-type and mutant Shp2 phosphatase activity in vivo.

Shp2 phosphatase activity in (A) cardiac tissue (n = 8 mice) and (B) spleen of WT (n = 4 mice) or Ptpn11^D61G(−/+) mice (n = 5 mice) that received SFX-01 in the drinking water for 10 days. (A, B) Bars represent mean activity ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. (C) Representative immunoblots showing the input and immunoprecipitated Shp2 protein and SFN-labeled Shp2 from cardiac tissue isolated from mice with or without oral SFX-01 administration as in (A). (D) Representative immunoblot of Shp2 immunoprecipitated from cardiac tissue of WT mice following treatment with SFX-01 for 4 days after incubation with biotin-iodoacetamide. (E) Representative far-western immunoblot of Shp2 immunoprecipitated from cardiac tissue of WT mice following treatment with SFX-01 for 4 days. The immunoblot was then exposed to biotin-phenylarsenic acid (PAA). Source data are available online for this figure.

Figure 4. SFX-01 is therapeutic against myeloid disease in Noonan syndrome mice.

(A) Changes in leukocyte cell populations in blood isolated from individual WT or Ptpn11^D61G(−/+) mice before and after 10 weeks of SFX-01 in drinking water. P values were calculated by paired t tests. For WT mice: n = 14 before and after vehicle treatment, n = 14 before SFX-01 treatment, and n = 15 after SFX-01 treatment. For Ptpn11^D61G(−/+) mice: n = 14 before vehicle treatment, n = 16 after vehicle treatment, n = 11 before SFX-01 treatment, and n = 14 after SFX-01 treatment. (B) Blood leukocyte cell population after 10 weeks SFX-01 treatment. (left) Bars represent mean leukocyte population ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 14 vehicle treatment, and n = 15 SFX-01 treatment. For Ptpn11^D61G(−/+) mice: n = 16 vehicle treatment, and n = 14 SFX-01 treatment. (right) Representative microphotographs of Wright-Giemsa-stained blood isolated from mice after 10 weeks SFX-01 treatment or controls. Scale bars show 200 µm. (C) Violin plots of blood myeloid-derived (CD11b⁺/Ly6G⁺ and CD11b⁺/Ly6C⁺) cell populations from WT or Ptpn11^D61G(−/+) mice after 10 weeks of SFX-01 in drinking water. Horizontal lines represent quartiles and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 13 vehicle or SFX-01 treatment. For Ptpn11^D61G(−/+) mice: n = 9 vehicle treatment, and n = 13 SFX-01 treatment. (D) Changes in spleen size from individual WT or Ptpn11^D61G(−/+) mice before and after 10 weeks of SFX-01 in drinking water. P values were calculated by paired t tests. For WT mice: n = 14 before and after vehicle treatment (P = 0.0102), n = 14 before and after SFX-01 treatment (P = 0.0890). For Ptpn11^D61G(−/+) mice: n = 13 before and after vehicle treatment (P = 0.0253), n = 15 before and after SFX-01 treatment (P = 0.1694) for each group. (E) Mean changes in spleen size in WT or Ptpn11^D61G(−/+) mice over 10 weeks of SFX-01 treatment compared to before treatment. Bars represent mean spleen size ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 14 vehicle or SFX-01 treatment. For Ptpn11^D61G(−/+) mice: n = 13 vehicle treatment, and n = 15 SFX-01 treatment. (F) Violin plots of spleen myeloid-derived (CD11b⁺/Ly6G⁺ and CD11b⁺/Ly6C⁺) cell populations from WT or Ptpn11^D61G(−/+) mice after 10 weeks of SFX-01 in drinking water. Horizontal lines represent quartiles and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 14 vehicle treatment, and n = 15 SFX-01 treatment (P = 0.6138). For Ptpn11^D61G(−/+) mice: n = 16 vehicle treatment, and n = 12 SFX-01 treatment (P = 0.0251). Source data are available online for this figure.

Figure 5. SFX-01 induces cell cycle arrest via hyperactive STAT1 signaling.

(A) Representative immunoblot of relative STAT1 phosphorylation and cyclin D1 expression in leukocytes isolated from WT or Ptpn11^D61G(−/+) mice after 10-week SFX-01 treatment. Graphs represent densitometric analyses of relative STAT1 (Ser 727) phosphorylation and cyclin D1 expression. Bars represent mean expression level ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. n = 5 mice for STAT1 (Ser 727) phosphorylation and n = 3 mice biological replicates for cyclin D1 expression. (B) Representative immunoblots of relative STAT1 phosphorylation and cyclin D1 expression in a Shp2-activating mutant expressing human GDM-1 cell line treated with SFX-01 or vehicle. Graph bars represent mean expression levels ( ± SEM; n = 4 biological replicates) and P values calculated by unpaired t test. (C) Cell cycle analysis of GDM-1 cells cultured with SFX-01. Bars represent mean percentage ( ± SEM), and significance compared to controls was calculated by one-way ANOVA with Dunnett’s test. n = 4 for vehicle or 250 µM SFX-01 treatment, n = 3 for 1, 5, 25, or 100 µM SFX-01 treatment, biological replicates. (D) Colony-forming ability of GDM-1 cells treated with SFX-01. Bars represent mean normalized values (± SEM), and because of the low sample size and not assuming a normal Gaussian distribution, significance was calculated using a Kruskal–Wallis test with an uncorrected Dunn’s test for comparison to the control group. n = 2 for vehicle treatment, n = 3 for 0.1 µM SFX-01 treatment (P = 0.3957), n = 3 for 1 µM SFX-01 treatment (P = 0.2167), n = 5 for 10 µM SFX-01 (P = 0.0245) and n = 3 for 100 µM SFX-01 (P = 0.0018) treatment, biological replicates. (E) Cell cycle analysis and (F) colony-forming ability of control (CB) or JMML patient-derived HSCs cultured with or without SFX-01. Clonogenicity of HSC was normalized to control cells, and bars represent mean ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. For (E): CB: n = 3 for vehicle, 5, or 25 µM SFX-01 treatment, and n = 2 for 100 µM SFX-01 treatment. JMML: n = 7 for vehicle or 5 µM SFX-01 treatment, and n = 8 for 25 or 100 µM SFX-01 treatment, biological replicates. For (F): CB: n = 14 for vehicle treatment, n = 4 for 10 µM SFX-01 treatment, n = 17 for 50 µM SFX-01 treatment, and n = 19 for 100 µM SFX-01 treatment. JMML: n = 6 for vehicle or 10 µM SFX-01 treatment, n = 11 for 50 µM SFX-01 treatment, and n = 12 for 100 µM SFX-01 treatment, biological replicates. (G) A heatmap displaying the significantly altered Shp2 substrates in CD11b+ myeloid bone marrow cells isolated from WT or Ptpn11^D61G(−/+) mice, under conditions with or without SFX-01 treatment based on data from the phosphoproteomics analysis (n = 3 biological replicates). Source data are available online for this figure.

Figure EV1. Combined treatment with SFX-01 and Shp2 did not accentuate their individual attenuation of proliferation.

CD11b+ bone marrow cells isolated from WT and Ptpn11^D61G(−/+) mice treated with SFX-01, SHP099 or both together. SFX-01 or SHP099 attenuated cell proliferation and this was not accentuated when these interventions were combined. Data are presented as means ( ± SEM; n = 5–6) with P values calculated by two-way ANOVA with Tukey’s multiple comparison test. *P < 0.00001 versus untreated WT or ^#P < 0.00001 versus untreated Ptpn11^D61G(−/+).