The HIV latency reversing agent HODHBt inhibits the phosphatases PTPN1 and PTPN2

Abstract

Nonreceptor tyrosine phosphatases (NTPs) play an important role in regulating protein phosphorylation and have been proposed as attractive therapeutic targets for cancer and metabolic diseases. We have previously identified that 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhanced STAT activation upon cytokine stimulation, leading to increased reactivation of latent HIV and effector functions of NK and CD8 T cells. Here, we demonstrate that HODHBt interacted with and inhibited the NTPs PTPN1 and PTPN2 through a mixed inhibition mechanism. We also confirm that PTPN1 and PTPN2 specifically controlled the phosphorylation of different STATs. The small molecule ABBV-CLS-484 (AC-484) is an active site inhibitor of PTPN1 and PTPN2 currently in clinical trials for advanced solid tumors. We compared AC-484 and HODHBt and found similar effects on STAT5 and immune activation, albeit with different mechanisms of action leading to varying effects on latency reversal. Our studies provide the first specific evidence to our knowledge that enhancing STAT phosphorylation via inhibition of PTPN1 and PTPN2 is an effective tool against HIV.

Keywords: AIDS/HIV; Cytokines; Phosphoprotein phosphatases; Signal transduction.

Figure 1. HODHBt modulates the thermal stability of PTPN1 and PTPN2 in vitro.

(A) Compressed CETSA-MS results indicating changes in thermal stability of protein in both PBMCs and K562. The x axis represents amplitude (log₂ fold change), and the y axis represents effect significance (–log₁₀ [P value]). Statistically significant (P < 0.01) proteins in common between PBMCs and K562 cells are indicated. (B) Thermal melting curves for PTPN1, PTPN2, CRKL, STAT5, RPL7A, and β-actin in CD4 T cell lysates after treatment with 100 μM HODHBt versus 100 μM HBt (n = 2–4). (C) Purified PTPN1 catalytic domain and purified commercial PTPN2 catalytic domain CETSA after treatment with DMSO, 100 μM HODHBt, or 100 μM HBt (n = 2–3).

Figure 2. HODHBt is a mixed inhibitor of PTPN1 and PTPN2.

(A) HODHBt directly inhibits the catalytic activity of the catalytic domain of PTPN1and the catalytic domain of PTPN2 using a fluorogenic assay (n = 2). Data are shown as mean ± SD. IC₅₀ values calculated for 2 independent experiments. (B) Effect of HODHBt on PTPN1- and PTPN2-catalyzed fluorogenic PTPN1 substrate. (C) Lineweaver-Burk plots. Lineweaver-Burk plots represent the Michaelis-Menten equation of enzyme kinetics.

Figure 3. Inhibiting PTPN1 and PTPN2 with HODHBt enhances activation of different STATs in a cytokine-dependent manner.

(A and B) Analysis of phosphorylation levels of STAT-1, -3, and- 5 (A) or STAT-2, -4, and -6 (B) in primary total CD4 T cells after treatment with DMSO, 100 ng/mL IL-15, 1 ng/mL IFN-α, 2 ng/mL IL-12, and 2 ng/mL IL-4 in the presence of 100 μM HODHBt or HBt for 24 hours. (C) Levels of pSTAT2⁺ cells in naive CD4 T cells treated with 100 μM HODHBt ± 10 ng/mL IFN-α for 24 hours (n = 3). (D) Levels of STAT2 phosphorylation after transfection of V5-STAT2 into 293FT cells and treatment with DMSO or 100 μM HODHBt. (E) Dose response of STAT1/2 transcriptional activity mediated by IFN-α (left) and IFN-β (right) in the presence of 1, 10, or 100 μM HODHBt in HEK-Blue IFN-α/β cells. The data represent the mean ± SD of an experiment performed in triplicate. (F) Dose response of STAT1 transcriptional activity mediated by IFN-γ in the presence of 1, 10, or 100 μM HODHBt in HEK-Blue IFN-γ cells. The data represent the mean ± the SD of an experiment performed in triplicate.

Figure 4. AC-484 promotes immune activation and synergizes with IL-15 to reactivate latent HIV.

(A) Structure comparison of HODHBt and AC-484. (B and C) Measurement of STAT5 transcriptional activity (B) and toxicity (C) after treatment with dose response of HODHBt and AC-484 in HEK–Blue–IL-2/IL-15 cells. The data represent the mean ± SD of an experiment performed in duplicate. (D) Reactivation of latent HIV in Tcm cells measured by flow cytometry after treatment with 100 μM HODHBt or 10 μM AC-484 ± 100 ng/mL IL-15, or αCD3/CD28 (n = 10). Dunnett’s multiple-comparison test was used to calculate P values (*P < 0.05; **P < 0.01; ****P < 0.0001). (E) Bliss independence synergy calculations for reactivation. Wilcoxon matched-pairs signed-rank test was used to calculate P values (**P < 0.01). (F) PBMCs were treated with 100 μM HODHBt and a dose response of AC-484 ± 1 ng/mL IL-15 for 48 hours. CD69 induction was analyzed by flow cytometry in CD4 T cells, CD8 T cells, and NK cells (n = 4–8). Data are the average effect from 4–8 donors. Tukey’s multiple-comparison test was used to calculate P values (*P < 0.05; ***P < 0.001; ****P < 0.0001). (G) Secretion of pro- and antiinflammatory cytokine was measured using a 10-plex cytokine ELISA in supernatants from F.

Figure 5. HODHBt and AC-484 have differing effects on STAT5 phosphorylation.

(A) Total CD4s were pretreated with 100 μM HODHBt, 10 μM AC-484, and 5 μM AC-484 for 2 hours prior to the addition of IL-2. pSTAT5 levels were measured by flow cytometry 1, 24, and 48 hours after stimulation with IL-2 (n = 6). Wilcoxon matched-pairs signed-rank test was used to calculate P values (*P < 0.05; **P < 0.01). (B) Total CD4 T cells were pretreated with 100 μM HODHBt or 10 μM AC-484 for 2 hours prior to the addition of IL-15. pSTAT5 levels were measured by flow cytometry 48 hours after stimulation with IL-2 (n = 3). Paired 2-tailed t test was used to calculate P values (*P < 0.05; **P < 0.01). (C and D) Protein levels of PTPN1, PTPN2, pSTAT5, and STAT5 were measured by Western blot in CD4 T cells pretreated with 100 μM HODHBt or 20 μM AC-484 for 2 hours before the addition of IL-2 (C) and IL-15 (D) for 24 hours (n = 3). Paired 2-tailed t test was used to calculate P values (*P < 0.05).

Figure 6. Proposed mechanism of action of HODHBt and AC-484.

Created with Biorender.com.