SHIPi improves hematologic recovery after chemotherapy

Abstract

The use of recombinant growth factors has led to improved hematopoietic function after chemotherapy, but hematologic and immune complications still occur after chemotherapy which can be life-threatening. Here we show that pan-SHIPi compounds can induce endogenous G-CSF and TPO production in vivo and can also increase host survival after a lethal fungal challenge. In addition, we show that consistent with their ability to induce G-CSF, pan-SHIPi compounds can promote increased granulopoiesis in normal mice and speed recovery of neutrophil counts after chemotherapy. We also report the identification of a novel SHIP1-selective inhibitor, A32, that also increases steady state production of both G-CSF and TPO. These findings indicate small molecule inhibitors of SHIP1 can promote hematologic recovery after myeloablative chemotherapy and are a unique means to induce endogenous production of multiple growth factors that promote hematologic recovery.

Keywords: 5-FU; A32; Candida albicans; Chemotherapy; K161; Neutrophils; SHIP1; SHIPi.

Fig. 1

Pan-SHIPi compounds of different chemical classes increase steady-state neutrophil production and function in control of Candida albicans infection. A-C All compounds were dosed at 10 mg/kg via i.p. injection for 7 days with neutrophils counts in peripheral blood determined on day 8 by automated Hemavet analysis. Each point on the scatterplots in A-C indicate a value for an individual mouse. D A novel SHIP inhibitor K185 protects mice from lethal i.v. C. albicans challenge (1 × 10.⁶ cfu). Mice were treated with K185 (10 mg/kg, i.p., n = 9) or Vehicle (saline:DMSO, n = 8) on days −8, −7 and −1 and on day 0 challenged with 1 million C. albicans by i.v. injection. The significance of survival difference between vehicle and K185 dosed mice was determined by a Mantel-Cox log-rank test. The data in all panels are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

K161 enhances TPO production and blood cell recovery after chemotherapy. Mice were treated daily for 5 days with 5-FU at 35 mg/kg by intraperitoneal injection, followed by 6 daily subcutaneous injections with 30 mg/kg of K161 or vehicle (H₂O). A Survival of the K161 and vehicle (H20) treated mice after a 5 day regimen of 5FU (35 mg/kg).(p > 0.05, Mantel-Cox log-rank test) (B) Plasma thrombopoietin (TPO) is significantly increased by K161 treatment at 3 and 5 days following 5FU treatment. C Neutrophils (NE) production is significantly increased on days 8 and 15 post-5FU with K161 treatment. Production of leukocytes (D, WBC), monocytes (E, MO), eosinophils (F, EO) and basophils (G, BA) are increased at 15 days post-5FU in the K161 treated group vs. vehicle controls. H Red blood cell (RBC) recovery is also significantly lower at day 8 post-5FU, but reduced at day 15 vs. vehicle controls. No significant effect was observed on lymphocyte (I, LY) or platelet counts (J, PLT) post-5FU. Significance of the above blood counts was assessed by a two-way ANOVA, *p < 0.05, ***p < 0.001, ****p < 0.0001, n = 18/group

Fig. 3

A32 is a potent SHIP1 inhibitor that induces G-CSF and TPO in vivo. A A32 chemical structure. B Computational docking (AutoDock) of A32 with the SHIP1 enzyme domain (PDB:6IBD) (A32 magenta, SHIP1 blue) and (C) the active site of SHIP1. Predicted hydrogen bonds of A32 with active site amino acid residues are indicated. Malachite Green enzyme assay data for A32 with (D) recombinant SHIP1 enzyme and (E) SHIP2 enzyme. The Malachite Green assays in C for SHIP activity are representative of 2–3 independent experiments performed independently by two different researchers. F G-CSF and (G) TPO are increased in plasma of A32 treated mice one day after IP administration of A32 (30 mg/Kg) as compared to vehicle-treated (DMSO) control mice. The data in (F) is representative of two independent experiments with 4–5 mice/group while the study in (G) was performed once with 5 mice/group. The AI screen that initially identified A32 as binding to the SHIP1 active site is based on neural net algorithm and screening approach described in (Atomwise 2024). Subsequent computational docking of A32 to the SHIP1 enzyme domain and active site was done using Autodock. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001