A first-in-class Wiskott-Aldrich syndrome protein activator with antitumor activity in hematologic cancers

Abstract

Hematologic cancers are among the most common cancers in adults and children. Despite significant improvements in therapies, many patients still succumb to the disease. Therefore, novel therapies are needed. The Wiskott-Aldrich syndrome protein (WASp) family regulates actin assembly in conjunction with the Arp2/3 complex, a ubiquitous nucleation factor. WASp is expressed exclusively in hematopoietic cells and exists in two allosteric conformations: autoinhibited or activated. Here, we describe the development of EG-011, a first-in-class small molecule activator of the autoinhibited form of WASp. EG-011 possesses in vitro and in vivo antitumor activity as a single agent in lymphoma, leukemia, and multiple myeloma, including models of secondary resistance to PI3K, BTK, and proteasome inhibitors. The in vitro activity was confirmed in a lymphoma xenograft. Actin polymerization and WASp binding were demonstrated using multiple techniques. Transcriptome analysis highlighted homology with drugs inducing actin polymerization.

Figure 1.

The chemical structure and synthetic route of EG-011. (A) The chemical structures of EG-011 and ibrutinib and the structural differences between them. (B) The synthetic route of EG-011.

Figure 2.

EG-011 has strong in vitro antilymphoma activity. (A) In vitro cell proliferation assay, represented as half maximal inhibitory concentration (IC₅₀) values, calculated after 72 h of treatment, in 62 lymphoma cell lines. Cell lines are colored differently based on the different histological subtype. On the right, a representative dose-response curve in one sensitive and one resistant cell line. (B) The distribution of IC₅₀ values after treatment with EG-011 among the different subtypes of lymphoma. Each dot represents one cell line with the respective IC₅₀. Numbers on the bottom represents the number of cell lines present in each subtype group. (C) Percentages of apoptotic cells, determined by annexin V staining, after 48 h or 72 h of EG-011 treatment (100 nM and 500 nM). The average and standard deviation of at least two independent experiments are shown. (D) Cell cycle changes after 72 h of EG-011 treatment (500 nM and 2 µM) in two sensitive lymphoma cell lines. The average and standard deviation of at least two independent experiments are shown. (E) Percentages of apoptotic cells, shown by annexin V staining, after 24 h and 48 h of EG-011 treatment in two primary cells from healthy controls. *P<0.05; **P<0.01. ABC: activated B-cell-like; DLBCL: diffuse large B-cell lymphoma; GCB: germinal center B-cell-like; MZL: marginal zone lymphoma; MCL: mantle cell lymphoma; ALCL: anaplastic large cell lymphoma; CTCL: cutaneous T-cell lymphoma; HL: Hodgkin lymphoma; PTCL: peripheral T-cell lymphoma; PLL: prolymphocytic leukemia; PMBCL: primary mediastinal B-cell lymphoma; ATCL: adult T-cell leukemia/lymphoma; DMSO: dimethylsulfoxide.

Figure 3.

EG-011 is active in models of secondary resistance to Food and Drug Administration-approved compounds. (A-F) EG-011 is active in cell lines derived from splenic marginal zone lymphomas (A, B) and multiple myeloma (C-F) showing secondary resistance to Food and Drug Administration (FDA)-approved compounds. Dose-response curves after 72 h of EG-011 treatment in cell lines with acquired resistance to FDA-approved compounds compared to parental cell lines. Splenic marginal zone lymphoma cell lines: (A) VL51, parental, resistant to idelalisib, ibrutinib, or copanlisib; (B) Karpas1718 parental or resistant to idelalisib. Multiple myeloma cell lines: (C) AMO-1; (D) L363; (E) ARH77; (F) RPMI-8226 parental, resistant to proteasome inhibitors carfilzomib and bortezomib. res: resistant; WT: wild-type; CFZ: carfilzomib; BTZ: bortezomib.

Figure 4.

EG-011 has in vivo antilymphoma activity. (A) In vivo activity of EG-011 (200 mg/Kg, intraperitoneal, 10 mice) compared to control vehicle (intraperitoneal, 9 mice) in NOD-SCID mice. The upper graph represents the mean tumor volume (mm) with standard error of mean for each day. The lower graph represents the growth of each individual animal under treatment with the vehicle control vehicle or EG-011. (B) Tumor weights (mg) of control- and EG-011-treated mice at the end of the treatment (day 9). P values are calculated with the Mann-Whitney test. *P<0.05; ***P<0.001.

Figure 5.

EG-011 is a first-in-class Wiskott-Aldrich syndrome protein activator. (A) Melting curves of Wiskott-Aldrich syndrome protein (WASp) under treatment with dimethylsulfoxide (DMSO) vehicle control (dashed lines) or EG-011 (solid lines). Protein lysate from an EG-011-sensitive cell line (REC1) was subjected to a thermal shift assay performed at temperatures between 37°C to 67°C in the presence or absence (vehicle) of EG-011. The unfolding profile shows a high decrease of solubility of WASp in the presence of EG-011 versus vehicle. The melting temperature is marked with an “X” for each melting curve. Two biological replicates. (B) Pyrene actin polymerization measured as fluorescence intensity. EG-011 was compared to actin only, actin plus Arp2/3 complex and WASp, and the negative control LV009. Data are displayed as normalized fluorescence with the standard error of mean. Average of at least two independent experiments. (C) Representative confocal images of sensitive lymphoma cell lines treated for 8 h with DMSO or EG-011 (500 nM and 5 µM). Quantification of mean fluorescence intensity (ImageJ) per cell line in a 20X image (Leica widefield microscope) with around 200 cells. Experiments were performed in at least duplicate. Cells were stained for actin filaments using Alexa Fluor™ 488-labeled phalloidin (green channel). Cells were counter-stained with DAPI (blue channel). (D) Phalloidin mean fluorescence intensity after EG-011 treatment (500 nM and 5 µM) at three timepoints (4, 8 and 24 h) compared to DMSO treatment in the sensitive cell line. The Kruskal-Wallis test, followed by Dunn multiple comparisons, was performed. (E) Number of high fluorescence intensity spots in a sensitive lymphoma cell line after EG-011 treatment (500 nM and 5 µM) at three timepoints (4, 8, and 24 h) compared to DMSO. Cells are stained with phalloidin in order to visualize filamentous actin (F-actin). A ratio paired t test was performed. (F) Baseline levels of active WASp in sensitive and resistant cell lines. Cells are stained with antibody recognizing the active form of WASp. (G) Increased levels of active WASp after 8 h of EG-011 treatment (500 nM and 5 µM). All experiments are performed in at least duplicate. The Kruskal-Wallis test, followed by Dunn multiple comparisons, was performed. NS: non-significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001