Preclinical efficacy of prexasertib in acute lymphoblastic leukemia

Abstract

The addition of molecularly targeted therapies to current chemotherapy regimens may improve acute lymphoblastic leukemia (ALL) outcomes and reduce acute and late toxicities. Checkpoint kinase 1 (CHK1) orchestrates cell cycle checkpoint control in the setting of DNA damage. CHK1 is expressed in both T- and B-ALL and represents a promising therapeutic target. Herein, we show that prexasertib, a targeted CHK1 inhibitor, exhibits significant single-agent efficacy in vivo using ALL patient-derived xenograft (PDX) models and synergizes in vitro with a nucleoside analog. These results support further clinical testing of prexasertib in ALL.

Figure 1:. Prexasertib inhibits the proliferation of ALL cells in vitro and in vivo.

A-B. Dose-response curves for leukemia cell lines treated with different concentrations of Prexasertib. Leukemia cell viability was assessed after 48 hours using the CellTiter-Glo Luminescent Cell Viability Assay and IC50 values were calculated from the dose-response curves using Prism 9.0. C-D. To assess apoptosis, leukemia cells were treated with prexasertib or DMSO control for 24 hours and cell death assessed by staining with annexin-V antibody and a viability dye followed by flow cytometry. A representative flow cytometry plot is shown in (C). The annexin-V and annexin-V/viability dye positive populations were then used to calculate the percentage of apoptotic cells (D). Error bars represent the mean ± SD of three technical replicates. P: ****, <0.0001 by t-test. E-F. Leukemia cell lines (E) and primary leukemia samples (F) were treated with prexasertib for 48 hour or 24 hours, respectively. Cellular lysates were then harvested for immunoblotting. For the primary leukemia cells, the name denotes the immunophenotype of the sample. G-H. Kaplan-Meier survival curves for B-ALL PDX mice treated with prexasertib or vehicle control. NSG mice were transplanted with primary B-ALL cells (PRoXe Samples CBAB-62871-V1 (G) and CBAB-29894-V2 (H); 2x10⁶ cells; N=5 per group). When leukemia cells were detectable in the peripheral blood, mice were randomized and treated with prexasertib (10 mg/kg subcutaneous) or vehicle control twice daily for 3 days followed by 4 days without treatment for 4-5 weeks as illustrated in the figure. The log-rank (Mantel-Cox) test was used to compare the survival curves (P: **, < 0.01 (G & H)).

Figure 2:. The combination of prexasertib and gemcitabine exhibit synergy in vitro in ALL.

A-C. CEM (A), REH (B) and Jurkat (C) leukemia cells were treated for 48 hours with a combination of prexasertib and gemcitabine. Survival was assessed with the Cell-Titer-Glo assay. Color bars indicate % inhibition normalized to untreated cells. These data were then analyzed with SynergyFInder(13) and calculated δ values, or the deviation between the measured and expected combination dose–response curves, were used to generate 3D drug interaction landscapes.