Prexasertib, a Chk1/Chk2 inhibitor, increases the effectiveness of conventional therapy in B-/T- cell progenitor acute lymphoblastic leukemia

Abstract

During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed for the treatment of different type of cancers. In this study we evaluated the efficacy of the Chk 1/2 inhibitor prexasertib mesylate monohydrate in B-/T- cell progenitor acute lymphoblastic leukemia (ALL) as single agent and in combination with other drugs. The prexasertib reduced the cell viability in a dose and time dependent manner in all the treated cell lines. The cytotoxic activity was confirmed by the increment of apoptotic cells (Annexin V/Propidium Iodide staining), by the increase of γH2A.X protein expression and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). Furthermore, the inhibition of Chk1 changed the cell cycle profile. In order to evaluate the chemo-sensitizer activity of the compound, different cell lines were treated for 24 and 48 hours with prexasertib in combination with other drugs (imatinib, dasatinib and clofarabine). The results from cell line models were strengthened in primary leukemic blasts isolated from peripheral blood of adult acute lymphoblastic leukemia patients. In this study we highlighted the mechanism of action and the effectiveness of prexasertib as single agent or in combination with other conventional drugs like imatinib, dasatinib and clofarabine in the treatment of B-/T-ALL.

Keywords: CHK1; DNA damage response; acute lymphoblastic leukemia; cell cycle; chemo-sensitizer agent.

Figure 1. Effect of prexasertib on cell viability, induction of apoptosis, inhibition of Chk1 pathway and cell cycle profile in B-/T-ALL cell lines

Graphical representation of the IC₅₀ values of the B-/T-ALL cell lines after 24 and 48 hours of incubation with prexasertib. The IC₅₀ values were obtained from two independent experiments A. Cell cycle profile of B-/T-ALL cell lines treated with or without prexasertib (IC₅₀ value) for 24 hours B. Graphical representation of apoptosis induction by prexasertib. BV-173, NALM-6 and REH cells were treated with increasing concentration of drug for 24 and 48 hours C. The blots show, for each cell lines, the expression of key elements of the Chk1 pathway after 24 hours of incubation with prexasertib (IC₅₀ value) D. In the figure the samples named Control were cells treated with 0.1 % of DMSO. In the Western blot analysis the homogeneity of the protein loaded (30 μg) was determined by using an internal control (β-actin).

Figure 2. Effect of prexasertib in combination with TKIs in Philadelphia-positive cell lines

Cell viability analysis of BV-173 and SUP-B15 cell lines incubated with prexasertib and TKIs (imatinib and dasatinib) for 24 and 48 hours. For each cell lines three different experiments have been performed A. The blot shows the expression different proteins of the Chk1 pathway after 24 hours of incubation with prexasertib in combination with the two TKIs. The cell line BV-173 was incubated with: prexasertib: 7nM; imatinib: 500nM; dasatinib: 50nM B. Combination index analysis of BV-173 and SUP-B15 cell lines incubated with increasing concentration of prexasertib (BV-173: from 10 to 0.6 nM, dilution rate 1:2; SUP-B15: from 100 to 6.25 nM, dilution rate 1:2) and two increasing concentrations of imatinib (BV-173: 250 and 500 nM; SUP-B15: 5 and 10 μM) for 24 and 48 hours. The black curve (control) represents the effect of the prexasertib alone while the two red curves represent the two combinations (full square prexasertib + 250 nM (BV-173) or +5 μM (SUP-B15) of imatinib; full triangle prexasertib + 500 nM (BV-173) or 10μM (SUP-B15) of imatinib). The curves represent the mean of two independent experiments C. Normalized Isobologram graphs represent the effect (Combination Index, C.I.) of the combination between prexasertib and imatinib on BV-173 and SUP-B15 cell lines after 24 and 48 hours of combination. Each combination is represented in the legend with a specific symbol. D. In the figure the samples named Control were cells treated with 0.1 % of DMSO. In the Western blot analysis the homogeneity of the protein loaded (30 μg) was determined by using an internal control (β-actin).

Figure 3. Effect of prexasertib in combination with clofarabine in Philadelphia-negative cell lines

Cell viability analysis of Philadelphia-negative ALL cell lines incubated prexasertib and clofarabine for 24 and 48 hours. For each cell lines three independent experiments have been performed A. The blots show the expression on NALM-6 and REH cell lines of different proteins of the Chk1 pathway after 24 hours of treatment with prexasertib in combination with clofarabine. The dosages of each drug have been chosen based on the IC₅₀ values after 24 hours of incubation B. Combination index assay of NALM-6 and REH cell lines treated with increasing concentration of prexasertib (from 1.5 to 100 nM) and sub-toxic concentration of clofarabine (5 and 10 nM). The black curve (Control) represents the effect of the prexasertib alone while the two red curves represent the effect of the combinations (full triangle prexasertib + 5 nM of clofarabine; full square prexasertib +10 nM of clofarabine) after 48 hours of incubation with the two drugs. The curves in graph are representative of the mean of two independent experiments C. Normalized Isobologram graphs represent the effect (Combination Index, C.I.) of the combination between prexasertib and clofarabine on NALM-6 and REH cell lines. Each combination is represented in the legend with a specific symbol D. In the figure the samples named Control were cells treated with 0.1 % of DMSO. In the Western blot analysis the homogeneity of the protein loaded (30 μg) was determined by using an internal control (β-actin).

Figure 4. Effect of prexasertib on primary leukemic cells isolated from adult ALL patients and on peripheral mononuclear cells isolated from healthy donors

Cell viability analysis on primary leukemia cells isolated from 9 adult ALL patients treated with increasing concentration of prexasertib (100, 200 and 500 nM) for 24 hours. A. Cell viability assay of mononuclear cells isolated form the peripheral blood of 5 healthy donors, treated with increasing concentration of prexasertib (100, 200 and 500 nM) B. The blot shows the induction of DNA damages on primary leukemic cells isolated from the peripheral blood of 4 newly diagnosed ALL patients treated for 24 hours with prexasertib (100nM) C. The blot shows the expression of different proteins of the Chk1 pathways after 24 hours of incubation with prexasertib (100nM) on the mononuclear cells isolated from the peripheral blood of 5 healthy donors D. Cell viability assay of primary leukemia cells isolated from 3 Philadelphia-positive ALL patients treated with prexasertib (100nM) and imatinib (5 μM) for 24 hours. The graph represents the main of response to the combination of the three patients E. The blot shows the induction of DNA damages on primary leukemia cells isolated from a Philadelphia-positive patient after 24 hours of treatment with prexasertib (100nM) in combination with imatinib (5μM) F. In the Western blot analysis the homogeneity of the protein loaded (30 μg) was determined by using an internal control (β-actin).

Figure 5. Schematic representation of the effect of prexasertib on leukemic blast after the exposure to different genotoxic agent

The left side of the cartoon hypothesizes how leukemic cell could survive to chemotherapy drugs and to other genotoxic agent, activating the cell cycle checkpoint and arresting the cell cycle progression. The right side of the cartoon hypothesizes the mechanism of action of the compound in enhancing cell death and inducing checkpoint override after the exposure to different DNA damaging agents.