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Defining a therapeutic window for kinase inhibitors in leukemia to avoid neutropenia - PubMed

  • ️Sun Jan 01 2017

Defining a therapeutic window for kinase inhibitors in leukemia to avoid neutropenia

Kate McArthur et al. Oncotarget. 2017.

Abstract

Neutropenia represents one of the major dose-limiting toxicities of many current cancer therapies. To circumvent the off-target effects of cytotoxic chemotherapeutics, kinase inhibitors are increasingly being used as an adjunct therapy to target leukemia. In this study, we conducted a screen of leukemic cell lines in parallel with primary neutrophils to identify kinase inhibitors with the capacity to induce apoptosis of myeloid and lymphoid cell lines whilst sparing primary mouse and human neutrophils. We have utilized a high-throughput live cell imaging platform to demonstrate that cytotoxic drugs have limited effects on neutrophil viability but are toxic to hematopoietic progenitor cells, with the exception of the topoisomerase I inhibitor SN-38. The parallel screening of kinase inhibitors revealed that mouse and human neutrophil viability is dependent on cyclin-dependent kinase (CDK) activity but surprisingly only partially dependent on PI3 kinase and JAK/STAT signaling, revealing dominant pathways contributing to neutrophil viability. Mcl-1 haploinsufficiency sensitized neutrophils to CDK inhibition, demonstrating that Mcl-1 is a direct target for CDK inhibitors. This study reveals a therapeutic window for the kinase inhibitors BEZ235, BMS-3, AZD7762, and (R)-BI-2536 to induce apoptosis of leukemia cell lines whilst maintaining immunocompetence and hemostasis.

Keywords: apoptosis; hematopoietic progenitor cells; kinase inhibitors; leukemia; neutropenia.

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Conflict of interest statement

Conflicts of Interest The authors declare no financial conflicts of interest.

Figures

Figure 1
Figure 1. Effects of kinase inhibition on the proliferation of leukemia cells

A. Human leukemia cell lines were treated with duplicate serial dilutions of kinase inhibitors and the effect on cell viability was determined by Cell Titer Glo assay after 48 hours. Data shown represents the mean IC50 of each condition from 2 independent experiments. HL-60 cells were treated with serial dilutions of mTor/PI3K B. and MEK C. inhibitors and the effect on cell viability was determined after 48 hours. Data shown is the mean ± SEM of 3 independent experiments. Note, in some instances error bars are smaller than symbols displayed.

Figure 2
Figure 2. Viability of neutrophils in response to kinase inhibitors

A. & B. Viability analysis from live cell imaging of primary murine bone-marrow neutrophils primed with either G-CSF (A) or GM-CSF (B) in response to a panel of kinase inhibitors [μM] over 24 hours. Bar graphs display percentage viability relative to G-CSF- or GM-CSF-only primed controls at 12 hours. Red bars denote significance, p < 0.05 (corrected by Hommel's modification of a Bonferroni t test). C. Summary of data in A) & B) highlighting the kinase inhibitors that significantly altered neutrophil viability. D. Viability of saline-treated primary murine bone-marrow neutrophils compared to neutrophils primed with GM-CSF or G-CSF for 21 hours. E. EC50 values for PIK-75 and SNS-032 as calculated from kinetic live cell imaging data of murine bone-marrow neutrophils shown in F., concentrations as marked. For all above, data represent mean ± SEM from three independent samples.

Figure 3
Figure 3. Assessment of human neutrophil viability in response to kinase inhibitor treatment

Human peripheral blood neutrophils from three healthy donors were primed with G-CSF or GM-CSF for 1 hour before addition of selected kinase inhibitors (as shown) or DMSO (control) at concentrations as indicated. Neutrophil viability was assessed after 18 hours of treatment, by propidium iodide exclusion measured by flow cytometry. Mean ± SEM shown.

Figure 4
Figure 4. Neutrophils heterozygous for the Mcl-1 allele are more sensitive to CDK inhibition

A. SNS-032 and PIK75 induce Bak/Bax-dependent apoptosis in primary murine bone marrow neutrophils that is sensitized by haplo-insufficiency of Mcl-1. Dose-dependent changes to neutrophil viability induced by PIK-75 or SNS-032 were assessed by live cell imaging over 20 hours, with genotypes and concentrations as marked. EC50 values were calculated from the 12 hour time-point, data represent mean ± SEM with n = 3 per genotype. B. Immunoblot of Mcl-1 protein in bone marrow neutrophils from Mcl-1+/- mice, and from mouse embryonic fibroblasts as a control.

Figure 5
Figure 5. Viability of hematopoietic myeloid progenitor cells in response to kinase inhibitors or chemotherapeutics

A. & B. Viability analysis from live cell imaging of primary murine myeloid HPCs primed with either GM-CSF (A) or G-CSF (B) in response to a panel of kinase inhibitors [μM] over 24 hours. Significant results (p < 0.05) denoted in red (when adjusted for multiple comparison testing) or orange (not adjusted for multiple comparison testing). (C & D) Dose-dependent effects of common chemotherapeutics on GM-CSF-primed primary murine myeloid HPCs C. or GM-CSF-primed primary murine bone-marrow neutrophils D. as assessed by live cell imaging analysis (concentrations as marked). E. Comparison of G-CSF (red/orange) or GM-CSF (blue) priming of myeloid HPCs before treatment with chemotherapeutics as assessed by live cell imaging (concentrations as marked). Bar graphs display percentage viability relative to G-CSF- or GM-CSF-only primed controls at 12 hours. Mean ± SEM with n = 3 independent samples. * p < 0.05. F. Venn diagram representing compounds that induce apoptosis of all leukemia cell lines tested (EC50 < 1 μM), and their apoptosis-inducing potential on hematopoietic progenitor cells and neutrophils.

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