Unraveling the impact of crizotinib to promote megakaryopoiesis for alleviating thrombocytopenia in myelodysplastic neoplasms

Abstract

Current therapeutic options for myelodysplastic neoplasms (MDS)-associated thrombocytopenia are limited. Megakaryocyte maturation might be an innovative therapeutic strategy because its dysregulation profoundly contributes to MDS pathogenesis. Here, we identified crizotinib, a clinically approved anti-cancer drug for anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer, as a potent inducer of megakaryocyte maturation. We demonstrated that crizotinib effectively induced polyploidization to increase the platelet-producing capacity of megakaryocytes derived from an MDS murine model and MDS patients by targeting Aurora kinases rather than its canonical targets, ALK/ROS1/c-MET. Importantly, crizotinib administration substantially ameliorated thrombocytopenia in our preclinical model. Our findings underscore the remarkable potential of crizotinib for drug repurposing and offer a novel therapeutic strategy for MDS patients with thrombocytopenia facing health-related quality of life concerns.

Fig. 1. MDS mice exhibit thrombocytopenia mainly related to defects in MK maturation.

A Platelet counts in peripheral blood from control mice (n = 11) and CBL^ΔE8/9/RUNX1^S291fs mice (n = 9). B Sizes of CD41⁺ cells in BM from the indicated mice (n = 8). C Ploidy of CD41⁺ cells in BM from the indicated mice (n = 8). D Histological analysis of MKs in BM from the indicated mice. BM sections stained with hematoxylin and eosin were imaged (left) and MK size was quantified by measuring the area (right). More than 200 cells were evaluated from the mice (n = 4). Scale bar, 25 µm. E Population of MKPs in BM from control mice (n = 12) and CBL^ΔE8/9/RUNX1^S291fs mice (n = 13). F Population of MEPs in BM from the indicated mice (n = 4). The gating strategies for B, C, E, and F were included in Supplementary Data 1. P-values were calculated using the Student’s two-tailed t-test (A–C, E, and F) and Welch’s two-tailed t-test (D).

Fig. 2. In vitro recapitulation of the disease phenotypes of MKs in MDS.

The phenotypes of MKs derived from ex vivo cultured HSPCs from MDS model mice. A Representative images of ex vivo cultured HSPCs from the indicated mice. Large cells in the upper panel were matured MKs derived from HSPCs. Scale bar, 400 µm. B Ploidy of MKs derived from ex vivo cultured HSPCs from the indicated mice. The data represent the mean ± s.d. of three independent experiments. C Diff-quik staining of ex vivo cultured HSPCs from the indicated mice (left). Scale bar, 50 µm. MK size was quantified using ImageJ (right). The data represent the mean ± s.d. of three independent experiments with >5 fields of view per sample. The gating strategy for B was included in Supplementary Data 2. P-values were calculated using the Student’s two-tailed t-test (B, C).

Fig. 3. Crizotinib promotes MK maturation in vitro.

Effects of crizotinib on phenotypes of MKs derived from ex vivo cultured HSPCs from MDS model mice and HSPCs transduced with CBL^ΔE8/9/RUNX1^S291fs. A Representative images of DMSO- or crizotinib-treated ex vivo cultured HSPCs from MDS mice. Scale bar, 400 µm. B Effect of crizotinib on the ploidy of MKs derived from ex vivo cultured HSPCs from MDS mice. The data represent the mean ± s.d. of three independent experiments. C Effects of crizotinib on the morphology of ex vivo cultured HSPCs from MDS mice. Scale bar, 50 µm. D The megakaryocyte colony-forming units (CFU-MK) in crizotinib-treated HSPCs from MDS mice. The data are from three independent mice. E Gene expression analysis of MK and platelet markers in CBL^ΔE8/9/RUNX1^S291fs–transduced HSPCs treated with DMSO or crizotinib for 48 h (n = 2, biological replicates). F Representative images of proplatelet formation in DMSO- or crizotinib- treated MKs derived from ex vivo cultured HSPCs from MDS mice (left; cyan, Hoechst 33342; red, beta I tubulin). Scale bar, 30 µm. Proplatelet-forming MKs were quantified across three independent experiments, analyzing more than 10 fields of view per condition (right). P-values were calculated using the paired two-tailed t-test (D) and the Student’s two-tailed t-test (B, F). n.s., not significant; **, P < 0.01.

Fig. 4. Crizotinib promotes MK maturation by inhibiting Aurora kinases, addressing the dysregulated cell cycling observed in MDS patients with thrombocytopenia.

A Effect of (S)-crizotinib on the ploidy of MKs derived from CBL^ΔE8/9/RUNX1^S291fs–transduced HSPCs. The data represent the mean ± s.d. of three independent experiments. B Effects of inhibitors against PLK1, and kinases to which crizotinib binds, on the ploidy of MKs derived from CBL^ΔE8/9/RUNX1^S291fs–transduced HSPCs. The data represent the mean ± s.d. of three independent experiments. C Effects of the indicated inhibitors on the phosphorylation levels of PLK1 T210, evaluated by flow cytometric analysis. The data represent the mean ± s.d. of five independent experiments. D The top 10 gene sets positively or negatively enriched in BM CD34⁺ cells from MDS patients with thrombocytopenia [PLT (platelet count) < 15 × 10⁴/µL, n = 10)] vs. those from HD (n = 5) by GSEA (Hallmark & Reactome gene sets). Gene sets related to cell cycle progression are highlighted in red. NES, normalized enrichment score. The top 100 lists were included in Supplementary Tables 1 and 2. (E) Platelet counts of the MDS patients and relative gene expression levels of AURKA, AURKB, PLK1, and PLK4 in cells analyzed in D. RPKM, reads per kilobase per million mapped reads. The blue-shaded area in the figure represents the normal range of platelet counts in healthy individuals. The gating strategy for C was included in Supplementary Data 3. P-values were calculated using one-way ANOVA with Tukey’s multiple comparison test [A (vs. DMSO), C (vs. DMSO)], one-way ANOVA with Dunnett’s multiple comparison test (B), and Welch’s two-tailed t-test (E). n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 5. Crizotinib promotes MK maturation in clinical human samples and MDS mice leading to increased platelet counts.

A Effect of crizotinib on the polyploidy of MKs derived from clinical human samples (n = 6). B Effects of vehicle (n = 6), crizotinib (n = 8), and alisertib (n = 6) on complete blood counts in MDS mice. WBC, white blood cell. RBC, red blood cell. C Flow cytometric analysis of ploidy of MKs in the BM of MDS mice administered vehicle (n = 10), crizotinib (n = 6), or alisertib (n = 9). D Flow cytometric analysis of the size of MKs in the BM of MDS mice administered vehicle (n = 16), crizotinib (n = 9), or alisertib (n = 9). E Histological analysis of hematoxylin and eosin-stained BM sections from MDS mice administered vehicle, crizotinib, or alisertib. MK size was quantified using the obtained images. Scale bar, 25 µm. More than 350 cells were evaluated from the indicated mice (n = 6). F Population of MKPs in the BM from MDS mice administered vehicle (n = 14), crizotinib (n = 10), or alisertib (n = 9). G Population of MEPs in the BM from MDS mice administered vehicle, crizotinib, or alisertib (n = 6). P-values were calculated using the paired two-tailed t-test (A), one-way ANOVA with Tukey’s multiple comparison test (B–E), and one-way ANOVA with Dunnett’s multiple comparison test (F, G).