Shikonin Overcomes Drug Resistance and Induces Necroptosis By Regulating the Mir-92a-1-5p/Mlkl Axis in Chronic Myeloid Leukemia Cells

BACKGROUND Resistance to cell death and metabolic reprogramming are common features of tumor cells. Although the introduction of selective BCR/ABL tyrosine kinase inhibitors (TKIs) has dramatically improved the outcomes and survival rates of chronic myeloid leukemia (CML) patients, some patients (20-30%) develop TKI resistance. The most aggressive and treatment-resistant CML is the subtype harboring BCR/ABL with the T315I mutation, and this subtype is refractory to nearly all TKI-induced apoptosis. Thus, alternative approaches that induce apoptosis-independent cell death are thought to compensate for apoptotic-resistant cells. Recently, necroptosis (also called programmed necrosis), which is generally driven by RIPK1/RIPK3/MLKL activation, has been demonstrated to be a new type of programmed cell death mode that is different from apoptosis. Thus, a deeper understanding of the molecular mechanisms regulating necroptosis might lead to the development of new therapeutic strategies that could remarkably improve the treatment-responses and outcomes of patients with TKI-resistant CML. RESULTS Shikonin, a compound purified from traditional Chinese medicine, has been reported to induce cell death in various tumor cell lines via a wide range of mechanisms. In our current study, we found that shikonin can effectively inhibit proliferation and induce necrosis-like morphological alterations (Fig. A and B) accompanied by RIPK1/RIPK3/MLKL signaling activation ((Fig. C) in CML cell lines, including the T315I mutant type (32Dp210-T315I). The effects of shikonin can be attenuated by the necroptosis-specific inhibitor (essentially a RIPK1 inhibitor) Nec-1, but not by the pan-apoptosis inhibitor z-VAD-fmk, indicating the occurrence of necroptosis in these cells ((Fig. B and C). Our data also show that shikonin has in vivo anti-CML activity via necroptosis induction in 32Dp210-T315I cells xenografted into NOD/SCID mice via subcutaneous injection ((Fig. D). miRNAs play an important role in tumorigenesis mainly via regulation of gene expression. Our next generation sequencing-based microRNA expression profiling showed significant dysregulation of miR-92a-1-5p expression in a shikonin-treated CML cell line (K562) (Fig. E). We then measured the miR-92a-1-5p expression levels in bone marrow samples from CML patients and patients with nonhematologic malignant diseases. The data showed that the miR-92a-1-5p expression level was higher in primary cells obtained from CML-BC patients than in those from non-CML-CP patients, suggesting that miR-92a-1-5p upregulation is correlated with poor outcomes (Fig. F). Bioinformatics analyses and a dual luciferase reporter gene assay proved that MLKL, a downstream factor in the necroptosis pathway that usually acts as the necroptosis executor, is a direct target of miR-92a-1-5p (Fig. G). Overexpression of miR-92a-1-5p in vitro led to decreased MLKL protein abundance in CML cells (Fig. G). Inhibition of miR-92a-1-5p via use of a specific antago-miRNA could inhibit CML xenograft tumor growth and induce necroptosis via MLKL upregulation in vivo (Fig. H). Hence, we believe that miR-92a-1-5p plays a role in promoting the proliferation and survival of CML via downregulating the abundance of MLKL, the necroptosis executor. CONCLUSIONS In conclusion, our study proves that shikonin can overcome TKI resistance and induce necroptosis in CML cells, mainly via a mechanism involving RIPK1/RIPK3/MLKL activation. Our study also suggests that miR-92a-1-5p is frequently overexpressed in CML patients with poor outcomes and that it can promote tumor survival by inhibiting MLKL expression. For the first time, we demonstrated that miR-92a-1-5p inhibition via antago-miRNA can potentially be used to treat CML via necroptosis induction. Since necroptosis has not yet been considered to be a therapeutic strategy for tumor treatment, our research confirms that it might indeed serve as a new modality to better control drug-resistant CML. Download : Download high-res image (1MB) Download : Download full-size image Figure . Disclosures No relevant conflicts of interest to declare.