Abstract
Although standard induction therapy initially elicits a promising response in the majority of acute myeloid leukemia (AML) patients, the majority relapse. Leukemia stem cells (LSCs) that survive chemotherapy are believed to be responsible for AML relapse. Therefore, new therapies that eliminate LSCs are desperately needed.
ONC201 is a TRAIL inducer and the founding member of the imipridone family. It has been shown to induce apoptosis in LSCs (Ishizawa et al, Science Signaling. 2016; 9:ra17). ONC201 was chemically modified to increase the potency and selectivity against cancer cells, resulting in the new analog ONC213. In this study, we investigated the antileukemic activity and the underlying molecular mechanism of ONC213 in preclinical AML models.
ONC213 activity in AML cell lines and primary AML patient samples was first tested in vitro. MTT assay results revealed that ONC213 IC50s ranged from 91.7 nM to 2.4 µM in AML cell lines and primary AML patient samples, which are achievable in vivo based on results from a PK study in mice (a single dose of 50 and 100 mg/kg ONC213 resulted in peak plasma concentrations of 3.7 μM and 8 μM, respectively). Annexin V/propidium iodide staining and flow cytometry analysis results showed variable responses for the AML cell lines tested. After 48 h treatment with 500 nM ONC213, striking induction of cell death in MOLM-13 and MV4-11 cells was detected (at least 72% Annexin V+ cells), while THP-1 and U937 cells showed little to no increase in Annexin V+ cells (6-11%). Similar results were obtained in primary AML patient samples. In contrast to the 48 h treatment of THP-1 and U937 cells, increasing the treatment duration to 120 h resulted in greater than 50% Annexin V+ cells, suggesting that a longer exposure time is necessary in some cell lines. In MV4-11 and MOLM-13 cells, initiation of cell death was detected 8 to 12 h post ONC213 treatment. Colony formation assays revealed that ONC213 treatment significantly reduced colony formation capacity of primary AML patient samples to less than 5% compared to vehicle control, while having no significant effect on normal hematopoietic progenitor cells. A primary AML patient sample was treated with or without ONC213 for 48 h, transplanted into NSG mice, and ten weeks later bone marrow was harvested and human CD45+ cells were measured. ONC213 treatment significantly reduced human AML engraftment compared to vehicle control (0.6% vs. 21.3%; p<0.05), demonstrating that ONC213 kills LSCs in vitro. Next, we examined in vivo efficacy of ONC213 against an AML cell line derived xenograft mouse model. MV4-11 cells were injected into NSGS mice through the tail vein. Three days post-injection, the mice were randomized into vehicle control or 125 mg/kg ONC213 cohorts (5 mice per cohort) and treated daily for 8 days. Modest weight loss was noted but was entirely manageable. ONC213 treatment extended the survival of mice by 88% (median survival 62 vs 33 days).
Unlike ONC201, ONC213 treatment of AML cells did not increase the expression of TRAIL. Interestingly, RNAseq results showed that 500 nM ONC213 treatment for 48 h downregulated 33 mRNAs in the oxidative phosphorylation (OXPHOS) pathway, suggesting that ONC213 treatment decreases OXPHOS in AML cells. Thus far, six of the downregulated mRNAs (UQCRQ, SDHA, COX6C, NDUFS5, ATP5D, and NDUFB1) were verified by real-time RT-PCR after both 8 h and 48 h ONC213 treatment. LSCs have been shown to be highly reliant on OXPHOS, while normal hematopoietic stem cells and some bulk AML cells can switch to glycolysis for ATP production during times of OXPHOS inhibition. Thus, ONC213 may kill LSCs through inhibition of OXPHOS.
In addition to downregulation of OXPHOS related genes, we found that ONC213 treatment downregulates Mcl-1. Since Mcl-1 mediates resistance to the promising Bcl-2-selective inhibitor ABT-199 (Venetoclax) and inhibition of Bcl-2 impairs OXPHOS, ONC213 would likely synergize with ABT-199 in AML cells. Indeed, combined treatment resulted in striking synergistic induction of apoptosis in both AML cell lines and primary patient samples. Enhanced cell death was detected 8 h post combination treatment in both MOLM-13 and MV4-11 cells. Results from colony formation assays revealed that the combination spares normal hematopoietic progenitor cells. Taken together, ONC213 is active as a single agent and in combination with ABT-199 in AML.
Disclosures
Allen: Oncoceutics: Employment. Stogniew:Oncoceutics: Employment. Prabhu:Oncoceutics: Employment. Ge:MEI Pharma: Research Funding.
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