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Synergy between IDH1 inhibitor and hypomethylating agent
Figure 3 on previous page. Combination treatment with BAY1436032 and azacitidine strongly depletes leukemia stem cells in vivo through inhibition of MAP- kinase signaling and activation of myeloid differentiation. (A) Limiting dilution transplantation of bone marrow cells from IDH1 mutant patient-derived xenograft (PDX) mice treated with vehicle, azacitidine (1 mg/kg, subcutaneously, days 1-5), BAY1436032 (150 mg/kg, orally [p.o.], daily [q.d.], 4 weeks) or the sequential or simultaneous combination of BAY1436032 and azacitidine with the same doses as in the single agent treated mice. 2,000,000, 200,000, 20,000, 2,000, 200 or 20 human acute myeloid leukemia (AML) cells per mouse were transplanted into three recipient mice per cell dose. Leukemia stem cells (LSC) frequencies are shown (mean ± standard error of the mean [SEM], n=3). Mice with hCD45+ cells in peripheral blood after 8 weeks (>0.1%) were scored positive. The log-fraction plot of the limiting dilution model has been generated using the ELDA software; the median LSC frequencies are shown by solid lines and the 95% confidence inter- vals (CI) by dotted lines. The fold change in LSC frequency normalized to vehicle treated control mice is shown as a bar graph. The fold decrease in LSC compared to vehicle treated mice is indicated on top of the bars. (B) Unsupervised hierarchical clustering using euclidean distance of cells from bone marrow of IDH1 mutant PDX mice treated with vehicle, azacitidine (1 mg/kg, subcutaneously, days 1-5), BAY1436032 (150 mg/kg, orally [p.o.], daily [q.d.], for 4 weeks) or the simultaneous combination of BAY1436032 and azacitidine. Cells were harvested from bone marrow at 4 weeks after treatment and sorted for hCD45+ cells. Gene expression pro- filing using RNA was performed on Affymetrix Human HG_U133 Plus 2.0 microarrays (n=3 per group). (C) Principal component analysis of all treatment groups using the top 4,000 differentially expressed genes. (D) Gene set enrichment analysis (MSigDB version 6.0) showing the most enriched transcription factor target gene sets from the indicated treatment comparisons. NES: normalized enrichment score; * gene sets involved in MAP Kinase signaling; ** gene sets involved in RB/E2F sig- naling (E) Heatmap from gene expression levels of MAP kinase signaling genes, RB/E2F signaling genes and myeloid differentiation genes from the bone marrow of IDH1 mutant PDX1 mice treated with vehicle, azacitidine (1 mg/kg, subcutaneous [s.c.] sq, days 1-5), BAY1436032 (150 mg/kg, p.o., q.d., 4 weeks) or the simul- taneous combination of BAY1436032 and azacitidine. Gene expression was determined by quantitative RT-PCR relative to the housekeeping gene ABL and was nor- malized to gene expression in vehicle-treated cells (mean ± SEM, n=3 independent experiments). (F) Representative western blots of in vitro cultured HT1080, a fibrosarcoma cell line with an endogenous heterozygous IDH1 R132C mutation treated with vehicle, azacitidine, BAY1436032 or the simultaneous combination of BAY1436032 and azacitidine using antibodies against the indicated signaling proteins. (G) Inhibition of colony formation by the MEK1/2 inhibitor trametinib in colony-forming cell assays using primary human AML cells with wild-type IDH1 or mutant IDH1 (mean ± SEM). (H) Inhibition of colony formation by the CDK4/6 inhibitor abemaciclib in colony-forming cell assays using primary human AML cells IDH1 wild-type or IDH1 mutant (mean ± SEM). From the six patients with IDH1 mutant AML, three harbored a IDH1 R132H mutation and one each an IDH1 R132C, IDH1 R132L and IDH1 R132G mutation. *P<0.5; ***P<0.001, wt: wild-type, mut: mutant: AZA: azacitidine.
combination treatment, supporting the inhibitory effect of the combination therapy on LSC (Online Supplementary Figure S7).14 Gene set enrichment analysis revealed that the suppressed transcription factors in the combination treat- ment group were linked to MAP kinase and retinoblas- toma/E2F (Rb/E2F) signaling (Figure 3D, Online Supplementary Table S1-3). We also performed epigenome wide asscociation studies by DNA methylation arrays and compared them with the transcriptome obtained by RNA sequencing (RNASeq) in IDH1 R132C cells isolated from AML-PDX1 mice and treated in vitro with either DMSO, 500 nM azacitidine (day 1-4), 50 nM of BAY1436032 (day 1-5), and sequential or simultaneous treatments with BAY1436032 and azacitidine. Principal component analysis showed that sequentially treated cells separated well from simultaneously treated cells (Online Supplementary Figure S8A-B). The simultaneous treatment induced strong syner- gistic gene expression (S-value >0), while the gene expres- sion was mostly antagonistic in the sequential treatment group (S-value <0; Online Supplementary Figure S8C). The simultaneous treatment overall led to increased methyla- tion compared to sequential treatment (Online Supplementary Figure S8D). Gene set enrichment analysis revealed that the suppressed transcription factors in the simultaneous treatment group were linked to MAP kinase signaling (RAS/RAF), while transcription factors enriched in single treatments were linked to Rb/E2F signaling (Online Supplementary Figure 8E). In concordance with gene expres- sion, an epigenome-wide association showed increased methylation of transcription factors involved in MAP kinase (RAS/RAF) signaling upon simultaneous treatment, while it showed decreased methylation of gene sets involved in RB/E2F signaling upon single agent treatment (Online Supplementary Figure 8F). Gene expression analysis by quantitative RT-PCR validated an additive suppression of MAP kinase (ELK1, ETS1 and CCND1) and E2F signaling (E2F1, CCNA2 and CCNE1) by the combination treatment, which are involved in cell survival and proliferation. Myeloid differentiation genes (PU.1, CEBPA and GABPA) were upregulated by the combination of BAY1436032 and azacitidine, which might be due to the higher proportion of differentiated human CD45+ cells and may not be a direct effect of the drugs. (Figure 3E, Online Supplementary Figure
S9A-C). We further validated our findings at protein level in HT1080, a fibrosarcoma cell line with an endogenous het- erozygous IDH1 R132C mutation. The phosphorylation of ERK1/2 and expression of its downstream target proteins ELK1 and CYCLIN D1 was decreased in cells simultaneous- ly treated with BAY1436032 and azacitidine. Importantly, the phosphorylation of retinoblastoma (pRB), which is nec- essary for E2F mediated transcription of cell cycle transition genes, was strongly decreased in cells treated with the com- bination of BAY1436032 and azacitidine, while E2F1 pro- tein levels were not affected (Figure 3F). We next evaluated if targeting the MEK pathway or cyclin dependent kinases alter the proliferation of IDH1mut AML cells using using the MEK1/2 inhibitor trametinib and the CDK4/6 inhibitor abemaciclib. Trametinib 4-fold more potently inhibited colony-forming potential in IDH1 mutant compared to IDH1 wild-type patients (Figure 3G). Similarly, the CDK4/6 inhibitor abemaciclib inhibited the colony-forming poten- tial 3-fold more potently in IDH1 mutant compared to IDH1 wild-type patients (Figure 3F). This data suggests syn- ergy between BAY1436032 and azacitidine in induction of differentiation through transcriptional activation of myeloid differentiation genes and inhibition of proliferation and self-renewal through inhibition of MAP-kinase and RB/E2F signaling.
Discussion
Here we provide preclinical evidence that the simultane- ous combination of a mIDH1 inhibitor with a hypomethy- lating agent significantly inhibits LSC in a synergistic man- ner. While the phase I dose-escalation study of mIDH1 inhibitor ivosidenib has shown encouraging results, 79% of IDH1 mutant AML patients who had a complete remis- sion or complete remission with partial hematologic recov- ery had detectable allele burden of mutant IDH1, suggest- ing that the IDH1 mutant clone was not eradicated.7 It was shown previously that BAY1436032 has antileukemic activity, induces differentiation and depletes LSC 100-fold during a 4-week treatment.10 However, BAY1436032 nei- ther has anti-leukemic activity nor induces myeloid differ- entiation in an IDH1 wild-type AML PDX model in vivo.10
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