Micro-RNA-125a mediates the effects of hypomethylating agents in chronic myelomonocytic leukemia

Background Chronic myelomonocytic leukemia (CMML) is an aggressive hematopoietic malignancy that arises from hematopoietic stem and progenitor cells (HSPCs). Patients with CMML are frequently treated with epigenetic therapeutic approaches, in particular the hypomethylating agents (HMAs), azacitidine (Aza) and decitabine (Dec). Although HMAs are believed to mediate their efficacy via re-expression of hypermethylated tumor suppressors, knowledge about relevant HMA targets is scarce. As silencing of tumor-suppressive micro-RNAs (miRs) by promoter hypermethylation is a crucial step in malignant transformation, we asked for a role of miRs in HMA efficacy in CMML. Results Initially, we performed genome-wide miR-expression profiling in a KrasG12D-induced CMML mouse model. Selected candidates with prominently decreased expression were validated by qPCR in CMML mice and human CMML patients. These experiments revealed the consistent decrease in miR-125a, a miR with previously described tumor-suppressive function in myeloid neoplasias. Furthermore, we show that miR-125a downregulation is caused by hypermethylation of its upstream region and can be reversed by HMA treatment. By employing both lentiviral and CRISPR/Cas9-based miR-125a modification, we demonstrate that HMA-induced miR-125a upregulation indeed contributes to mediating the anti-leukemic effects of these drugs. These data were validated in a clinical context, as miR-125a expression increased after HMA treatment in CMML patients, a phenomenon that was particularly pronounced in cases showing clinical response to these drugs. Conclusions Taken together, we report decreased expression of miR-125a in CMML and delineate its relevance as mediator of HMA efficacy within this neoplasia.


Supplementary
Graphs denote the mean +/-SD of at least three independent experiments. Comparisons against the control condition were performed using a one-sample t test against a reference value of 1.

Supplementary Fig S5.
The miR-125a upstream/promoter region is demethylated after HMA treatment in primary patient specimens. Database analysis of genome-wide DNA methylation profiling after treating eight primary acute myeloid leukemia (AML) patient specimens with either cytarabine (AraC) or decitabine (https://www.ncbi.nlm.nih.gov/geo/; GSE40870). In more detail, Klco et al. [2] performed genome-wide DNA methylation profiling using an Illumina HumanMethylation450 BeadChip in cytarabine/decitabine-treated specimens and compared the results to control-treated samples. Methylation of each CpG-site was displayed as methylation β-value, a value ranging from 0 to 1 (with 1 representing the maximum methylation of a CpG-site). Three CpG-sites within the upstream/promoter region of miR-125a were identified (cg25417766, cg02476580, and cg06529181); however, only one of them Subsequently, apoptosis was assessed by Annexin-V/7AAD assay. The respective control situations (treated with the empty dissolvent only) were set at a value of 1, and the relative increase of apoptosis in the Aza-treated conditions was calculated using the ratio Aza-treated to control-treated cells. Cells were considered apoptotic when they stained positive for Annexin-V or Annexin-V/7AAD. Graphs represent the mean +/-SD of three independent experiments. Comparisons against the control condition were performed using a one-sample t test against a reference value of 1.
In combination with the data from Supplementary Fig S4, these data demonstrate that lower concentrations of Aza cause the rapid increase of miR-125a expression as well, but fail to have an immediate effect on apoptosis. Importantly, however, the effects on apoptosis are seen at a later time-point in this situation. As miR-125a overexpression causes apoptosis as well Cells were seeded at a density of 1x10 5 /ml in serum-starved media containing 5% FBS.

Subsequently, the number of viable cells was counted on four consecutive days. (B)
BrdU/7AAD cell cycle/proliferation assays were performed to assess the percentage of cells in

S-phase (top gate), G0/G1-phase (left bottom gate), and G2/M-Phase (right bottom gate). (C)
Annexin-V/7AAD apoptosis assays after pre-treatment with 0.5µM staurosporine for 4h. Cells were considered apoptotic when they stained positive for Annexin-V (upper left gate) or Annexin-V/7AAD (upper right gate). The graphs represent the mean +/-standard deviation (SD) of at least three independent experiments. Statistical differences were assessed by paired t test. * denotes P < 0.050, ** denotes P < 0.010. BrdU, Bromodeoxyuridine; 7AAD, 7-Aminoactinomycin. Supplementary Fig S9. THP1 knockdown of miR-125a and CRISPR/Cas9 mediated knockout of miR-125a in U937. (A) THP1 cells were transiently transfected with a miR-125a hairpin inhibitor (THP1 miR-125a KD) and miR-125a expression was assessed by qPCR three days after transfection. THP1 cells transfected with a scrambled control (THP1 control KD) were chosen as the control situation and set to a value of 1. miR-125a in U937 was deleted by the CRISPR/Cas9 methodology (U937 miR-125a KO) and miR-125a expression was controlled by qPCR. Parental U937 were chosen as the control situation and set to a value of 1. The relative expression in THP1 miR-125a KD and in U937 miR-125a KO was calculated as the ratio of the target condition to the control condition. Graphs denote the mean +/-SD of at least three independent experiments. Statistical significance was evaluated by one-sample t test against a reference value of 1. (B) Sequencing results of U937 miR-125a KO demonstrate a complete deletion of the miR-125a region on chromosome 19.

Supplementary Fig S10. The Aza-mediated increase of miR-125a expression is inhibited by
miR-125a shRNA-knockdown. THP1 cells were transiently transfected with a miR-125a specific shRNA (THP1 miR-125a KD) and scrambled control shRNA (THP1 control KD), respectively. The expression of miR-125a was measured after 24h treatment with 2.5µM Aza (indicated as +) or empty dissolvent (indicated as -) by qPCR. THP1 control KD treated with empty dissolvent was chosen as the control and set to a value of 1. The relative expression in all other conditions was calculated as the ratio of the target condition to the control condition.
Graphs denote the mean +/-SD of at least three independent experiments. Statistical significance was evaluated by one-sample t test against a reference value of 1 (THP1 control KD), and unpaired t test (THP1 miR-125a KD), respectively. Aza, azacitidine

Supplementary Fig S11. Synergistic/additive effects of Aza treatment and miR-125a
transduction on miR-125a expression. THP1 cells with stable overexpression of miR-125a (THP1 miR-125a OE) were treated with 2.5µM Aza. Importantly, this increased the miR-125a expression even further, which suggests a synergistic or additive effect. miR-125a expression was assessed after 24h using qPCR. THP1 miR-125a OE treated with empty dissolvent was chosen as the control and set to a value of 1. The relative expression in the other condition was calculated as the ratio of the target condition to the control condition. Graphs denote the mean +/-SD of at least three independent experiments. Comparisons against the control condition were performed using a one-sample t test against a reference value of 1. Aza, azacitidine. S12. Synergistic/additive effects of Aza and miR-125a overexpression on the apoptosis of myeloid cells. We demonstrated that both miR-125a overexpression and Azatreatment induce the apoptosis of THP1 cells within this study. To reveal a potential synergism or additive effects, we treated THP1 miR-125a OE cells with 2.5µM Aza. Apoptosis was measured after 24h by Annexin-V/7AAD assay. The control condition (treated with the empty dissolvent only) was set at a value of 1, and the relative increase of apoptosis in the Aza-treated condition was calculated as the ratio of Aza-treated to control-treated cells. Cells were considered apoptotic when they stained positive for Annexin-V or Annexin-V/7AAD. Graphs represent the mean +/-SD of at least three independent experiments. Comparisons against the control condition were performed using a one-sample t test against a reference value of 1. Aza,  Wt, wildtype; WBC, white blood cells; Hb, haemoglobin; Plt, platelets, PB, peripheral blood; BM, bone marrow; g, grams; cm, centimetres.

Clinical characteristics of CMML patients
Association with miR-125a expression