C/EBPβ is a critical mediator of IFN-α–induced exhaustion of chronic myeloid leukemia stem cells by Asumi Yokota, Hideyo Hirai, Ryuichi Sato, Hiroko Adachi, Fumiko Sato, Yoshihiro Hayashi, Atsushi Sato, Naoka Kamio, Yasuo Miura, Masakazu Nakano, Daniel G . Tenen, Shinya Kimura, Kei Tashiro, and Taira Maekawa BloodAdv Volume 3(3):476-488 February 12, 2019 © 2019 by The American Society of Hematology

Asumi Yokota et al. Blood Adv 2019;3:476-488 © 2019 by The American Society of Hematology

IFN-α upregulates C/EBPβ expression irrespective of the presence of BCR-ABL signaling. IFN-α upregulates C/EBPβ expression irrespective of the presence of BCR-ABL signaling. (A) Schematic illustration of the experimental protocol. EML cells were treated with 100 U/mL IFN-α for 0.5 or 3 hours, with or without 12-hour treatment with 1 μM imatinib (IM). (B) Western blotting analysis of EML cells transduced with empty or BCR-ABL expression vector. Representative data from 3 independent experiments are shown. Cebpb is a single-exon gene; the 3 isoforms, liver activating protein (LAP)*, LAP, and liver inhibitory protein (LIP), are translated from different start codons. These blots are representative of at least 3 independent experiments. (C) Blot intensity was quantified using ImageJ and normalized against the corresponding level of GAPDH. The level of the LAP isoform in nontreated, empty vector-transduced EML cells was defined as 1.0, and the average relative expression levels of LAP under each condition from 4 different blots were plotted. Data are means ± standard deviation (SD) of 3 independent experiments. Results were normalized against the corresponding level of GAPDH protein. (D) IFN-α–induced Cebpb mRNA expression in EML cells transduced with empty or BCR-ABL expression vectors. Data are means ± SD of 3 independent experiments. Results were normalized against the corresponding level of Gapdh mRNA. *P < .05; **P < .01. Asumi Yokota et al. Blood Adv 2019;3:476-488 © 2019 by The American Society of Hematology

STAT1 and STAT5 are recruited to a 3′ distal enhancer of Cebpb that is required for upregulation of Cebpb in response to IFN-α. STAT1 and STAT5 are recruited to a 3′ distal enhancer of Cebpb that is required for upregulation of Cebpb in response to IFN-α. (A) EML cells transduced with empty (top lane) or BCR-ABL (bottom lane) expression vectors were subjected to ChIP-seq analysis using anti-STAT5 antibody. Panels show recruitment of STAT5 around the Cebpb locus, as displayed by the Integrative Genomics Viewer. Representative data from 2 independent samples are shown. (B) Enrichment of STAT5 activated by BCR-ABL on the enhancer region of Cebpb gene identified in panel A, as confirmed by ChIP-PCR. Enrichment levels of STAT5 are shown as red (promoter region) and blue (enhancer region) bars. Data are means ± standard error (SE) of 3 independent experiments. (C) Enrichment of H3K27Ac in the presence or absence of BCR-ABL to the enhancer region of Cebpb. Data are means ± SE of 3 independent experiments. (D) Effect of CRISPR/dCas9-mediated targeting of STAT5 binding sites on Cebpb mRNA expression in EML cells. The gRNA sequence was designed to target STAT5 binding sites in the distal region, as shown in supplemental Figure 3B. The Cebpb mRNA level was normalized against the corresponding level of Gapdh mRNA. The normalized Cebpb mRNA level in dCas9 (+) gRNA (−) EML cells transduced with MIG-empty was defined as 1.0. mRNA expression levels are shown as red (MIG-empty) or blue (MIG-BCR-ABL) bars. Data are means ± SD of 3 independent experiments. (E) dCas9-mediated repression of the Cebpb enhancer impaired BCR-ABL–induced myeloid differentiation. Percentage of c-kit− CD11b+ myeloid cells in BCR-ABL–expressing EML cells transduced with dCas9 and a vector control for gRNA (upper left) or with dCas9 and a vector expressing gRNA targeting the STAT5 binding sites (lower left). Data are means ± SD of 2 independent experiments (right). Representative flow cytometric data are shown. (F) Chromatin obtained from EML cells treated with IFN-α was subjected to ChIP-PCR using anti-STAT1 (blue bars; top) and anti-STAT5 (blue bars; bottom) antibodies. Normal immunoglobulin G (red bars) was used as a control for the ChIP experiments. Data are means ± SD of 3 independent experiments. (G) Induction of Cebpb mRNA expression by IFN-α in EML cells was significantly impaired by a combination of dCas9 and gRNA targeting the STAT5 binding sites. Cells were treated with 100 U/mL IFN-α for 3 hours. Cebpb mRNA level was normalized against the corresponding level of Gapdh mRNA. Normalized Cebpb mRNA levels are shown relative to the level in untreated dCas9 (+) gRNA (−) cells, shown as red (untreated) or blue (IFN-α–treated) bars. (H) Involvement of STAT1 and STAT5 in IFN-α–mediated upregulation of Cebpb mRNA. EML cells stably expressing BCR-ABL were retrovirally transduced with the indicated dn mutants of STAT1 and STAT5. On day 3, transduced cells were purified and subjected to quantitative RT-PCR before and after a 3-hour treatment with IFN-α. Expression level of Cebpb mRNA in IFN-α–treated cells is shown relative to the level in nontreated cells. Data are means ± SD of 3 independent experiments. *P < .05; **P < .01. Asumi Yokota et al. Blood Adv 2019;3:476-488 © 2019 by The American Society of Hematology

IFN-α induces differentiation and exhaustion of CML stem cells via induction of C/EBPβ in vitro. IFN-α induces differentiation and exhaustion of CML stem cells via induction of C/EBPβ in vitro. (A) Experimental protocol of serial replating assay. Lin− c-kit+ Sca-1+ GFP+ cells were purified from WT or C/EBPβ KO BM cells transduced with MIG-BCR-ABL, and then subjected to a colony-forming assay in the presence or absence of 100 U/mL IFN-α. (B) Numbers of colonies at the indicated rounds of plating. First-round colonies were counted on day 7; second- and third-round colonies were counted on days 10 and 14, respectively. Colony numbers are normalized against the input (10 000 cells per dish). Data are means ± SD of triplicates. Representative data from 2 independent experiments are shown. (C) May-Giemsa staining of cells harvested from second-round colonies derived from WT and C/EBPβ KO cells transduced with BCR-ABL (left and right panels, respectively) (scale bars, 20 μm). (D) Representative flow cytometric data of cells recovered from the second-round colonies. CD11b expression levels are shown as histograms with red (control) or blue (+IFN-α) lines. Asumi Yokota et al. Blood Adv 2019;3:476-488 © 2019 by The American Society of Hematology

The IFN-α-C/EBPβ axis induces exhaustion of LSCs in vivo. The IFN-α-C/EBPβ axis induces exhaustion of LSCs in vivo. (A) Schematic illustration of experimental procedures for serial BM transplantation. Primary recipient mice were intraperitoneally injected with vehicle or poly I:C at day 9 after BM transplantation. BM cells were harvested from the primary recipient mice at day 17, and then subjected to flow cytometric analysis or serial transplantation into secondary recipient mice. (B-C) Representative flow cytometric data of BM cells obtained from primary recipient mice at day 17; (right) frequencies of GFP+ leukemic cells in lin− c-kit+ and CD150+ immature populations. Data are means ± SD of 7 or 8 recipients from 2 independent experiments. *P < .05; **P < .01. (D) Survival curve of secondary recipient mice. Representative data from 3 independent experiments are shown. Asumi Yokota et al. Blood Adv 2019;3:476-488 © 2019 by The American Society of Hematology

IFN-α upregulates CEBPB and promotes exhaustion of CD34+ CML stem cells derived from CML-CP patients. IFN-α upregulates CEBPB and promotes exhaustion of CD34+CML stem cells derived from CML-CP patients. (A) Effects of IFN-α on CEBPB and CEBPA mRNA expression in lin− CD34+ cells obtained from 5 CML patients. CEBPB and CEBPA mRNA levels were normalized against the corresponding level of GAPDH mRNA. Purified cells were subjected to quantitative RT-PCR after IFN-α treatment of 0.5 or 3 hours in vitro. Data from 5 independent experiments are shown as scatter plot. Bold horizontal lines, mean values from 3 or 5 samples; vertical lines, SD of each group of data. *P < .02; **P < .01. (B) Number of total colonies derived from lin− CD34+ BM cells from 5 CML patients. IFN-α was added at the indicated concentrations shown as red (control), blue (100 U/mL), or green (500 U/mL) bars. Experiments were all performed in triplicate; data are means ± SD. Differences between controls (IFN-α = 0 U/mL) and test samples were statistically analyzed. *P < .05; **P < .01. (C) Changes in colony types in response to IFN-α shown as pie charts: blue, myeloid colonies; red, erythroid colonies; and green, mixed colonies. Data are means of triplicates. Actual mean and SD values and statistical analysis of the percentage of each classification are shown in supplemental Table 7. (D) Flow cytometric analysis of the first-round colonies. Representative dot plots for UPN3 are shown. Dot plots indicate the percentage of CD66b+ cells in each sample. Data are means ± SD. *P < .05; **P < .01. n.s., not significant; UPN, unique patient number. Asumi Yokota et al. Blood Adv 2019;3:476-488 © 2019 by The American Society of Hematology