Supplementary Figures S1-S5 and Legends from AMPK–ULK1-Mediated Autophagy Confers Resistance to BET Inhibitor JQ1 in Acute Myeloid Leukemia Stem Cells

Supplementary figure S1. Differential sensitivity of human AML LSCs to JQ1. Supplementary figure S2. Relative levels of the autophagy pathway effectors LC3-II, beclin-1, and pULK1 (S555). Supplementary figure S3. Autophagy induction in KG1 and KG1a cells and effects of autophagy inhibition on JQ1-induced apoptosis in LSCs. Supplementary figure S4. JQ1-induced apoptosis through intrinsic apoptosis pathway in JQ1-sensitive LSCs. Supplementary figure S5. Diagram illustrating the potential effects of the BET inhibitor JQ1 in JQ1-resistant LSCs.</p

Supplemental Table 2 from AMPK–ULK1-Mediated Autophagy Confers Resistance to BET Inhibitor JQ1 in Acute Myeloid Leukemia Stem Cells

Patient characteristics according to JQ1 sensitivity in primary acute myeloid leukemia blasts</p

Supplemental Table 1 from AMPK–ULK1-Mediated Autophagy Confers Resistance to BET Inhibitor JQ1 in Acute Myeloid Leukemia Stem Cells

Effects of JQ1 on phenotypes and apoptosis of primary acute myeloid leukemia stem cells from 13 patietns</p

Additional file 1 of PERK/NRF2 and autophagy form a resistance mechanism against G9a inhibition in leukemia stem cells

Additional file 1: Table S1. Synergistic effects of the G9a and PERK inhibitor on apoptosis of primary acute myeloid LSCs. Figure S1. Effects of treatment with the PERK inhibitor GKS2606414 for 48 h in the presence or absence of 10 μM BIX-01294, on apoptosis. Figure S2. Effect of PERK inhibition on BIX-01294- induced apoptosis in KG1a cells. (A) KG1a cells were treated with 10 μM BIX-01294 in the presence or absence of the PERK inhibitor GSK260641 at 5 μM. After incubation for 48 h, the apoptotic fraction was measured using flow-cytometric analysis. (B) KG1a cells were transfected with PERK siRNA or scrambled siRNA as described in the Materials and Methods and then treated with 10 μM BIX-01294 for 48 h. Figure S3. Effects of treatment for 48 h with the NRF2 inhibitor brusatol in the presence or absence of 10 μM BIX-01294 on apoptosis. Figure S4. Effects of PERK inhibition in the absence or presence of 2 nM bafilomycin A1 on autophagy induction

Receiver operating characteristics (ROCs) curve analysis for the diagnostic value of miR-21.

<p>The area under the ROC curve (AUC) was 0.648 (95% CI: 0.49 to 0.72).</p

Differences in baseline serum miR-21 expression levels.

<p>(A) Difference in serum miR-21 levels between healthy donors and patients with MDS prior to HMA therapy. Expression levels of serum miR-21 were normalized to the reference gene, miR-192, which was selected as described in Design and Methods. (B) Difference in baseline serum miR-21 levels between responders to HMA therapy and non-responders. Bar graph and whisker indicate the median value and standard deviation of serum miR-21 levels, respectively. Significance is indicated by the <i>P</i> value.</p

Overall characteristics of patients and stratification according to serum miR-21 level.

<p>Abbreviations: miR-21, microRNA-21; WHO, World Health Organization; RA, refractory anemia;</p><p>RARS, RA with ring sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia; RCMD-RS, RCMD with ring sideroblasts; RAEB, refractory anemia with excess blasts; MDS-U, myelodysplastic syndromes unclassifiable; PB, peripheral blood; BM, bone marrow; HMA, hypomethylating agent; IPSS, International Prognostic Scoring System; NA, not available; WPSS, WHO classification-based Prognostic Scoring System; alloHCT, allogeneic hematopoietic stem cell transplant, INT, intermediate.</p>a<p>Cytogenetic: Good = normal, -Y alone, del(5q) alone, del(20q) alone; Poor = complex (≥3 abnormalities) or chromosome 7 anomalies; Intermediate = other abnormalities.</p>b<p>At least 2 units of red blood cell transfusion during 8 weeks prior to HMA initiation.</p

Multivariate analysis for overall response to HMA and progression-free survival.

<p>Abbreviations: ORR, overall response rate; PFS, progression-free survival; HR, hazard ratio; CI, confidence interval; IPSS, International Prognostic Scoring System; miR-21, microRNA-21.</p

Expression and stability of reference gene candidates in the sera of healthy donors and patients with MDS.

<p>(A) Quantification (Ct) of candidate reference miRNAs (miR-192, miR-16, and miR-93) in serum samples of healthy donors and MDS patients corrected for efficiency and two interpolate controls are shown. <i>Box plots</i> represent lower and upper quartiles with the median depicted with a horizontal line. Whiskers depict the 10th and 90th percentiles. Differences in serum levels of candidate miRNAs were not found between healthy donors (white box), MDS patients prior to HMA therapy (gray box), and MDS patients treated with four courses of HMA therapy (black box). (B) Average expression stability values for candidate reference miRs in MDS patients, which were calculated by the <i>geNorm</i> algorithm, are shown as a bar graph and with actual values. High expression stability is indicated by a low stability value. MDS, myelodysplastic syndromes; miRNAs, microRNAs; HMA, hypomethylating agents.</p

Response to HMA therapy according to serum miR-21 level.

<p>Abbreviations: miR-21, microRNA-21; HMA, hypomethylating agent; IWG, International Working Group; CR, complete response; PR, partial response; mCR, marrow CR; SD, stable disease; HI, hematologic improvement; PD, progressive disease; ORR, overall response rate.</p><p>Results are shown as n (%) or median value.</p