Additional file 1 of Repressing HIF-1α-induced HDAC9 contributes to the synergistic effect of venetoclax and MENIN inhibitor in KMT2Ar AML
Creators: name: Ling, Qing
name: Zhou, Yutong
name: Qian, Yu
name: Qian, Jiejing
name: Zhang, Yi
name: Wang, Jinghan
name: Zhu, Yanan
name: Zhou, Yile
name: Wei, Juying
name: Yang, Chunmei
name: Sun, Jie
name: Yu, Wenjuan
name: Jin, Jie
name: Zhang, Xiang
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Physical Sciences not elsewhere classified
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Pharmacology
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology
subject: Computational Biology

description: Additional file 1: Supplementary Information. Materials and Methods, Figure S1-S18. Figure S1. KMT2Ar-AML was sensitive to MI-503. The IC50 of MI-503 in AML cell lines (72 h). Figure S2. MEN1i inhibited the proliferation of KMT2Ar-AML cell lines. (A-C) IC50 of MENis, such as MI-503 (A), MI-403 (B), and VTP50469 (C), in THP-1, MV4-11, and MOLM13 cells (7 days). Figure S3. OCI-AML3 was relatively less sensitive to MEN1i than KMT2Ar-AML. (A, B) The IC50 of MI-463 (A) and VTP50469 (B) in OCI-AML3 and KMT2Ar-AML cell lines (72 h). Figure S4. No significant synergistic effects of VEN plus MEN1i were observed in non-KMT2Ar-AML cell lines. Growth inhibition and synergistic index of VEN plus MI-503 in HL-60, OCI-AML2, OCI-AML3, KG-1, and U937 cells (72 h). Figure S5. VEN plus MI-463 or VTP-50469 also cooperated in the inhibition of KMT2Ar-AML cell lines. (A-B) Growth inhibition and synergistic index of VEN plus MI-403 (A) or VTP-50469 (B) in THP-1, MV4-11, and MOLM13 cells (72 h). Figure S6. VEN plus MI-503 did not influence cell cycle distribution and cell differentiation of KMT2Ar-AML cell lines. (A) Cell cycle analysis for THP-1, MV4-11, and MOLM13 cells after single-agent or combinatorial treatment with VEN and MI-503 (72 h); (B) Cell differentiation was determined by CD11b and CD14 staining in THP-1, MV4-11, and MOLM13 cells after single-agent or combinatorial treatment with VEN and MI-503 (72 h). Figure S7. No significant synergistic effects of VEN plus MEN1i were observed in primary bone marrow MNCs from non-KMT2Ar-AML patients or healthy donors. (A-B) Growth inhibition and synergistic index of VEN plus MI-503 in primary bone marrow MNCs from non-KMT2Ar-AML patients (A) or healthy donors (B) (72 h). Figure S8. Leukemic burden in treated MOLM13 xenotransplantation model. The relative luminescence unit (RLU) value was calculated for indicating leukemic burdens of treated mice (14 days[A] and 21 days[B]). Figure S9. The synergistic mechanism of VEN plus MI-503 was not related to further downregulation of HOXA9 and MEIS1 or disruption of BCL2/BCL-XL balance. (A) The mRNA expression of HOXA9 and MEIS1 in THP-1, MV4-11, and MOLM13 after treatments with DMSO, VEN, MI-503 and their combination (72 h) [two-tailed Student's t-tests; *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001 ****P &lt; 0.0001]; (B-C) The mRNA (B) and protein (C) expression of BCL2, BCL-XL and MCL-1 in THP-1, MV4-11, and MOLM13 after treatments with DMSO, VEN, MI-503 and their combination (72 h). Figure S10. Global transcriptional regulation of VEN plus MI-503 in KMT2Ar-AML cell lines. DEGs in three KMT2Ar-AML cell lines. Figure S11. VEN plus MI-503 specifically downregulated HDAC9. The mRNA expression of HDAC family in THP-1, MV4-11, and MOLM13 after treatments with DMSO, VEN, MI-503 and their combination (72 h). Figure S12. HDAC9 was not consistently down-regulated by VEN plus MI-503 until 72 treatments. (A-C) HDAC9 mRNA was detected under treatments for 12 h (A), 24 h (B), and 48 h (C), respectively. Figure S13. HDAC9 was not repressed by VEN plus MI-503 in OCI-AML3. (A, B) OCI-AML3 was treated by DMSO, VEN, MI-503, or VEN plus MI-503 at 72 h, and HDAC9 mRNA (A) and protein (B) were detected. Figure S14. HDAC9 knockdown was displayed in KMT2Ar-AML cell lines. HDAC9 was detected in KMT2Ar-AML cell lines after shRNA-mediated knockdown. Figure 15. HDAC9 expression in AML cell lines. (A-B) HDAC9 expression was determined in our AML cell lines (A) and obtained from Cancer Cell Line Encyclopedia (CCLE) database (B). Figure S16. VEN plus MI-503 inhibited the expression of hypoxia pathway. (A-B) HALLMARK_HYPOXIA (A) and LEONARD_HYPOXIA (B) gene sets for MI-503, VEN, or VEN plus MI-503 compared to DMSO, respectively. Figure S17. VEN plus MI-503 repressed the targets of HIF-1A. The mRNA expression of ALDOC, ADM, ENO3, PNRC1 and TMEM45A in THP-1, MV4-11, and MOLM13 after treatments with DMSO, VEN, MI-503 and their combination (72 h) [two-tailed Student's t-tests; *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001 ****P &lt; 0.0001]. Figure S18. The band quantification for western blot. (A-C) The band was quantified for Fig. 2C (A), 2H (B), and Figure S9C (C), respectively.