Fig. 6
HOXA9 is a functional target gene of RBM5 in AML. a Scatter plot analysis comparing RBM5 mRNA levels and HOXA9 mRNA levels across acute myeloid leukemia cell lines (data was obtained from the Depmap). P-value is calculated by linear regression. b Immunoblotting of RBM5 and HOXA9 in various leukemia cell lines and normal bone marrow (NBM) samples (left panel). The bar chart displays the quantitative protein levels of RBM5 and HOXA9 in various cell lines depicted in the left panel (right panel). c Real-time-qPCR and immunoblotting analysis was conducted on the RBM5-sg1, RBM5-sg2, and sgNT targeted MOLM13 and OCIAML2 to monitor the expression of HOXA9. Data shown are means ± SEM from three independent experiments. *P < 0.05, unpaired Student’s t-test. d Diagram of auxin-induced degron (AID) system to degrade endogenous RBM5. Endogenous RBM5 N-terminus AID knock-in KMT2A-r leukemia lines (SEM homozygous clones and MOLM13 bulk cells) were followed by constitutively expressing OsTIR1(F74G). With the presence of small molecule 5-Ph-IAA, the OsTIR1(F74G), as a substrate receptor in Skp1, Cullin, and F-box (SCF) complex, could target and mediate specific AID-tagged RBM5 proteins for ubiquitination and degradation. e The protein level of endogenous HA-miniAID-RBM5 of SEM homozygous clones can be acutely degraded with 5-Ph-IAA through degron-mediated proteasome degradation after 2-h post-treatment (left panel). HOXA9 mRNA level was significantly reduced after 2 h, 4 h, 6 h, and 24 h of treatment in SEM cells (right panel). f After 24 h of treatment with 5-Ph-IAA in HA-miniAID-RBM5 knock-in MOLM13 cells, the protein level of HA-miniAID-RBM5 decreased (left panel), and there was also a significant reduction in HOXA9 mRNA level (right panel). g The doxycycline-Tet-On system for ectopic overexpression of RBM5-HA in the MOLM13 cell line, after a 2-h treatment with doxycycline, a significant upregulation in HOXA9 mRNA levels was observed (right panel), accompanied by an increase in RBM5 expression (left panel). h Real-time-qPCR analysis was conducted in OCIAML2 cells transduced with RBM5 cDNA wild-type (WT) or six individual truncated mutants to validate the expression of HOXA9. Data shown are means ± SEM from three independent experiments. *P < 0.05, unpaired Student’s t-test. i ChIP-qPCR with anti-MYC tag antibody near H3K4me3-bound HOXA9 downstream region (Chr7:27,199,969–27,200,853) in OCIAML2-RBM5-MYCtag cells (n = 3). The red arrows indicate the primer 1 and primer 2 located position, and the blue arrow indicates the negative control (NC) primer located position, where there is no H3K4me3 signal. Statistical analysis (P-value) was performed using an unpaired Student’s t-test. All error bars represent mean ± SEM. j Rescued competitive proliferation assay was conducted by infecting OCIAML2-Cas9 and MOLM13-Cas9 cells overexpressing ectopic RBM5-wild-type cDNA (WT), RBM5-sgRNA1-resistant mutant cDNA (PAM-MUT), and HOXA9 cDNA (linked to Venus reporter) with lentiviral-sgRNAs against non-target (sgNT) and RBM5 (RBM5-sg1) at about 50% efficiency (all monitored by Venus reporter). Data shown are means ± SEM from three independent experiments. **P < 0.01, unpaired Student’s t-test