Ikaros‐Mediated Regulation of the Rab20 GTPase in B‐ALL is PP1 Dependent

Abstract

Rab20 is a member of the Rab GTPase family, a group of over 60 small GTPases that play critical roles in membrane trafficking and vesicular transport in all eukaryotic cells. Rab20 has been found to play a critical role in the immune response and is highly expressed in macrophages where it plays a role in phagosome and endosome formation. RAB20 overexpression has been observed in various human malignancies. However, the role of RAB20 in leukemia is unknown. Here we present data that RAB20 expression is repressed by the Ikaros tumor suppressor protein in B‐cell acute lymphoblastic leukemia (B‐ALL). Additionally, we report that protein phosphatase 1 (PP1) regulates Ikaros’ function as a repressor of RAB20 transcription. Global chromatin immunoprecipitation coupled with next‐generation sequencing (ChIP‐Seq) demonstrated that Ikaros binds to the promoter of RAB20 in B‐ALL. Ikaros binding to the RAB20 promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in both B‐ALL cell lines and primary B‐ALL. The role of Ikaros in regulating RAB20 expression in B‐ALL was dissected using gain‐of‐function and loss‐of‐function experiments. Ikaros overexpression resulted in increased Ikaros binding to the RAB20 promoter and decreased RAB20 expression. shRNA‐mediated knockdown of Ikaros abolished Ikaros binding at the RAB20 promoter and resulted in increased RAB20 expression. A luciferase reporter assay demonstrated that Ikaros directly represses RAB20 promoter activity. The role of Ikaros in regulating the chromatin signature at the RAB20 promoter was examined by combining Ikaros overexpression and knockdown in B‐ALL with qChIP against markers of active chromatin (H3K9ac & H3K4me3) and repressive chromatin (H3K9me3). Ikaros overexpression results in an increase in H3K9me3 and a decrease in both H3K9ac and H3K4me3 at the RAB20 promoter—indicating formation of repressive chromatin. Ikaros knockdown resulted in an increase in H3K9ac and H3K4me3—indicating formation of active chromatin. Since our previous studies showed that protein phosphatase 1 (PP1) can regulate Ikaros function, we tested whether PP1 affects Ikaros‐mediated regulation of RAB20 expression. Treatment of B‐ALL cells with the calyculin and tautomycin PP1 inhibitors resulted in decreased Ikaros binding at the RAB20 promoter and increased RAB20 expression. qChIP demonstrated that mutating the PP1‐interacting site of Ikaros—IKZF1(465‐7A)—ablated Ikaros’ RAB20 promoter binding. Consequently, using an Ikaros mutant with both a mutated PP1‐interacting site and phosphoresistant mutants (simulating active PP1 dephosphorylation) restored Ikaros binding to the RAB20 promoter. Together, these data demonstrate that Ikaros transcriptionally represses RAB20 expression in B‐ALL in a PP1‐dependent manner. The results provide a novel insight into intersection of PP1 and Rab GTPase signaling pathways and suggest a potential mechanism by which PP1 regulates Rab20 activity in leukemia.Support or Funding InformationNIH R01 CA213912; NIH F30 CA221109; The Four Diamonds Fund; St. Baldrick’s Foundation; Hyundai Hope on Wheels; Alex’s Lemonade Stand; Bear Necessities Pediatric Cancer Foundation