RAB27B controls palmitoylation-dependent NRAS trafficking and signaling in myeloid leukemia
Jian-Gang Ren, … , Mark R. Philips, Wei Tong
Jian-Gang Ren, … , Mark R. Philips, Wei Tong
Published June 15, 2023
Citation Information: J Clin Invest. 2023;133(12):e165510. https://doi.org/10.1172/JCI165510.
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Research Article Cell biology Hematology

RAB27B controls palmitoylation-dependent NRAS trafficking and signaling in myeloid leukemia

Abstract

RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation. Our proteomic studies revealed RAB27B upregulation in CBL- or JAK2-mutated myeloid malignancies, and its expression correlated with poor prognosis in acute myeloid leukemias (AMLs). RAB27B depletion inhibited the growth of CBL-deficient or NRAS-mutant cell lines. Strikingly, Rab27b deficiency in mice abrogated mutant but not WT NRAS–mediated progenitor cell growth, ERK signaling, and NRAS palmitoylation. Further, Rab27b deficiency significantly reduced myelomonocytic leukemia development in vivo. Mechanistically, RAB27B interacted with ZDHHC9, a palmitoyl acyltransferase that modifies NRAS. By regulating palmitoylation, RAB27B controlled c-RAF/MEK/ERK signaling and affected leukemia development. Importantly, RAB27B depletion in primary human AMLs inhibited oncogenic NRAS signaling and leukemic growth. We further revealed a significant correlation between RAB27B expression and sensitivity to MEK inhibitors in AMLs. Thus, our studies presented a link between RAB proteins and fundamental aspects of RAS posttranslational modification and trafficking, highlighting future therapeutic strategies for RAS-driven cancers.

Authors

Jian-Gang Ren, Bowen Xing, Kaosheng Lv, Rachel A. O’Keefe, Mengfang Wu, Ruoxing Wang, Kaylyn M. Bauer, Arevik Ghazaryan, George M. Burslem, Jing Zhang, Ryan M. O’Connell, Vinodh Pillai, Elizabeth O. Hexner, Mark R. Philips, Wei Tong

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Figure 3

RAB27B regulates NRAS activity and signaling.

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RAB27B regulates NRAS activity and signaling. (A–E) RAB27B was stably de...
(A–E) RAB27B was stably depleted via lentiviral-shRNA–mediated knockdown (KD) in TF-1 DKO cells, with shRNA against Luciferase (shLuc) used as a control. (A and B) KD efficiency of shRNA-RAB27B constructs was determined by WB (A) and qRT- PCR (B). (C) Cells were cultured in triplicates in different concentrations of human GM-CSF. Cell growth after 3 days in culture was determined by MTT absorbance. (D) TF-1 Ctrl or DKO cells (left), and TF-1 DKO cells with or without RAB27B depletion (right), were cultured in media containing serum only or serum and GM-CSF. Cell lysates were subjected to WB analysis with indicated antibodies to examine RAF/MEK/ERK activation. (E) TF-1 cells as described in (D) were cultured in media containing serum only. RAS GTPase activities were measured by RAS GTP pulldowns using RAF-1 RBD agarose beads, followed by WB with indicated antibodies. GTP-bound RAS represents active RAS. Input lysates were subjected to WB analysis with indicated antibodies as controls. (F–H) RAB27B was stably depleted via lentiviral-shRNA mediated KD in OCI-AML3 cells, with shLuc used as a control. (F) Cells were plated at equal cell numbers and cell growth was determined by counting of live cells. (G) ERK activation was determined by WB. (H) NRAS activity was determined by RAF-1 RBD agarose bead pulldown followed by WB using anti-NRAS antibodies. Input lysates were subjected to WB analysis with the indicated antibodies as controls. In all relevant panels, data are represented as mean ± SD, and 2-way ANOVA was used for comparing cell growth; *P < 0.05; **P < 0.01; ***P < 0.001.