Structural Determinants of the Gain-of-Function Phenotype of Human Leukemia-associated Mutant CBL Oncogene

Abstract

Mutations of the tyrosine kinase-directed ubiquitin ligase CBL cause myeloid leukemias, but the molecular determinants of the dominant leukemogenic activity of mutant CBL oncogenes are unclear. Here, we first define a gain-of-function attribute of the most common leukemia-associated CBL mutant, Y371H, by demonstrating its ability to increase proliferation of hematopoietic stem/progenitor cells (HSPCs) derived from CBL-null and CBL/CBL-B-null mice. Next, we express second-site point/deletion mutants of CBL-Y371H in CBL/CBL-B-null HSPCs or the cytokine-dependent human leukemic cell line TF-1 to show that individual or combined Tyr → Phe mutations of established phosphotyrosine residues (Tyr-700, Tyr-731, and Tyr-774) had little impact on the activity of the CBL-Y371H mutant in HSPCs, and the triple Tyr → Phe mutant was only modestly impaired in TF-1 cells. In contrast, intact tyrosine kinase-binding (TKB) domain and proline-rich region (PRR) were critical in both cell models. PRR deletion reduced the stem cell factor (SCF)-induced hyper-phosphorylation of the CBL-Y371H mutant and the c-KIT receptor and eliminated the sustained p-ERK1/2 and p-AKT induction by SCF. GST fusion protein pulldowns followed by phospho-specific antibody array analysis identified distinct CBL TKB domains or PRR-binding proteins that are phosphorylated in CBL-Y371H-expressing TF-1 cells. Our results support a model of mutant CBL gain-of-function in which mutant CBL proteins effectively compete with the remaining wild type CBL-B and juxtapose TKB domain-associated PTKs with PRR-associated signaling proteins to hyper-activate signaling downstream of hematopoietic growth factor receptors. Elucidation of mutant CBL domains required for leukemogenesis should facilitate targeted therapy approaches for patients with mutant CBL-driven leukemias.

Keywords: CBL; E3 ubiquitin ligase; leukemia; mutagenesis; oncogene; proline-Rich region; receptor tyrosine kinase; tyrosine kinase binding domain.

FIGURE 1.

Analysis of gain-of-function of CBL-Y371H in CBL-null and CBL/CBL-B-null primary mouse HSPCs. A, schematic illustration of various second-site mutants of CBL-Y371H mutants used in this study. B, mouse primary HSPCs were isolated and infected with the indicated retroviruses as described under “Experimental Procedures.” Data shown are representative FACS plot of the GFP+ population from Cbl null and Cbl/Cbl-b DKO HSPCs. C–H, lineage-negative HSPCs were purified by immuno-depletion of lineage-positive mature cells from bone marrow mononuclear cells collected from CBL-null (C–E) or CBL/CBL-B-null (DKO) mice (F–H). The cells were retrovirally infected to express the indicated constructs carrying an IRES-GFP, and infected (GFP+) cells obtained by FACS sorting were assessed in three replicates for cytokine-independent (C and F), SCF-stimulated (D and G), or TPO-stimulated (E and H) proliferation as described under “Experimental Procedures.” For each in C–H, left is one representative experiment with six replicates (mean ± S.D.); right is pooled data of three independent experiments shown as percentage of uninfected control (mean ± S.E.). *, p < 0.05.

FIGURE 2.

Proline-rich region of CBL-Y371H mutant is essential for its gain-of-function phenotype in HSPCs. A, CBL/CBL-B DKO mouse HSPCs were isolated, retrovirally infected with the indicated CBL constructs, and infected (GFP+) cells FACS-sorted as in Fig. 1, followed by analysis of SCF-induced proliferation (1 ng/ml) using the Cell TiterGlo assay. B–D, CBL-null mouse HSPCs were retrovirally infected with the indicated CBL constructs, and GFP+ cells were assessed in three replicates for cytokine-independent (B) or SCF-stimulated proliferation (C) (Cell TiterGlo), or for SCF-stimulated colony forming ability (D). Proliferation based on luminescence arbitrary unit in A–C was normalized to the vector-only control. Shown is pooled data of three independent experiments demonstrated as percentage to vector-only control (mean ± S.E.).

FIGURE 3.

Expression of CBL-Y371H in TF-1 leukemic cell line enhances cytokine-independent as well as cytokine-dependent proliferation. A, TF-1 cells were retrovirally infected with the vector or the indicated HA-tagged CBL constructs, the GFP+ population sorted and maintained continuously in GM-CSF-containing media. The expression of ectopic CBL constructs was verified using immunoprecipitation of cell lysates with anti-HA antibody. Reactivity with the culture medium-derived IgG heavy chain bands in the lysates provides a loading control. B and C, TF-1 cell lines expressing the indicated CBL constructs were GM-CSF deprived for 24 h, followed by culture in medium without GM-CSF (B) or supplemented with the indicated concentrations of GM-CSF (C). D, TF-1 cell lines expressing the indicated CBL constructs were GM-CSF deprived for 24 h, followed by culture in medium supplemented with the indicated concentrations of SCF. Proliferation was assessed after 72 h, using the Cell TiterGlo assay. B–D, left is one representative experiment with six replicates (mean ± S.D.); right is pooled data of three independent experiment shown as percentage to vector only control (mean ± S.E.). *, p < 0.05.

FIGURE 4.

TKB domain and proline-rich region of CBL-Y371H mutant are essential for the enhanced cytokine-independent and cytokine-dependent proliferation of TF-1 cell lines. A, stable retroviral expression of the indicated CBL constructs was verified using Western blotting with anti-HA antibody, with IgG heavy chain serving as a loading control as in Fig. 3A. B and C, TF-1 cell lines expressing the indicated ectopic CBL constructs were analyzed for SCF-stimulated (B) or cytokine-independent (C) proliferation as in Fig. 3. Shown are pooled data from three independent repeats (mean ± S.E.). *, p < 0.05.

FIGURE 5.

Proline-rich region of CBL-Y371H mutant essential for enhanced signaling downstream of SCF-induced c-KIT activation. TF-1 cell lines expressing the indicated CBL mutant constructs were GM-CSF-deprived and then either left unstimulated (time 0) or stimulated with SCF (100 ng/ml) for the indicated time points (minutes). A–C, cell lysates were subjected to Western blotting for the indicated proteins: p-ERK, total ERK, and p-AKT (A) and total c-KIT and p-c-KIT (B). Shown is a representative experiment of two independent repeats with similar results. C, phospho-c-KIT signals in Western blottings were quantified using ImageJ analysis of scanned images, and the signals were first normalized to HSC-70 and then expressed as phospho-c-KIT signals relative to those in vector-transfected cells at 5 min, which were assigned a value of 1. Shown are pooled data of three independent repeats (mean ± S.E.). Red asterisks (*) indicate significance between CBL-Y371H and CBL-Y371-ΔPRR, and black asterisks (*) show significance between CBL-Y371H and vector cells (D). The indicated live TF-1 cell lines were analyzed by FACS for surface levels of c-KIT at various points after SCF stimulation. Mean fluorescence intensity relative to unstimulated controls is plotted. Shown are pooled data of three independent repeats. *, p < 0.05. E, THP-1 and GDM-1 cell lines were serum-deprived for 24 h and then left unstimulated or stimulated with SCF (100 ng/ml) for the indicated time points (minutes). Cell lysates were Western-blotted for phospho-c-KIT, total c-KIT, phospho-CBL, total CBL, and total CBL-B with HSC-70 used as a loading control. Shown is a representative of two independent repeats with similar results.

FIGURE 6.

Proline-rich region of CBL-Y371H is required for hyper-activation of basal and SCF-induced signaling. A, TF-1 cell lines expressing indicated CBL mutant constructs were GM-CSF deprived and either left unstimulated or stimulated with SCF for the indicated time points. Anti-HA immunoprecipitations were carried out followed by anti-Tyr(P), anti-CBL-Tyr(P)-700, or anti-CBL-Tyr(P)-774 immunoblotting to visualize the phosphorylation of ectopic overall and site-specific phosphorylation of CBL proteins. Anti-HA blotting shows comparable loading. Shown is a representative experiment of two independent repeats with similar results. B, GM-CSF-deprived TF-1 cells expressing CBL-Y371H were stimulated with SCF for 15 min, and 5 mg of cell lysate protein aliquots used for pulldown with 50 μg of GST (lane 1, control), GST-CBL-N (lane 2), or GST-CBL-C (lane 3) fusion proteins non-covalently bound to glutathione-Sepharose beads. After washing, the bound proteins were visualized anti-Tyr(P) (4G10) immunoblotting, and the membrane was re-probed with an anti-phospho-c-KIT antibody. The whole cell lysate (25 μg) was concurrently resolved (lane 4). The molecular mass markers (in kilodaltons) are indicated on left. C, antibody array analysis of phosphoproteins in whole cell lysates. TF-1 cells expressing empty vector, CBL-Y371H, or CBL-Y371H-ΔPRR were GM-CSF-deprived and stimulated with SCF for 15 min. Cell lysates were collected in the antibody array lysis buffer supplied with the kit and incubated with antibody array filters and developed using enhanced chemiluminescence, followed by densitometry analysis of phosphoprotein signals. Signals were expressed relative to those in unstimulated vector control TF-1 cell lysates. D, antibody array analysis of preferential binding of cellular phosphoproteins with PRR-containing GST-CBL-C versus GST-CBL-N (TKB domain). TF-1 cells expressing CBL-Y371H were GM-CSF-deprived and stimulated with SCF for 15 min. 5-mg aliquots of cell lysate protein were incubated with 50 μg of glutathione-Sepharose-bound GST-CBL-N or GST-CBL-C fusion proteins. Bound proteins were eluted in 1% SDS-containing lysis buffer by a 5-min incubation at 95 °C, cooled to room temperature, diluted 10-fold in antibody array buffer, and analyzed for phosphoprotein signals using antibody arrays as in C. Data are presented as a ratio of GST-CBL-C pulldown signals over GST-CBL-N pulldown signals for each phosphoprotein. Boxes indicate preferential binding to GSCT-CBL-C or GST-CBL-N.

FIGURE 7.

Schematic presentation of the model of mutant CBL gain-of-function based on present studies. TKB, tyrosine kinase-binding domain; RF, RING finger domain; PRR, proline-rich region; CTR, C-terminal region; Ub, Ubiquitin; P, phosphorylation.