Farnesyltransferase inhibitor tipifarnib inhibits Rheb prenylation and stabilizes Bax in acute myelogenous leukemia cells

Abstract

Although farnesyltransferase inhibitors have shown promising activity in relapsed lymphoma and sporadic activity in acute myelogenous leukemia, their mechanism of cytotoxicity is incompletely understood, making development of predictive biomarkers difficult. In the present study, we examined the action of tipifarnib in human acute myelogenous leukemia cell lines and clinical samples. In contrast to the Ras/MEK/ERK pathway-mediated Bim upregulation that is responsible for tipifarnib-induced killing of malignant lymphoid cells, inhibition of Rheb-induced mTOR signaling followed by dose-dependent upregulation of Bax and Puma occurred in acute myelogenous leukemia cell lines undergoing tipifarnib-induced apoptosis. Similar Bax and Puma upregulation occurred in serial bone marrow samples harvested from a subset of acute myelogenous leukemia patients during tipifarnib treatment. Expression of FTI-resistant Rheb M184L, like knockdown of Bax or Puma, diminished tipifarnib-induced killing. Further analysis demonstrated that increased Bax and Puma levels reflect protein stabilization rather than increased gene expression. In U937 cells selected for tipifarnib resistance, neither inhibition of signaling downstream of Rheb nor Bax and Puma stabilization occurred. Collectively, these results not only identify a pathway downstream from Rheb that contributes to tipifarnib cytotoxicity in human acute myelogenous leukemia cells, but also demonstrate that FTI-induced killing of lymphoid versus myeloid cells reflects distinct biochemical mechanisms downstream of different farnesylated substrates. (ClinicalTrials.gov identifier NCT00602771).

Figures

Figure 1.

Tipifarnib inhibits proliferation and induces apoptosis in AML cell lines. (A–C) Aliquots containing 1−2×105 HL-60, U937, and ML-1 cells/mL were treated with the indicated concentrations of tipifarnib or diluent continuously, with cells being counted and diluted into fresh medium with drug every 2 days. Graphs on the left indicate cumulative cell number/mL after taking into account dilution. Graphs on the right show cell number on Day 6 relative to diluent-treated controls. (D–F) DNA histograms of cells treated with diluent or the indicated tipifarnib concentration for 6 days. Double-headed arrows indicate events with <2n DNA content. G-I, percentage of subdiploid events observed on Day 6 after analysis of FL2 height as illustrated in Online Supplementary Figure S2A. Results in all panels are representative of at least 3 independent experiments.

Figure 2.

Low expression of RasGRP1 in myeloid cell lines and AML specimens. (A) Whole cell lysates (50μg protein) from the indicated lymphoid and AML lines were subjected to immunoblotting with a previously characterized anti-RasGRP1 antibody. c-Raf served as a loading control. (B and C) Aliquots containing 3×105 bone marrow mononuclear cells from pre-treatment bone marrow aspirates of AML patients enrolled on a recent tipifarnib/etoposide phase II trial were subjected to SDS-PAGE followed by immunoblotting for the indicated antigens. Jurkat cells, which served as a positive control for RasGRP1 expression, were loaded at 3×105/lane (lane 1, panels B and C) or 1×105 and 0.3×105/lane (B, lanes 2 and 3, respectively). Number above each lane indicates RasGRP1:aprataxin ratio previously reported for the corresponding mRNA sample. NA, not assayed due to lack of RNA sample.

Figure 3.

Role of Rheb in tipifarnib-induced apoptosis. (A) After U937 cells were treated for 24 h with the indicated tipifarnib concentration, whole cell lysates were subjected to immunoblotting with antibodies that recognize the indicated antigens. GAPDH served as a loading control. In this and subsequent figures, gray arrow indicates farnesylated antigen and black arrow indicates unfarnesylated antigen. (B) U937 cells stably transduced with empty vector or HA-tagged Rheb M184L were treated for 24 h with diluent (0.1% DMSO) or 800 nM tipifarnib. Whole cell lysates were then probed with antibodies to the indicated antigen. Note that S6 phosphorylation diminishes less after tipifarnib treatment in cells transduced with HA-tagged Rheb M184L than in cells transduced with empty vector. (C) U937 cells stably transduced with empty vector or HA-tagged Rheb M184L were treated for 6 days with the indicated tipifarnib concentration, stained with propidium iodide and subjected to flow microfluorimetry as depicted in Online Supplementary Figure S2A.

Figure 4.

Tipifarnib-induced apoptosis in U937 cells is Bax- and Puma-dependent. (A) After U937 cells were treated for 6 days with the indicated tipifarnib concentration, whole cell lysates were subjected to immunoblotting with antibodies that recognize the indicated polypeptides. (B) After U937 cells were treated with the indicated tipifarnib concentration for 6 days, cell lysates were subjected to immunoprecipitation (IP) with 6A7 antibody, which recognizes Bax only in its active conformation. Blots were then probed with an antibody that recognizes total Bax. GAPDH served as a loading control for the input. (C) After U937 cells were treated for the indicated length of time with 800 nM, whole cell lysates were subjected to immunoblotting. (D) Independent clones of U937 cells stably transduced with empty vector (C2, C3), Puma shRNA (H4, H5) or Bax shRNA (A2, A7) were treated for 6 days with the indicated tipifarnib concentration, lysed, and stained with propidium iodide as illustrated in Online Supplementary Figure S2A. Inset in (D), immunoblot showing whole cell lysates probed for Puma (left panels) or Bax (right panels). β-actin served as a loading control.

Figure 5.

Posttranslational stabilization of Bax and Puma. (A and B) after U937 cells were treated with 800 nM tipifarnib for the indicated time in the presence of 5 μM Q-VD-OPh, a broad spectrum caspase inhibitor, RNA was isolated and subjected to semiquantitative (A) or quantitative RT-PCR (B) using GAPDH as a loading control. (C) After treatment for 3 days with diluent (lanes 1–4) or 800 nM tipifarnib (lanes 5–8), cells were supplemented with 30 μM cycloheximide for 0–48 h in the continued presence of diluent or tipifarnib, then subjected to SDS-PAGE followed by immunoblotting with antibodies that recognize the indicated antigens. GAPDH served as a loading control. The similar levels of Bax and Puma in lanes 1 and 5 are consistent with time course experiments in which a 6-day tipifarnib treatment up-regulated Bax or Puma (Figure 4A) but a 3-day treatment (Figure 4C) did not.

Figure 6.

Characterization of tipifarnib-resistant U937 cells. (A and B) parental U937 cells or cells growing continuously in 800 nM tipifarnib were washed free of drug, diluted to 20,000 cells/mL and incubated for 6 days in the presence of the indicated concentration of tipifarnib (A) or lonafarnib (B) before incubation with MTS. (C) Whole cell lysates prepared from parental U937 cells treated for 48 h with diluent or 800 nM tipifarnib (lanes 2 and 3, respectively) or from tipifarnib-resistant cells growing in 800 nM tipifarnib (lane 1) were subjected to SDS-PAGE followed by immunoblotting with antibodies that recognize the indicated antigen. (D) Immunoblots (left) or semiquantitative PCR (right) using protein or RNA, respectively, from parental or tipifarnib-resistant U937 cells. For the immunoblot, parental cells were treated with diluent or 800 nM tipifarnib for 48 h. HSP90β and GAPDH served as loading controls, respectively.

Figure 7.

Correlation between tipifarnib-induced Bax upregulation and apoptosis. A. After ML-1 cells were treated for 6 days with the indicated tipifarnib concentration, samples were stained with propidium iodide and subjected to flow microfluorimetry. B. Summary of subdiploid events observed after U937, ML-1 or HL-60 cells were analyzed as illustrated in panel A. C. After ML-1 cells were treated with diluent (lane 1) or tipifarnib at 100, 200, 400 or 800 nM (lanes 2–5, respectively) for 6 days, whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies to the indicated antigen. D. After ML-1 (lanes 1–3) or HL-60 (lanes 4–6) were treated for 48 h with 0.1% DMSO (lanes 1, 4), 800 or 1600 nM tipifarnib (lanes 2 and 5, respectively) or 100 nM rapamycin (lanes 3, 6), whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies to the indicated antigen. For each antigen, all lanes were derived from corresponding signals on a single piece of x-ray film but were rearranged for clarity. E. After HL-60 cells were treated for 6 days with diluent (lane 1) or tipifarnib at 200, 400, 800 or 1600 nM (lanes 2–5, respectively), whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies to the indicated antigens. The nucleoskeletal protein lamin B1 served as a loading controls in panels (D and E). (F and G). Aliquots containing 5 × 105 marrow mononuclear cells harvested prior to treatment (Day 0) or after 14 doses of tipifarnib (on Day 8 before further drug administration) on a phase II trial of tipifarnib (Days 1–14) + etoposide (Days 1–3 and 8–10) (F) or a phase II trial of single-agent tipifarnib (G) were subjected to SDS-PAGE followed by immunoblotting for the indicated antigens. Number below panel in F indicates RasGRP1:aprataxin ratio previously reported for the corresponding mRNA sample. NA: not assayed due to lack of RNA sample.