Active oral regimen for elderly adults with newly diagnosed acute myelogenous leukemia: a preclinical and phase 1 trial of the farnesyltransferase inhibitor tipifarnib (R115777, Zarnestra) combined with etoposide

Abstract

The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results, we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age, 77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600, etoposide 100) and 8A (tipifarnib 400, etoposide 200). In vivo, tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as #NCT00112853.

Figure 1

Antiproliferative effects of tipifarnib-containing combinations. (A,B) Log-phase HL-60 cells were treated with the indicated concentration of etoposide (○, A), tipifarnib (○, B), or the combination at a 2:1 ratio (●, A,B). (C) Combination index values calculated from the data shown in panels A and B when data were analyzed by the median effect method as previously described in detail. (D) Summary of results obtained when tipifarnib was combined with multiple orally bioavailable antileukemic agents. Results are expressed as the mean ± SD of combination index values at the IC₅₀ and IC₉₀ in the indicated number of independent experiments.

Figure 2

Tipifarnib enhances induction of apoptosis by etoposide in AML cell lines. (A) Log-phase HL-60 cells were treated for 24 hours with diluent, 1.5 μM etoposide, 1 μM tipifarnib, or the combination of 1.5 μM etoposide plus 1 μM tipifarnib. At the completion of the incubation, cells were fixed, stained with Hoechst 33258, and examined by fluorescence microscopy. Arrow represents apoptotic cells. (B) Samples shown in panel A and additional samples treated with differing concentrations of etoposide in the absence or presence of 1 μM tipifarnib were examined by fluorescence microscopy (> 500 cells/sample) by an investigator blinded to the treatment, and the percentage of cells displaying apoptotic morphologic changes was recorded. Inset in panel B: summary of 4 independent experiments in which HL-60 cells were treated with diluent, 2 μM etoposide, 1 μM tipifarnib, or 2 μM etoposide plus 1 μM tipifarnib. Error bars represent ± 1 SD. *P = .015 by paired t test. (C) Log-phase U937 cells were treated for 24 hours with diluent, 0.375 μM etoposide, 1 μM tipifarnib, or the combination of 0.375 μM etoposide plus 1 μM tipifarnib. At the completion of the incubation, cells were permeabilized, stained with propidium iodide, and examined by flow microfluorimetry. (D) Samples shown in panel C and additional samples treated with differing concentrations of etoposide in the absence or presence of 1 μM tipifarnib were examined by flow cytometry, and the percentage of cells with less than 2n DNA content was recorded. (D inset) Summary of 4 independent experiments in which U937 cells were treated with diluent, 0.375 μM etoposide, 1 μM tipifarnib, or 0.375 μM etoposide plus 1 μM tipifarnib. Error bars represent ± 1 SD. *P = .008 by paired t test.

Figure 3

Tipifarnib preferentially inhibits signaling downstream of mTOR. (A) HL-60 cells treated with 0, 62.5, 125, 250, 500, or 1000 nM tipifarnib (lanes 1-6, respectively) were washed and examined for topoisomerase IIα content by immunoblotting. Numbers at right represent migration of molecular markers in kilodaltons. The same blot was probed with anti-Hsp90 as a loading control. (B) Alkaline elution to evaluate the possibility that tipifarnib enhances etoposide uptake and/or trapping of covalent topoisomerase II-DNA complexes. After log-phase HL-60 cells were treated for 24 hours with 1 μM tipifarnib or diluent, etoposide was added for 30 minutes. The ability of etoposide to stabilize covalent protein-DNA covalent complexes was quantitated as indicated. (C) HL-60 cells were treated with diluent (lane 1), 62.5 (lane 2), 125 (lane 3), 250 (lane 4), 500 (lane 5), or 1000 nM (lane 6) tipifarnib for 24 hours. Whole-cell lysates were blotted with antibodies to phospho-Thr³⁸⁹-p70S6 kinase, phospho-Ser^235/236-S6, p70S6 kinase, and total S6 protein. (D) U937 cells were treated with diluent (lane 1), 125 (lane 2), 250 (lane 3), 500 (lane 4), or 1000 nM (lane 5) tipifarnib for 24 hours. Whole-cell lysates were blotted with antibodies to phospho-Ser^235/236-S6, total S6, the farnesylated protein HDJ-2 and, as a loading control, heat shock protein 90.

Figure 4

Effect of tipifarnib on antiproliferative effects of etoposide in clinical AML isolates. (A-C) Freshly isolated mononuclear cells from 3 newly diagnosed AML patients were plated in methylcellulose in diluent, 25 nM tipifarnib plus 50 to 200 nM etoposide, or 25 nM tipifarnib plus 50 to 200 nM etoposide as indicated. Bars represent relative colony counts observed after the indicated treatment. ○ represents relative colony counts predicted from the effects of the individual agents using the fractional product method. (D) Freshly isolated mononuclear cells from a newly diagnosed AML patient were plated in methylcellulose in the presence of etoposide alone (○), tipifarnib alone (inset), or 25 nM tipifarnib plus the indicated concentration of etoposide (●). (E) Combination index values calculated from the data in panel D (●) and assays in samples from 3 other AML patients (○ and △) under conditions that are render the analysis equivalent to isobologram analysis. Note that a combination index less than 1.0 indicates synergy.

Figure 5

Survival curves of patients treated with T + E. (A) DFS (median, —; 95% CI, – –) for 21 patients who achieved complete remission in response to T + E. (B) OS (median, —; 95% CI, ) for 84 adults treated with T + E. (C) OS for 21 patients achieving complete remission (CR, —), 13 patients achieving partial remission or hematologic improvement (PR/HI, ), and 49 patients who did not achieve response (NR/NE, – –).

Figure 6

Estimated dose-response relationship between combinations of etoposide and tipifarnib. The height of the surface and its shading indicate the response rate for each dose combination.

Figure 7

Assessment of pathway inhibition in situ. Bone marrow mononuclear cells harvested before institution of therapy (day 0) and on day 8 before drug administration were subjected to immunoblotting with antibodies that recognize phosphor-Ser^235/236 ribosomal protein S6, GSK3β phosphorylated by Akt on Ser and total GSK3β. The shift in HDJ-2 and appearance of prelamin A on day 8 served to confirm FTase inhibition,^, whereas Bak served as a loading control. Note that S6 phosphorylation was inhibited in 2 patients (patients A and B) but not the third (patient C).