Cytotoxicity of farnesyltransferase inhibitors in lymphoid cells mediated by MAPK pathway inhibition and Bim up-regulation

Abstract

The mechanism of cytotoxicity of farnesyltransferase inhibitors is incompletely understood and seems to vary depending on the cell type. To identify potential determinants of sensitivity or resistance for study in the accompanying clinical trial ( IntroductionFarnesyltransferase inhibitors (FTIs) are currently undergoing extensive clinical testing in various hematologic malignancies. [1][2][3] These agents inhibit farnesyltransferase, an enzyme that transfers the 15-carbon farnesyl group from farnesyl pyrophosphate to a variety of polypeptide acceptors, including the chaperone heat shock protein 40/HDJ-2; the nuclear intermediate filament proteins prelamin A and lamin B; the centromere protein CENP E; and small GTP-binding proteins of the Ras, Rho, and Rheb families. 4,5 Collectively, inhibition of farnesylation of these polypeptides leads to diminished cell proliferation. In addition, FTIs induce cell death in some model systems under certain conditions. These cytotoxic effects have been attributed to FTI-induced inhibition of prosurvival signaling by Akt,6,7 signal transducers and activators of transcription, [8][9][10] mitogen-activated protein kinases (MAPKs), 9,[11][12][13] or the Rheb target mammalian target of rapamycin. 14 Recent work has especially emphasized the role of Rheb inhibition as a mechanism of FTI-induced antilymphoma effects in murine lymphomas and leukemia. 15 Alternatively, it has been suggested that FTIs induce apoptosis by causing up-regulation of the proapoptotic Bcl-2 family members Bax, 16 Bak, 17 or Puma. 18 Although FTIs were initially developed based on the premise that inhibition of farnesylation would abrogate signaling by mutant Ras proteins, 19 these agents have demonstrated little efficacy in solid tumors. [20][21][22] In contrast, tantalizing activity was observed in several hematologic malignancies. [1][2][3] In particular, the orally bioavailable nonpeptidimimetic FTI tipifarnib 23 demonstrated activity in adults with acute leukemia. The initial phase 1 trial not only established a maximum tolerated dose in patients with relapsed and refractory acute leukemias but also determined that tipifarnib levels in bone marrow were 1.6-8 nmol/mg of tissue at this dose, demonstrated FT inhibition in leukemia cells in situ, and provided evidence of activity in relapsed AML. 24 Subsequent phase 2 and phase 3 studies have demonstrated response rates of 11%-23% in elderly patients with previously untreated poor risk acute myeloid leukemia (AML). 25,26 In an effort to select the subset of AML patients most likely to respond, Raponi et al empirically identified a 2-transcript signature, characterized by a high ratio of mRNA encoding the Ras guanine nucleotide exchange factor RasGRP1 27 relative to mRNA encoding the repair protein aprataxin, that had a 92% negative predictive value and a 28% positive predictive value in 2 single-agent phase 2 tipifarnib AML trials. 28 Based on these results, gene signature-guided trials of tipifarnib in acute leukemia are being initiated.Tipifarnib also has...

Measurement of BH3-only protein tolerance

“…Moreover, the fact that analyses involving endogenous Noxa (Figures 1 and 2) and EGFP-Noxa (Figure 3) yield comparable quantative results suggests that the actions of EGFP-Noxa and native endogenous Noxa are similar. Similarly, histograms exploring sensitivity to EGFP-BIM EL (2% survival at 120 000 molecules per cell, 60% survival at 25000 molecules per cell, Figure 3d) concur with results showing that 200 nM tipifarnib, which induces upregulation of BIM EL to roughly 25 000 molecules per cell (Figures 1d and 2b), kills 30-40% of cells (Supplementary Figure S1 and Ding et al 34 ), again showing agreement between the separate methods of quantitation and suggesting that the actions of BIM EL and EGFP-BIM EL are similar.…”

“…As indicated in Figure 7a, transfection with BIM diminished the tolerance for EGFP-NOXA. Accordingly, when Jurkat cells were treated with tipifarnib, which upregulates BIM, and pevonedistat, which upregulates NOXA (Figures 1b and d), 34,38 low concentrations of tipifarnib synergistically enhanced pevonedistat-induced killing (Figures 7b and c).…”

“…After identifying drug concentrations that induce apoptosis in the sensitive Jurkat cell line (Supplementary Figure S1), we examined changes in BH3-only proteins previously shown to be critical for killing by these agents. 34,35,37,38 The proteasome inhibitor bortezomib induced 3-fold increases in BIM and 8-to 16-fold increases in NOXA ( Figure 1a); the Nedd8-activating enzyme inhibitor pevonedistat-induced negligible to 3-fold increases in BIM and 4-fold increases in NOXA ( Figure 1b); the mTOR dual inhibitor TAK-228 induced 2-to 3-fold increases in BIM and 2-to 10-fold increases in PUMA ( Figure 1c); and the farnesyltransferase inhibitor tipifarnib induced 2-to 3-fold increases in BIM in the sensitive Jurkat line but no discernible increase in BIM or NOXA in the resistant SKW6.4 line (Figure 1d). Given the critical role of these BH3-only protein increases in drug-induced killing, 34,35,37,38 these results suggested that cells can only tolerate a certain degree of BH3-only protein upregulation before dying.…”

“…For example, p53-dependent and -independent transcriptional processes upregulate NOXA and PUMA, 31,32 inhibition of mitogen activated protein kinase signaling enhances BIM stability, 33,34 inhibitors of the mechanistic target of rapamycin (mTOR) induce both BIM and PUMA, 35 and inhibition of the proteasome pathway upregulates NOXA through multiple mechanisms. [36][37][38] Importantly, downregulation of the indicated BH3-only proteins impairs killing by each of the indicated treatments, [32][33][34][35]37,38 indicating the critical role of BH3-only protein upregulation in the cytotoxicity of these stresses. Despite extensive qualitative observations such as these, however, quantitative information regarding the extent of BH3-only protein upregulation required to induce apoptosis and the ability of different cells to survive this upregulation is limited.…”

Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm

“…Interestingly, the frequency of these vesicles in sections of apoptotic cells was comparable for all three lines (Figure 2f). Similar cytoplasmic vesicles were observed in Jurkat cells undergoing apoptosis in response to etoposide, an agent previously shown to induce apoptosis in these cells through the mitochondrial pathway, 20 as well as etoposide-treated HL-60, HCT116 and T98G cells; TRAIL-treated D32 and camptothecin-treated Daudi cells (Supplementary Figures S6a, b and d and data not shown). 21 Vesicle formation was not affected by EGTA (data not shown) but was inhibited by Q-VD-OPh (Supplementary Figures S6e and f), suggesting that this process is caspasedependent.…”