Potent Farnesyltransferase Inhibitor ABT-100 Abrogates Acute Allograft Rejection

TRANSPLANTATION IMMUNOLOGY

Potent Farnesyltransferase Inhibitor ABT-100 Abrogates Acute Allograft Rejection Ming-Sing Si, MD,a Ping Ji, MD,a Michael Lee, BS,a Jennifer Kwok, BS,a Jamie Kusumoto, BS,a Eric Naasz, BS,a Shi-Chung Ng, PhD,b and David K. Imagawa, MD, PhDa Background: Farnesyltransferase inhibitors (FTI’s) inhibit the function of Ras, a GTPase involved in carcinogenesis and T cell activation. We evaluated the in vitro and in vivo immunomodulatory properties of a rationally designed FTI, ABT-100. Methods: The effects of ABT-100 on human peripheral blood mononuclear cell (PBMC) proliferation and the expression of the T cell activation markers CD25 and CD69 were studied. In a Wistar to Lewis rat heterotopic cardiac transplant model, ABT-100 was orally dosed alone or with a subtherapeutic course of cyclosporine (CsA). The degree of graft immune cell infiltrate was determined. Results: ABT-100 potently inhibited PBMC proliferation, but did not decrease expression of CD25 and CD69 during activation. Treatment with 25, 50 and 100 mg/kg ABT-100 BID increased allograft mean survival time (MST) to 12.8⫾3 days, 13.5⫾5 days and 13.8⫾3 days, respectively (vs 6.5⫾3 days for controls, p⬍0.001 by log rank). A subtherapeutic course of CsA increased MST to 12.7⫾3 days (p⬍0.001 vs control). Combination with ABT-100 at 25 and 100 mg/kg BID improved MST to 18.7⫾5 days and 19.5⫾4 days (both p⬍0.001 vs control and respective monotherapy groups). ABT-100 treatment at 100 mg/kg BID significant decreased the amount of graft infiltrate (2.5⫾4 mononuclear cells/high power field (hpf) vs 29⫾11 cells/hpf, p⬍0.001). Conclusions: This is the first report that a specific FTI delays the development of acute rejection and supports the strategy of inhibiting Ras to impart immunomodulation. The antirejection and anticarcinogenic effects make FTI’s a potentially useful adjunct in the antirejection regimens of malignancy-prone organ transplant recipients. J Heart Lung Transplant 2005;24:1403–9. Copyright © 2005 by the International Society for Heart and Lung Transplantation.

Ras, a small guanosine triphosphate-binding protein, mediates important cellular signaling events that regulate activation, proliferation, motility, and other key cellular functions.1–3 Ras is a 21-kD protein that undergoes a post-translational modification known as farnesylation.4 This post-translational lipid modification is carried out by the enzyme farnesyltransferse and allows Ras to insert into lipid bilayers. The localization of Ras to specific cellular compartments facilitates its activaFrom the aUCI Transplantation Laboratory, Department of Surgery, University of California Irvine College of Medicine, Irvine, California and bCancer Research, Global Pharmaceutical and Development, Abbott Laboratories, Abbott Park, Illinois Submitted January 15, 2004; revised May 19, 2004; accepted June 3, 2004. This work was in part supported by a National Institutes of Health National Research Service Award and an American College of Surgeons Residents Research Scholarship awarded to M.S.I. Reprint requests: David K. Imagawa, MD, PhD, FACS, Section of Hepatobiliary and Pancreas Surgery and Islet Cell Transplantation, Department of Surgery, UCIMC, 101 The City Drive, Bldg 56, Rm 202, Orange, CA 92868. Telephone: 714-456-8341. Fax: 714-456-8437. E-mail: [email protected] Copyright © 2005 by the International Society for Heart and Lung Transplantation. 1053-2498/05/$–see front matter. doi:10.1016/ j.healun.2004.06.006

tion and the subsequent activation of effector molecules such as Raf-1.5 The lack of this post-translational modification leads to the loss of function of Ras. Constitutive Ras activation is characteristic of most cancers, prompting the development of farnesyltransferase inhibitors as anti-cancer agents.6,7 Farnesyltransferase inhibitors have shown significant efficacy in pre-clinical cancer studies and several candidate compounds are currently undergoing clinical trial evaluation.8 –11 Ras also plays an important role in T-cell activation and function. The importance of Ras in immune cell physiology was evident when it was discovered that ligation of the T-cell receptor led to Ras activation in T cells.12 Subsequent studies demonstrated that Ras, along with calcineurin, mediates signals from the T-cell receptor to activate the nuclear factor of activated T cells (NFAT).13 Further, Ras was also found to have an important role in T cell interleukin-2 (IL-2) production.14 Early work suggested that Ras had a mandatory role in T-cell activation; however, it was later shown that in some situations T cells could undergo Rasindependent activation.15 Given the importance of Ras in T-cell activation and function, we hypothesized that farnesyltransferase in1403

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hibitors would have immunomodulatory effects. Indeed, we were able to show that A-228839, a potent farnesyltransferase inhibitor, inhibited lymphocyte activation and cytokine production in vitro.16 In light of the results of our in vitro studies, we describe here our efforts to determine the potential anti-rejection properties of ABT-100, an orally available farnesyltransferase inhibitor. It has an inhibitory concentration of 50% (IC50) of 0.13 nmol/liter toward bovine farnesyltransferase and selectivity vs geranylgeranyltransferase (IC50 ⬎ 10,000 nmol/liter).17 More significantly, ABT-100 has also been shown to block Ras processing in several cell lines at sub-nanomolar concentrations.17 We first confirmed the in vitro immunomodulatory effects of ABT-100 on human peripheral mononuclear blood cell (PBMC) proliferation and T-cell activation and then evaluated its efficacy in the prevention of acute allograft rejection in a rat model of heterotopic cardiac transplantation.

markers as described elsewhere, with minor modifications.19 –21 Briefly, PBMCs isolated from 4 different donors were placed in complete media (1 ⫻ 106 cells/ml) and incubated with incremental concentrations of ABT-100 for 30 minutes before stimulation with PHA (7 ␮g/ml) for 72 hours in growth conditions. The PBMCs were then washed with phosphate-buffered saline (PBS) and subsequently stained with antiCD3⑀-PE, anti-CD69-FITC and anti-CD25-CyChrome monoclonal anti-bodies (BD Biosciences Pharmigen, San Diego, CA) for 15 minutes at room temperature shielded from light. After incubation with these anti-bodies, cells were washed in PBS and fixed with PBS-buffered 1% formalin (vol/vol) before analysis on a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA). Appropriate isotype controls were also obtained with each experiment. Data were analyzed using CellQuest Pro (BD Biosciences Immunocytometry Systems, San Jose, CA).

MATERIALS AND METHODS Lectin-Induced PBMC Proliferation Assay Human PBMCs were isolated from fresh buffy coats obtained from the UCI Medical Center Blood Bank by Ficoll centrifugation under an approved institutional human subjects protocol. PBMCs were placed in RPMI media supplemented with 10% fetal bovine serum (FBS, Life Technologies, Rockville, MD), 2 mmol/liter L-glutamine (Sigma, St. Louis, MO), 100 U/ml penicillin (Life Technologies), 100 ␮g/ml streptomycin (Life Technologies), 1% non-essential amino acids (Sigma), 1 mmol/ liter sodium pyruvate (Sigma) and 100 mmol/liter ␤-mercaptoethanol (Sigma). The PBMCs were then dispensed into 96-well microtiter plates at a concentration of 1 ⫻ 106 cells/ml at a volume of 100 ␮l/well. ABT-100 stock solution was freshly made by dissolving the compound in dimethylsulfoxide (DMSO). Serial dilutions of the ABT-100 stock solution were made and then added to the cells to obtain incremental concentrations of drug. The total amount of DMSO present in the proliferation assays was less than 0.1% by volume, and did not affect the proliferative and activation capacity of the PBMCs (data not shown). The PBMCs were incubated with ABT-100 for 30 minutes before stimulation with phytohemagglutinin (PHA, Calbiochem-Novabiochem Corp., San Diego, CA) at a 7 ␮g/ml-concentration. The PBMCs were allowed to proliferate for 72 hours at 37°C, 10% CO2 in humidified air before quantification by 3[H]-thymidine uptake as described elsewhere.18

Rat Transplant Model

T Cell Activation Marker Measurement To assess the effects of ABT-100 on T-cell activation, we measured the expression of cell surface activation

Animals Inbred 200 to 250 g male Lewis rats were the recipients and Wistar rats (allografts) or Lewis rats (isografts) were the cardiac donors. The rats were purchased from Harlan Sprague-Dawley, Indianapolis, IN. They were kept in conventional housekeeping facilities and allowed free access to food and water. Animal care and procedures were conducted under approved institutional protocols. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86 –23, revised 1985). Surgical Procedure Donor and recipient animals underwent induction anesthesia with isoflurane (via calibrated vaporizer), and anesthesia was maintained with pentobarbital (50 mg/kg intraperitoneal). Heart transplantation was performed according to the modified technique of Ono and Lindsey.22 Cardiac allograft survival was determined by daily abdominal palpation, and rejection was considered complete at the time of heart beat cessation. Experimental Design ABT-100 was prepared in carboxymethylcellulose and ethanol. The animals were divided into 7 groups (n ⫽ 6/group, unless specified otherwise). Group 1 (n ⫽ 8) received vehicle only twice daily by gavage. Group 2, 3, and 4 (n ⫽ 8) animals received ABT-100 at a dose of 25 mg/kg, 50 mg/kg, and 100 mg/kg, respectively, twice daily by gavage. Group

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Figure 1. Farnesyltransferase inhibitor ABT-100 abrogates peripheral blood mononuclear cell proliferation. Treatment of human peripheral blood mononuclear cells with incremental concentrations of ABT-100 30 minutes prior and during lectin stimulation for 72 hours resulted in potent inhibition of proliferation. Proliferation was measured by [3H]thymidine uptake. ABT-100 significantly inhibited proliferation at all concentrations tested. Shown in this graph is the average of 3 independent experiments (⫾SD). CPM, counts per minute; PHA, phytohemagglutinin.

5, 6, and 7 animals all received a sub-therapeutic course of cyclosporine A (CsA) (10 mg/kg daily for 3 days by gavage). Group 6 and 7 animals were also given ABT-100 at respective doses of 25 mg/kg and 100 mg/kg twice daily to determine if any synergism existed between ABT-100 and CsA. Transplanted hearts were harvested at the time of rejection, and allografts from 2 animals in groups 1 and 4 were harvested on Day 5. Multiple mid-ventricular sections were performed for histologic analysis. Histologic Analysis Graft samples obtained for histologic analysis were fixed in 10% formalin solution, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. Inflammatory infiltrate in Day 5 grafts from Groups 1 and 4 were quantified by a blinded investigator who counted the number of mononuclear inflammatory cells per high power field; at least 20 random high power fields were studied per graft. Statistical Analysis Results from individual in vitro experiments were averaged, and the 2-tailed Student’s t-test was used to compare different treatment concentrations. Analysis of variance (ANOVA) was also used to analyze surface activation marker expression of drug-treated cells and untreated control cells. Graft survival was analyzed by the Kaplan-Meier method, and graft survival comparisons between animal groups were evaluated with the log-rank test. A p value of less than 0.05 was considered to be statistically significant. RESULTS ABT-100 Inhibits PBMC Proliferation Figure 1 summarizes the results of 4 PBMC proliferation experiments in which incremental concentrations of

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Figure 2. ABT-100 only moderately affects activation surface marker expression on T cells. Peripheral mononuclear blood cell PBMCs were treated with incremental concentrations of ABT-100 before stimulation with phytohemagglutinin (PHA) for 72 hours. Cells were incubated with fluorophore conjugated monoclonal anti-bodies specific for CD3, CD25 and CD69, fixed and then analyzed on a flow cytometer. Results are represented as the average (⫾SD) of 4 independent experiments and are expressed as percentages of all CD3 positive cells analyzed. ABT-100 at the higher concentrations tested only moderately decreased the population of CD3 cells expressing both CD25 and CD69. There was a mild, yet significant, increase in the number of CD3 cells expressing CD69 at the lowest concentration of ABT-100 tested.

ABT-100 were incubated with the cells for 30 minutes before stimulation with PHA. ABT-100 potently inhibited lectin-induced PBMC proliferation with an IC50 determined graphically to be less than 1 ␮mol/liter. Inhibition of PBMC proliferation was statistically significant at all concentrations tested (p ⬍ 0.05). Near maximal drug effect was seen at concentrations near 1 ␮mol/liter, although complete suppression of proliferation was not seen at the highest concentration of ABT-100 tested (20 ␮mol/liter). ABT-100 Does Not Affect the Expression of T Cell Activation Markers CD25 and CD69 To determine the effect of ABT-100 on T-cell activation, we measured T-cell expression of the surface activation markers CD25 and CD69 during activation. Treatment of human CD3 T cells with ABT-100 did not significantly decrease the expression of these activation surface markers during lectin-induced activation, even at the highest concentration tested (10 ␮mol/liter, Figure 2). When the CD25 and CD69 double-positive CD3 T cells were analyzed, a moderate, yet significant, decrease was noted in this sub-set at the higher concentrations of ABT-100 tested. At 0.5 ␮mol/liter, ABT-100 mildly increased the expression of CD69 on CD3 T cells (9% vs 10%; p ⬍ 0.01). When the surface marker expression data was analyzed with ANOVA, we found no differences between untreated controls and ABT-100 treatment.

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Figure 4. ABT-100 inhibits immune cell infiltration into cardiac allografts. Wistar rat allografts harvested from Lewis rat recipients treated with ABT-100 (100 mg/kg twice daily) had a significant reduction in the number of inflammatory mononuclear cells. Shown are representative photomicrographs of hematoxylin and eosin preparations of control and treatment allografts (⫻200 magnification). (A) Control grafts at Day 5 post-transplant had an average (⫾SD) of 29 ⫾ 11 mononuclear cells per high power field. (B) Grafts from ABT-100 treated animals at Day 5 post-transplant had an average of 2.5 ⫾ 1.4 mononuclear cells per high power field. (C) Control grafts at rejection had an average of 78 ⫾ 5.7 mononuclear cells per high power field. (D) Grafts from ABT-100 treated animals at rejection had an average of 73 ⫾ 8.4 mononuclear cells per high power field. ABT-100 treatment significantly decreased the number of inflammatory mononuclear cells at both time points (p ⬍ 0.001 vs control).

Figure 3. ABT-100 significantly abrogates acute allograft rejection. Treatment of Lewis rat recipients of Wistar rat cardiac allografts with ABT-100 resulted in abrogation of acute allograft rejection. (A) ABT-100 was given at the indicated doses twice daily by oral gavage and significantly improved allograft survival (p ⬍ 0.001 vs vehicle control). (B) Combination with a sub-therapeutic course of cyclosporine A (CsA) led to further improvements in allograft survival (p ⬍ 0.001 vs vehicle control and respective ABT-100 monotherapy groups). Survival curves were generated by Kaplan-Meier analysis and comparisons made with the log-rank test. BID, twice a day.

ABT-100 Inhibits Acute Allograft Rejection To determine the in vivo anti-rejection effects of ABT100, we evaluated this compound in a rat heterotopic heart transplant model. Vehicle control animals consistently rejected their allografts by Day 7, yielding a mean graft survival time of 6.5 ⫾ 0.29 (SE) days. Treatment of allograft recipients with ABT-100 at doses of 25 mg/kg, 50 mg/kg, and 100 mg/kg twice daily resulted in significantly improved mean graft survival times of 12.8 ⫾ 0.3 days, 13.5 ⫾ 0.5 days, and 13.8 ⫾ 0.3 days, respectively (Figure 3A, p ⬍ 0.001 by log-rank test). Treatment of cardiac allograft recipients

with a sub-therapeutic regimen of CsA resulted in a mean allograft survival time of 12.7 ⫾ 0.3 days (p ⬍ 0.001 vs vehicle control). The combination of this sub-therapeutic course of CsA with ABT-100 at 25 mg/kg and 100 mg/kg twice daily improved the graft mean survival time to 18.7 ⫾ 0.5 days and 19.5 ⫾ 0.4 days, respectively (Figure 3B, both p ⬍ 0.001 vs vehicle control). The addition of sub-therapeutic CsA to ABT-100 treatment at both doses led to significant improvements in allograft survival time compared with the respective ABT-100 monotherapy groups (p ⬍ 0.001). ABT-100 Significantly Decreases Immune Cell Infiltrate in Cardiac Allografts ABT-100 treatment significantly abrogated the presence of immune cell infiltrate in cardiac allografts. Vehicle control recipients had an average of 29 ⫾ 11 mononuclear cells per high power field at 5 days post-transplant (Figure 4A). Recipients treated with ABT-100 at 100 mg/kg twice daily had an average of 2.5 ⫾ 1.4 mononuclear cells per high power field at 5 days posttransplant (Figure 4B, p ⬍ 0.001 vs control). Vehicle control recipients had an average of 78 ⫾ 5.7 mononu-

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clear cells per high power field at rejection (Figure 4C) while ABT-100 – treated animals had a slight yet significant decrease in immune cell infiltrate at rejection (73 ⫾ 8.4 immune cells per high power field, p ⬍ 0.001 vs control) (Figure 4D). DISCUSSION Because current immunosuppressive anti-rejection therapies, which include calcineurin inhibitors, anti-proliferative agents, and corticosteroids, have significant side effects, there is a need for the discovery and development of new agents to be used as monotherapy or as an adjunct in a strategy to reduce the dosage of current immunosuppressive agents needed to prevent rejection. Because Ras plays a significant role in T-cell activation, we postulated that farnesyltransferase inhibitors, such as anti-Ras agents, would have immunomodulatory activity. Furthermore, other methods of interfering with Ras function in vitro have resulted in the abrogation of T-cell activation and function. For instance, transfection of T cells with dominant negative Ras gene resulted in the inability of IL-2 gene induction after T-cell receptor activation.14 We explored our hypothesis that farnesyltransferase inhibitors would have immunomodulatory properties by investigating the effects of the potent farnesyltransferase inhibitor A-228839 on lymphocytes in vitro. We found that A-228839 potently inhibited lymphocyte proliferation and cytokine production, interfered with intracellular calcium homeostasis, and promoted activation-induced apoptosis.16 Here we confirmed that ABT-100, another selective farnesyltransferase inhibitor, also potently inhibits human PBMC proliferation. Like A-228839, ABT-100 has potent activity against lymphocyte proliferation at concentrations near 1 ␮mol/liter yet fails to completely suppress lymphocyte proliferation even at high concentrations. These results suggest the presence of Rasindependent mechanisms during lymphocyte activation and proliferation that farnesyltransferase inhibitors do not have activity against. Further, the lack of strong activity against the expression of activation markers CD25 and CD69 support the presence of Ras-independent mechanisms during T-cell activation. Alternative explanations to the incomplete suppression of proliferation and expression of surface activation markers include the presence of a sub-set of lymphocytes that are in a state where Ras function is not imperative for activation and effector function or that polyclonal activation with lectin may not be as dependent on Ras as activation via the T-cell receptor. Previous investigation by D’Ambrosio and colleagues demonstrated the involvement of Ras in CD69 expression on T cells.23 These investigators determined that transfection of constitutively active Ras in T cells led to

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increased surface expression of CD69 and that transfection of a dominant negative Ras led to decreased surface expression of this activation marker. However, their investigation did not exclude the possibility that CD69 expression could be increased by Ras-independent signals, and thus our results that farnesyltransferase inhibition did not decrease CD69 expression may not be contradictory in this situation. We believe that ABT-100 does not have a physiologically significant effect on T-cell CD69 expression even though there was a minimal but statistically significant increase in the expression of this activation marker at 0.5 ␮mol/liter ABT-100. That the inflammatory infiltrate was nearly absent in ABT-100 –treated recipients 5 days post-transplant may be the result of inhibited lymphocyte activation, impaired chemokine production, and/or impaired inflammatory cell chemotaxis. It has been shown recently that chemokine receptor binding of monocyte chemoattractant protein-1 on monocytes leads to the activation of several signaling molecules including Ras.24 It has also been suggested that Ras is also involved in chemokineinduced integrin-dependent adhesion of T cells at sites of inflammation.25 Furthermore, the farnesylated guanosine triphosphatase Rho that may also be inhibited by ABT-100 is involved in regulating lymphocyte motility.26,27 Indirect evidence that points to a possible effect of farnesyltransferase inhibitors on immune cell motility includes the findings that the farnesyltransferase inhibitor L-744,832 inhibits breast carcinoma cell motility in vitro and that the relatively non-specific prenylation inhibitor perillyl alcohol arrests T-cell motility in vitro.28,29 We are currently determining the effects of ABT-100 on lymphocyte and immune cell motility and adhesion. We determined that a specific farnesyltransferase inhibitor delays the development of acute allograft rejection in an animal model of organ transplantation. Although it prevented the development of an inflammatory infiltrate relatively early post-transplant, ABT100 treatment of rat recipients of cardiac allografts did not result in indefinite graft survival. Moreover, dose escalation did not result in significant gains in anti-rejection activity. This in vivo result parallels those seen in our in vitro proliferation assays: lack of complete suppression of immune cell activation and function, even at high drug exposure, thus highlighting the significance of Ras-independent pathways in the activation of immune cells or the presence of a population of lymphocytes that do not rely on Ras for robust activation. In vitro studies have demonstrated that Ras and calcineurin synergize to activate NFAT in T cells.13 Hence we expected that blockade of Ras with ABT-100 and calcineurin with CsA would result in synergistic

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inhibitory activity of the 2 agents. In the animal model that we used here, the anti-rejection activity of ABT-100 is additive to that of CsA. A potential explanation to the lack of synergistic activity in our animal model is our dosing regimen of CsA. CsA was only given for 3 days, thus resulting in potential drug synergism for this limited to this time interval. However, in our in vitro studies with A-228839, additive (not synergistic) activity against lymphocyte proliferation was seen when it was combined with constant exposure to CsA.16 Defects in Ras activation have also been proposed to be an underlying mechanism of T-cell anergy.30,31 T-cell anergy is a strategy for obtaining tolerance. Although not specifically measured here, it is not unreasonable to speculate that ABT-100 interference with Ras function did not result in effective induction of long-term anergy as allografts did not survive indefinitely. Although current immunosuppressive regimens are effective in preventing acute allograft rejection, organ transplant recipients are at risk for lymphomas and skin cancers, which are a major cause of morbidity and mortality.32 Treatment of post-transplant malignancies includes tapering or even withdrawal of immunosuppressive medications, thus placing the allograft at risk for rejection. A farnesyltransferase inhibitor has been recently shown to be effective in treating lymphoma in a murine model.33 The potential anti-rejection and anti-carcinogenic effects of farnesyltransferase inhibitors make this class of agents a potential adjunct in the treatment and prevention of malignancies in organ transplant recipients. REFERENCES 1. Downward J. Targeting RAS signaling pathways in cancer therapy. Nat Rev Cancer 2003;3:11–22. 2. Hancock JF. Ras proteins: different signals from different locations. Nat Rev Mol Cell Biol 2003;4:373– 84. 3. Oxford G, Theodorescu D. Ras superfamily monomeric G proteins in carcinoma cell motility. Cancer Lett 2003;189: 117–28. 4. Casey PJ, Solski PA, Der CJ, Buss JE. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A 1989;86: 8323–7. 5. Kikuchi A, Williams LT. The post-translational modification of ras p21 is important for Raf-1 activation. J Biol Chem 1994;269:20054 –9. 6. Prendergast GC, Oliff A. Farnesyltransferase inhibitors: antineoplastic properties, mechanisms of action, and clinical prospects. Semin Cancer Biol 2000;10:443–52. 7. Prendergast GC, Rane N. Farnesyltransferase inhibitors: mechanism and applications. Expert Opin Investig Drugs 2001;10:2105–16. 8. Awada A, Eskens FA, Piccart M, et al. Phase I and pharmacological study of the oral farnesyltransferase in-

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29. Wei X, Si MS, Imagawa DK, Ji P, Tromberg BJ, Cahalan MD. Perillyl alcohol inhibits TCR-mediated [Ca(2⫹)](i) signaling, alters cell shape and motility, and induces apoptosis in T lymphocytes. Cell Immunol 2000;201:6 –13. 30. Fields PE, Gajewski TF, Fitch FW. Blocked Ras activation in anergic CD4⫹ T cells. Science 1996;271:1276 – 8. 31. Rapoport MJ, Lazarus AH, Jaramillo A, Speck E, Delovitch TL. Thymic T cell anergy in autoimmune nonobese diabetic mice is mediated by deficient T cell receptor regulation of the pathway of p21ras activation. J Exp Med 1993;177:1221– 6. 32. Vial T, Descotes J. Immunosuppressive drugs and cancer. Toxicology 2003;185:229 – 40. 33. Mishra S, Zhang B, Groffen J, Heisterkamp N. A farnesyltransferase inhibitor increases survival of mice with very advanced stage acute lymphoblastic leukemia/lymphoma caused by P190 Bcr/Abl. Leukemia 2004;18:23– 8.