research paper
`
`Dual targeting of Bruton’s tyrosine kinase and Janus kinase 3
`with rationally designed inhibitors prevents graft-versus-host
`disease (GVHD) in a murine allogeneic bone marrow
`transplantation model
`
`Marina Cetkovic-Cvrlje1,2
`and Fatih M. Uckun1,2,3
`1Experimental BMT Program, Parker Hughes
`Cancer Center, 2Departments of Immunology and
`3Drug Discovery Program, Parker Hughes
`Institute, St Paul, MN, USA
`
`Received 26 May 2004; accepted for publication
`
`29 June 2004
`
`Correspondence: Marina Cetkovic-Cvrlje,
`
`Parker Hughes Institute, 2720 Patton Road, St
`
`Paul, MN 55113, USA.
`
`E-mail: mcetkovic@ih.org
`
`Summary
`
`The purpose of the present study was to evaluate the effectiveness of targeting
`Bruton’s tyrosine kinase (BTK) with a specific BTK inhibitor, a-cyano-
`b-hydroxy-b-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13), for
`prevention of acute fatal graft-versus-host disease (GVHD) in a murine
`model of allogeneic bone marrow transplantation (BMT). Vehicle-treated
`control C57BL/6 mice receiving bone marrow/splenocyte grafts
`from
`allogeneic BALB/c donors developed severe multi-organ acute GVHD and
`died after a median survival time (MST) of 40 d. LFM-A13 treatment
`(25 mg/kg/d) significantly prolonged the MST of the BMT recipients to 47 d.
`The probability of survival at 2 months after BMT was 2 ± 2% for vehicle-
`treated control mice and 22 ± 6% for mice treated with LFM-A13
`(P ¼ 0Æ0008). Notably, the combination regimen of LFM-A13 plus the
`standard anti-GVHD drug methotrexate (MTX) (10 mg/m2/d) was more
`effective than LFM-A13 alone, while the combination regimen of LFM-A13
`plus the novel anti-GVHD drug JANEX-1 (60 mg/kg/d), targeting Janus
`kinase 3, was more effective than LFM-A13, JANEX-1 or MTX alone. More
`than 70% of recipients receiving this most effective GVHD prophylaxis
`(LFM-A13 + JANEX-1) remained alive throughout the 80-d observation
`period with an MST of >80 d. Taken together, these results indicate that
`targeting BTK with the chemical
`inhibitor LFM-A13 may attenuate the
`severity of GVHD, especially when it is combined with other anti-GVHD
`drugs, such as MTX and JANEX-1.
`
`Keywords: Bruton’s tyrosine kinase inhibitor, LFM-A13, bone marrow
`transplantation, graft-versus-host disease, mouse.
`
`Graft-versus-host disease (GVHD) remains a significant cause
`of morbidity and mortality after allogeneic bone marrow
`transplantation (BMT) (Ferrara, 2003). GVHD occurs when
`alloreactive donor T cells become sensitized to the host, then
`expand and mediate host tissue destruction (Ferrara, 2003).
`Decades of experimental and clinical research have demon-
`strated that donor T cells are the principal mediators and
`effectors of GVHD. However, in addition to donor T cells,
`elements of
`the non-T-cell compartment have also been
`implicated in the pathogenesis of GVHD (Sprent et al, 1990,
`
`1995; Mielcarek et al, 1997; Koh et al, 2000; Aranha et al, 2002;
`Rao et al, 2003, Tanaka et al, 1999).
`Our earlier studies in a well-established murine model of
`allogeneic BMT (Cetkovic-Cvrlje et al, 2001, 2002) have
`revealed an important role for Janus kinase 3 (JAK3 kinase),
`which is abundantly expressed in T cells, in the pathogenesis of
`acute GVHD (Cetkovic-Cvrlje et al, 2002; Cetkovic-Cvrlje &
`Tibbles, 2004; Cetkovic-Cvrlje & Uckun, 2004). Targeting
`JAK3 with the specific tyrosine kinase inhibitor JANEX-1 was
`shown to prevent severe GVHD in mice undergoing allogeneic
`
`ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 821–827
`
`doi:10.1111/j.1365-2141.2004.05126.x
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`M. Cetkovic-Cvrlje et al
`
`the main major histocompatibilty complex
`BMT across
`(MHC) barriers (Cetkovic-Cvrlje et al, 2001; Uckun et al,
`2002a). The purpose of the present study was (a) to evaluate
`the effects of inhibiting Bruton’s tyrosine kinase (BTK) within
`the non-T-cell compartment on development of severe GVHD
`after allogeneic BMT in mice and (b) to determine whether
`dual
`targeting of BTK and JAK3 kinases with rationally
`designed inhibitors would further improve the survival out-
`come of allogeneic BMT.
`Bruton’s tyrosine kinase (Steller, 1995), a member of the
`BTK/Tec family of protein tyrosine kinases that includes TecI,
`TecII, Itk, Bmx/Etk, and DSrc28C (found in Drosophila; Mano,
`1999),
`is a cytoplasmic protein tyrosine kinase of non-T
`lineage haematopoietic cells which plays a pivotal role in
`regulatory signal transduction pathways (Mukhopadhyay et al,
`1999, 2002; Kurosaki, 2000; Horwood et al, 2003, Rawlings &
`Witte, 1994; Smith et al, 1994; Uckun, 1998). BTK is involved
`in signal transduction pathways that regulate growth, differ-
`entiation, and survival of B-lineage lymphoid cells (Rawlings &
`Witte, 1994; Uckun, 1998; Kurosaki, 2000). Recent studies
`have revealed that BTK is involved in induction of macrophage
`effector functions (Mukhopadhyay et al, 1999, 2002). Further-
`more, BTK was found to be a key element of lipopolysaccha-
`ride-induced tumor necrosis factor production in monocytes
`(Horwood et al, 2003).
`The structure-based design of specific inhibitors of BTK has
`been reported recently (Mahajan et al, 1999). Advanced
`docking procedures were used for the rational design of LFM
`analogues with a high likelihood to bind favourably to the
`catalytic site within the kinase domain of BTK. The lead
`a-cyano-b-hydroxy-b-methyl-N-(2,5-dibromo-
`compound
`phenyl)-propenamide
`(LFM-A13),
`inhibited recombinant
`BTK with an IC50 value of 2Æ5 lmol/l (Mahajan et al, 1999).
`Besides its remarkable potency in BTK kinase assays, LFM-A13
`is also a highly specific inhibitor of BTK. Even at concentra-
`tions as high as 300 lmol/l, LFM-A13 did not affect the
`enzymatic activity of other protein tyrosine kinases, including
`the Janus family tyrosine kinases JAK1 and JAK3, SYK, Src
`family tyrosine kinases HCK and LYN, or the receptor family
`tyrosine kinases EGF-Receptor Kinase (EGFR) and Insulin-
`Receptor Kinase (IRK) (Mahajan et al, 1999).
`Here, we showed that a combination of the JAK3 inhibitor
`JANEX-1 and BTK inhibitor LFM-A13 confers a significant
`survival advantage for mice undergoing allogeneic BMT and is
`superior to GVHD prophylaxis using the standard anti-GVHD
`drug methotrexate (MTX). Notably, more than 70% of BMT
`recipients treated with JANEX-1 plus LFM-A13 remained alive
`throughout the 80-d observation period.
`
`Material and methods
`
`C57BL/6 and BALB/c mice
`
`Eight to 10-week-old C57BL/6 (H-2b) male mice and 6–8-
`week-old BALB/c (H-2d) male mice were purchased from
`
`Taconic, Germantown, NY, USA. Mice were housed in a
`controlled environment (12-h light/12-h dark photoperiod,
`22 ± 1°C, 60 ± 10% relative humidity), which is fully accred-
`ited by the United States Department of Agriculture (USDA).
`All husbandry and experimental contact made with the mice
`maintained specific pathogen-free (SPF) conditions. All mice
`were kept
`in Micro-Isolator cages
`(Lab Products,
`Inc.,
`Maywood, NY, USA) containing autoclaved food (Harlan
`Teklad LM-485), water and bedding. Animal studies were
`approved by the Parker Hughes Institute Animal Care and Use
`Committee, and all animal care procedures conformed to the
`Principles of Laboratory Animal Care (National Institutes of
`Health no. 85–23, revised 1985).
`
`Pretransplant total body irradiation
`
`recipient C57BL/6 mice,
`For pretransplant conditioning,
`positioned in a pie-shaped Lucite holder (Braintree Scientific
`Inc., Boston, MA, USA) underwent total body irradiation
`(TBI) (7Æ5 Gy) 1 d prior to BMT, which was delivered by a
`Cesium Instrument (JL Sheppard Labs, 47Æ08 rad/min). Recip-
`ients were given antibiotic-supplemented water (sulphameth-
`oxazole/trimethoprim, Hi-Tech Pharmacal, Amityville, NY,
`USA) starting the day before transplantation.
`
`Bone marrow transplantation
`
`Donor bone marrow (BM) was collected into Roswell Park
`Memorial Institute (RPMI) 1640 medium supplemented with
`l-glutamine (Cellgro) (Mediatech, Hendon, VA, USA) by
`flushing the shafts of femurs and tibias and cell suspensions
`were prepared as previously described (Cetkovic-Cvrlje et al,
`2001, 2002, Uckun et al, 2002a).
`In parallel,
`single cell
`suspensions of donor splenocytes (S) were prepared from
`minced spleens as a source of GVHD-causing T cells. The cells
`were washed and resuspended for i.v. injection via the caudal
`vein. The standard BM/S inoculum consisted of 25 · 106 BM
`cells and 25 · 106 splenocytes in 0Æ5 ml of RPMI 1640
`medium.
`
`Graft-versus-host disease monitoring
`
`Bone marrow transplantation recipients were monitored daily
`for any clinical evidence of GVHD (weight loss, manifestations
`of skin erythema, allopecia, hunching, diarrhoea) and survival
`(Cetkovic-Cvrlje et al, 2001, 2002, Uckun et al, 2002a) during
`the 80-d observation period. Survival times were measured
`from the day of BMT (day 0).
`
`Evaluation of engraftment status after BMT
`
`The allo-engraftment was documented by flow cytometric
`(FACScan, Becton Dickinson, Mountain View, CA, USA)
`H-2Dd typing of peripheral blood nucleated cells using
`fluorescein isothiocyanate (FITC)-labelled anti-H-2Dd antibody
`
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`(clone 34-2-12; Pharmingen, San Diego, CA, USA), which
`marks BALB/c cells. Immunofluorescent staining of cells and
`flow cytometry were performed using standard procedures
`(Cetkovic-Cvrlje et al, 2001; Uckun et al, 2002a).
`
`Drug treatments
`
`For GVHD prophylaxis – injections of LFM-A13, JANEX-1,
`MTX (Immunex Corporation, Seattle, WA, USA) or vehicle
`control were administered to recipient mice. The BTK- and
`JAK3-inhibitory compounds LFM-A13 (25 mg/kg/d) and
`JANEX-1 (60 mg/kg/d), respectively, were administered daily
`starting on day 0 of BMT. These compounds were synthesized
`and characterized as previously described in detail (Mahajan
`et al, 1999; Sudbeck et al, 1999). The standard anti-GVHD
`drug MTX (10 mg/m2/d) was used for comparison. MTX was
`administered i.p. on days 1, 3, 6 and 11 post-BMT. All drugs
`were administered intraperitoneally (i.p.) in a volume of
`200 ll.
`
`Histopathologic examination of tissues
`
`Recipient mice were electively sacrified at specified time
`points and necropsied. Tissues were removed and fixed in
`10% neutral-buffered formalin. Fixed tissues were embedded
`into 4 lm sections and stained with
`in paraffin,
`cut
`haematoxylin and eosin. All histopathology slides were
`blindly coded and graded by a veterinary pathologist. Livers
`were scored positive for GVHD when there was a periportal
`infiltrate, lungs were scored positive when there was evidence
`of vasculitis with a lymphocytic infiltrate, skin was scored
`positive when there was single cell necrosis, and colon was
`scored positive when there was single cell necrosis or crypt
`dropout. After initial scoring, all slides were reviewed for
`GVHD grading, as described previously by Bryson et al
`(1997). Our GVHD grading system was as follows: Liver:
`grade 0Æ5, focal portal
`lymphoid infiltrate; grade 1, wide-
`spread portal
`lymphoid infiltrate; grade 2, focal bile duct
`invasion or cellular injury; grade 3, multiple foci of bile duct
`injury and regeneration; grade 4, widespread injury and
`destruction of bile ducts; Small and large intestine: grade 0Æ5,
`occasional or rare necrotic cells in glands or crypts; grade 1,
`multiple foci of necrotic cells in glands or crypts; grade 2,
`necrosis involving several crypts or glands with focal abscess
`formation in crypts; grade 3, widespread crypt abscesses with
`focal glandular destruction; grade 4,
`loss of mucosa with
`granulation tissue response; Skin-ear: grade 0Æ5, occasional or
`rare single basal vacuolar necrosis; grade 1, several foci of
`single basal vacuolar necrosis; grade 2, contiguous single cell
`necrosis or multiple necrotic cells in proximity of lymphoid
`infiltrates; grade 3, confluent loss of cells with cleft formation
`or loss of skin appendages with extensive lymphoid infiltrates;
`grade 4,
`loss of epidermis or epithelium with or without
`granulation tissue response.
`
`BTK Inhibitor LFM-A13 in Prevention of Mouse GVHD
`
`Statistical analysis
`
`Group comparisons of continuous variables were done using
`Student’s t-tests. The survival data were analysed by life-table
`less than 0Æ05 (log-rank test) were
`methods. P-values of
`considered significant.
`
`Results
`
`Targeting BTK with the chemical inhibitor LFM-A13
`attenuates fatal acute GVHD across the major
`histocompatibility barrier in mice
`
`Severe GVHD, associated with overt diarrhoea, hunching,
`weight loss and ruffled fur, was induced in lethally irradiated
`(7Æ5 Gy TBI) C57BL/6 mice (H-2b) across the MHC barrier by
`injection of BM/S grafts from BALB/c mice (H-2d). In an
`attempt aimed at preventing the development of fatal GVHD,
`recipient mice were treated with LFM-A13 (25 mg/kg/d) every
`day from the day of BMT until the end of the 80-d observation
`period. Control mice were treated with vehicle alone. All of the
`TBI-conditioned,
`vehicle-treated control C57BL/6 mice
`(n ¼ 43) receiving BM/S grafts from BALB/c mice developed
`severe multi-organ GVHD, as clinically signaled by develop-
`ment of overt diarrhoea, hunching, weight loss and ruffled fur
`within 2–3 weeks and died with a median survival time (MST)
`of 40 d (Table I, Fig 1). Histopathological examination of
`multiple organs from seven control mice that either died or
`were terminated in moribund condition between the weeks 4
`and 6 post-BMT confirmed the diagnosis of multi-organ
`GVHD (Table II). The average GVHD scores in these mice
`were 3Æ0 ± 0Æ1 for the liver, 1Æ9 ± 0Æ1 for the skin, 1Æ1 ± 0Æ1 for
`intestine, and 1Æ4 ± 0Æ2 for the large intestine
`the small
`(Table II).
`LFM-A13 treatment significantly improved the survival of
`BMT recipients (n ¼ 50, P ¼ 0Æ0008 compared with vehicle-
`treated control recipients) and prolonged the MST to 47 d
`(Table I). The probability of survival at 2 months post-BMT
`was 2 ± 2% for vehicle-treated control mice and 22 ± 6% for
`mice treated with LFM-A13 (Table I, Fig 1). However, only
`2 ± 2% of LFM-A13-treated mice survived the experimental
`period of 80 d (Table I, Fig 1).
`The standard anti-GVHD drug MTX (10 mg/m2/d) and the
`recently described experimental
`anti-GVHD compound
`JANEX-1 targeting JAK3 (Cetkovic-Cvrlje et al, 2001; Uckun
`et al, 2002a) were used for comparison. Both drugs were found
`to have comparable efficacy in attenuating severe acute GVHD
`after allogeneic BMT in a murine models of GVHD (Cetkovic-
`Cvrlje et al, 2001; Table I). JANEX-1 and MTX significantly
`improved the survival of BMT recipients (P < 0Æ0001 and
`P < 0Æ0001 respectively), and prolonged the MST to 57 and
`63 d respectively (Table I). The probability of survival at 80 d
`after BMT was 31 ± 12% for MTX-treated and 31 ± 9% for
`recipients treated with JANEX-1, compared with 0 ± 0% for
`
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`
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`M. Cetkovic-Cvrlje et al
`
`Cumulative proportion
`surviving (% ±,SEM)
`
`P-value (log rank)
`
`lethal GVHD in
`Table I. Attenuation of
`murine allogeneic BMT recipients by tar-
`geting BTK with LFM-A13.
`
`Treatment protocol
`
`n MST (d)
`
`40 d
`
`60 d
`
`80 d
`
`vs. (A)
`
`vs. (B)
`
`0Æ0008
`
`–
`
`–
`
`(A) Vehicle
`40
`43
`47 ± 8
`2 ± 2
`0 ± 0
`0Æ0008
`(B) LFM-A13
`47
`50
`74 ± 6
`22 ± 6
`2 ± 2
`0Æ0006
`<0Æ0001
`(C) JANEX-1
`57
`26
`69 ± 9
`46 ± 10
`31 ± 9
`0Æ0004
`31 ± 12 <0Æ0001
`(D) MTX
`63
`16
`88 ± 8
`56 ± 12
`73 ± 13 <0Æ0001 <0Æ0001
`(E) LFM-A13 + JANEX-1*,**
`11 >80
`100±0
`82±12
`58 ± 11 <0Æ0001 <0Æ0001
`(F) LFM-A13 + MTX
`19 >80
`84 ± 8
`68 ± 11
`C57BL/6 (H-2b) recipients were lethally irradiated (TBI ¼ 7Æ5 Gy) and transplanted with BM/S
`grafts from MHC-disparate BALB/c (H-2d) mice and subjected to the treatment regimens presented
`above (the details of these treatment regimens are given in Materials and Methods); *P < 0Æ05
`compared with group D, and **P ¼ 0Æ0008 compared with group C; statistically significant differ-
`ences obtained by life table analysis (logrank test).
`
`vehicle-treated mice (Table I). Table I, as well as Fig 1A and B
`emphasize that both JANEX-1 (P ¼ 0Æ0006) and MTX
`(P ¼ 0Æ0004) were more effective than LFM-A13 in improving
`the survival outcome postallogeneic BMT.
`
`under these experimental conditions, donor cell engraftment
`was not prevented and the attenuation of GVHD in LFM-
`A13 + MTX-treated recipient mice was not due to lack of
`donor cell engraftment with concomitant autologous recovery.
`
`Efficacy of combined LFM-A13 plus MTX treatment in
`prevention of fatal acute GVHD across the major
`histocompatibility barrier in mice
`
`GVHD Prophylaxis with LFM-A13 plus JANEX-1
`markedly improves the survival outcome of recipient mice
`postallogeneic BMT
`
`We next sought to identify an effective GVHD prevention
`regimen that employs the BTK inhibitor LFM-A13 in combi-
`nation with a standard immunosuppressive agent. As shown
`previously (Cetkovic-Cvrlje et al, 2001; Uckun et al, 2002a),
`MTX is a potent anti-GVHD drug. MTX was more potent than
`LFM-A13 in the attenuation of acute GVHD (P ¼ 0Æ0004,
`Table I and Fig 1A). The combination regimen LFM-A13 plus
`MTX was more effective than LFM-A13 alone (P < 0Æ0001,
`Table I and Fig 1A). However, the combination regimen LFM-
`A13 plus MTX was not statistically more effective than MTX
`alone, despite the prolongation of MST to >80 d compared
`with 63 d in the MTX only group (Table I). More than half of
`the C57BL/6 recipients receiving this (LFM-A13 + MTX)
`GVHD prophylaxis remained alive and healthy throughout
`the 80-d observation period with a cumulative survival
`probability of 58 ± 11% compared with 31 ± 12% in MTX-
`treated group (Table I, Fig 1A).
`The histopathologic examination of organs of five repre-
`sentative long-term survivors of the LFM-A13 + MTX-treated
`group revealed that average liver, skin and large intestine
`scores in these mice were significantly lower than the GVHD
`scores of vehicle-treated control mice (Table II). According to
`the scoring system, the histologic GVHD grades were 2Æ2 ± 0Æ3
`for the liver, 0Æ9 ± 0Æ3 for the small intestine, 0Æ5 ± 0Æ0 for the
`large intestine, and 0Æ6 ± 0Æ3 for the skin (Table II).
`The long-term survival of LFM-A13 + MTX-treated mice
`was not due to poor engraftment of donor cells. Notably,
`98Æ5 ± 1Æ1% H-2Dd-positive donor cell engraftment was
`observed in LFM-A13 + MTX-treated mice, indicating that,
`
`We showed previously that the JAK3 kinase inhibitor JANEX-1
`is a potent anti-GVHD agent (Cetkovic-Cvrlje et al, 2001;
`Uckun et al, 2002a). Here we set out to determine if the
`survival outcome of allotransplanted bone marrow recipients
`could be further improved by using a combination of JANEX-1
`(60 mg/kg/d) plus LFM-A13 (25 mg/kg/d) for GVHD pro-
`phylaxis. As shown in Table I and Fig 1B, the combination
`treatment of LFM-A13 plus JANEX-1 was highly effective in
`the attenuation of acute GVHD, and significantly better than
`the treatment with either LFM-A13 alone (P < 0Æ0001) or
`JANEX-1 alone (P ¼ 0Æ0006). While the MST of recipients
`treated with LFM-A13 alone and JANEX-1 alone increased to
`47 and 57 d, respectively, compared with the MST of 40 d in
`vehicle controls,
`the combination treatment (LFM-A13 +
`JANEX-1) increased the MST to >80 d. Notably, 73 ± 13% of
`recipients treated with this combination treatment survived
`beyond the 80-d observation period, compared with only
`2 ± 2% and 31 ± 12% recipients of LFM-A13- and JANEX-1-
`treated
`groups
`respectively
`(Table I). Histopathologic
`examination of the organs of LFM-A13 + JANEX-1-treated
`long-term survivors (n ¼ 7) showed that average liver, skin,
`small and large intestine GVHD scores in these mice were
`significantly lower than those of vehicle-treated control mice
`(Table II). Average GVHD scores of long-term survived LFM-
`A13 + JANEX-1-treated BMT recipients were 0Æ9 ± 0Æ2 for the
`liver, 0Æ3 ± 0Æ1 for the skin, 0Æ6 ± 0Æ1 for the small intestine
`and 0Æ4 ± 0Æ1 for the large intestine. Flow cytometric H-2Dd-
`typing of splenocytes obtained from seven long-term H-2Db-
`positive survivors treated with this effective two-drug combi-
`
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`BTK Inhibitor LFM-A13 in Prevention of Mouse GVHD
`
`& Papadopoulos, 1997). To date, most therapeutic approaches
`designed to reduce acute GVHD,
`including ex vivo T cell
`depletion of marrow grafts (Martin et al, 1988; Poynton, 1988;
`Marmont et al, 1991), use of positively selected CD34+
`haematopoietic precursor cells (Berenson et al, 1996), and
`systemic immunosuppression (O’Reilly, 1983) are associated
`with an increased rate of graft rejection, more severe immu-
`nosuppression, and higher relapse rate of the original malig-
`nancies. Therefore, novel anti-GVHD agents with potent anti-
`leukaemic activity are urgently needed for effective prevention
`of GVHD after BMT without facilitating the recurrence of
`leukaemia.
`in area of BMT have
`the studies
`Several decades of
`demonstrated that donor T cells are the principal mediators
`of GVHD. However, recent data obtained from clinical and
`experimental transplantation indicated the role of non-T-cell
`compartment in development of GVHD (Sprent et al, 1990,
`1995; Mielcarek et al, 1997; Koh et al, 2000; Aranha et al, 2002;
`Rao et al, 2003, Tanaka et al, 1999). Here, we show that
`targeting BTK within the non-T-cell compartment using the
`rationally designed BTK inhibitor LFM-A13 attenuated the
`severity of GVHD postallogeneic BMT in an acute murine
`GVHD model. However, LFM-A13 was less effective than the
`standard anti-GVHD drug MTX or the recently described anti-
`GVHD agent JANEX-1. Therefore we investigated whether the
`inhibition of BTK in combination with the inhibition of JAK3
`could have beneficial effects on GVHD development. Indeed,
`combined prophylactic treatment with LFM-A13 and JANEX-
`1 exhibited potent
`in vivo biologic activity. Notably,
`the
`combination regimen LFM-A13 + JANEX-1 was more effect-
`ive than LFM-A13 alone or JANEX-1 alone (73% recipients
`treated with LFM-A13 + JANEX-1 survived throughout 80-d
`observation period, with a MST of >80 d, compared with 2%
`and MST of 47 d in LFM-A13-treated, and 25% and MST of
`56 d in JANEX-1-treated recipients). The combination regi-
`men LFM-A13 + MTX was as effective as LFM-A13 + JANEX-
`1 (58% recipients survived on day 80 post-BMT with a MST of
`>80 d). However, LFM-A13 + MTX treatment was not signi-
`ficantly more effective than treatment with MTX alone. In
`contrast, LFM-A13 + JANEX-1 treatment was more effective
`than each of the drug treatments alone (either LFM-A13,
`JANEX-1, or MTX alone). The H-2D typing of nucleated
`peripheral blood cells from LFM-A13 + JANEX-1- and LFM-
`A13 + MTX-treated recipients confirmed that >90% of the
`circulating cells were of donor origin. Thus, the attenuation of
`GVHD in recipient mice treated with both dual treatments
`LFM-A13 + JANEX-1- and LFM-A13 + MTX was not due to
`lack of donor cell engraftment with concomitant autologous
`recovery.
`LFM-A13 was described as the first BTK-specific tyrosine
`kinase inhibitor and the first anti-leukaemic agent targeting BTK
`(Uckun & Zheng, 2000, 2001). Notably, treatment of leukaemic
`cells with LFM-A13 resulted in abrogation of BTK activity at a
`concentration of 10 lmol/l (Mahajan et al, 1999). LFM-A13
`disrupted BTK-Fas association and rendered resistant leukaemic
`
`Fig 1. Effects of the BTK inhibitor LFM-A13 in combination with
`methotrexate (MTX) (A) and JANEX-1 (B) on the post-BMT survival
`outcome in a murine model of acute GVHD. Irradiated (7Æ5 Gy)
`C57BL6 (H-2b) recipients were given BM and splenocytes (25 · 106 of
`each) from BALB/c (H-2d) mice. LFM-A13 was administered i.p. at
`25 mg/kg/d from day 0 to day 80. Methotrexate (MTX) was used at a
`dose level of 10 mg/m2/d and administered i.p. on days 1, 3, 6 and 11
`post-BMT. JANEX-1 was administered i.p. at 60 mg/kg/d from day 0
`to day 80. P-values obtained in comparison with control group by the
`life table analysis (log-rank test). See Table I for details of the life table
`analysis.
`
`nation showed 95Æ6 ± 1Æ8% H-2Dd donor cell engraftment
`(Table III).
`
`Discussion
`
`Acute graft-versus-host disease is a major cause of morbidity
`and mortality in patients undergoing allogeneic BMT (O’Reilly
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`M. Cetkovic-Cvrlje et al
`
`Group
`
`Time of analysis
`(weeks)
`
`Organ score (mean ± SEM)
`
`Table II. Severity of multi-organ GVHD in
`BMT recipients.
`
`n
`
`Liver
`
`Skin
`
`Small intestine Large intestine
`
`4–6
`Vehicle
`12
`MTX
`12
`LFM-A13 + MTX
`LFM-A13 + JANEX-1 12
`
`1Æ1 ± 0Æ1
`1Æ9 ± 0Æ1
`7 3Æ0 ± 0Æ1
`0Æ8 ± 0Æ2
`0Æ9 ± 0Æ3*
`4 1Æ9 ± 0Æ4*
`0Æ9 ± 0Æ3
`0Æ6 ± 0Æ3*
`5 2Æ2 ± 0Æ3*
`7 0Æ9 ± 0Æ2** 0Æ3 ± 0Æ1** 0Æ6 ± 0Æ1*
`
`1Æ4 ± 0Æ2
`0Æ5 ± 0Æ2*
`0Æ5 ± 0Æ0*
`0Æ4 ± 0Æ1*
`
`Scoring was performed as described in Materials and Methods. Data are presented as mean ± SEM.
`Statistical analysis of the differences between the drug-treated and vehicle-treated group was done by
`Student’s t-test; *P < 0Æ05-0Æ0005; **P < 0Æ0001.
`
`Table III. Donor cell engraftment.
`
`References
`
`Group
`
`LFM-A13 + MTX
`LFM-A13 + JANEX-1
`MTX
`
`n
`
`10
`7
`10
`
`H-2Dd (%)
`
`98Æ5 ± 1Æ1
`95Æ6 ± 1Æ8
`99Æ0 ± 2Æ4
`
`Recipient mice were electively sacrificed and analysed at the end of the
`80-d observation period. Data are presented as mean ± SEM values for
`the percentage of H-2Dd positive donor cells in the nucleated per-
`ipheral blood cell population.
`
`cells sensitive to Fas-mediated apoptosis (Vassilev et al, 1999). In
`accordance with the antiapoptotic function of BTK, treatment of
`BTK-positive B-lineage leukaemia cells with LFM-A13 enhanced
`their sensitivity to vincristine- and ceramide-induced apoptosis
`(Mahajan et al, 1999). Therefore, LFM-A13 shows potential as
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`beneficial function of allogeneic BMT, the effects of LFM-A13
`treatment (alone and in combination with MTX or JANEX-1)
`on the GVL function of the bone marrow allografts remains to be
`determined in our future studies.
`Taken together, our results indicate that targeting BTK with
`a chemical inhibitor, such as LFM-A13, may be useful in the
`prevention of severe GVHD after allogeneic BMT, especially
`when it is combined with other potent anti-GVHD prophy-
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`A13 are dual function anti-GVHD agents with anti-leukaemic
`properties, GVHD prophylaxis using a combination of these
`tyrosine kinase inhibitors may be particularly helpful
`in
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