Patent Application: US-54934706-A

Abstract:
the invention provides a method for treating cancers that are dependent on cyclin d1 for proliferation , survival , metastasis and differentiation , involving administering effective amount of arsenic trioxide to an affected patient .

Description:
reference will now be made in detail to the presently preferred embodiments of the invention , which , together with the examples following the detailed description , serve to explain the principles of the invention . the inventors discovered that as 2 o 3 induced apoptosis in mcl lines at 2 - 4 μm , which was within the plasma levels achieved after as 2 o 3 therapy . as 2 o 3 induced a dose and time dependent suppression of cyclin d1 . the suppression of cyclin d1 restored rb to a hypophosphorylated state , in parallel with a change in cell cycle . these biologic changes were consistent with the apoptosis observed upon as 2 o 3 treatment . the inventors further showed that the down - regulation of cyclin d1 mediated by as 2 o 3 occurred at a post - transcriptional level . this might be expected , as cyclin d1 is under the transcriptional control of the immunoglobulin heavy chain gene enhancer in mcl , which is unlikely to be affected by as 2 o 3 . furthermore , in physiologic conditions , the control of cyclin d1 during the cell cycle is also mediated in part via alteration in the stability of cyclin d1 . 7 this process is controlled by phosphorylation of cyclin d1 at thr - 286 , a process mediated by gsk - 3β . 8 - 11 gsk - 3β is itself tightly regulated . mitogens inactivate gsk - 3β by a pathway involving ras , phosphatidylinositol 3 kinase ( pi3k ), and protein kinaseb / akt . 25 , 26 ras activates pi3k , which in turn activates akt . akt inactivates gsk - 3β by phosphorylating it at serine residue 9 . 27 this removes the inhibition of gsk - 3β on cyclin d1 , allowing cyclin d1 to accumulate and thus activate cell cycling . on the other hand , gsk - 3β can also be activated by phosphorylation at a tyrosine residue 216 ( try - 216 ) in the kinase domain . 28 little is known , though , of the physiologic mechanisms controlling gsk - 3β phosphorylation at tyr - 216 . there is some evidence that gsk - 3β might autophorphorylate . 29 in d . discoideum , the tyrosine kinase zak1 phosphorylates gsk - 3β in response to camp . 30 in mammalian cells , the tyrosine kinases fyn , 31 csk 32 and pyk2 33 , 34 have been implicated in gsk - 3β phosphorylation at tyr - 216 . therefore , an important novel observation in this study is the as 2 o 3 - mediated increase of gsk - 3β try - 216 phosphorylation . how an inorganic molecule as 2 o 3 might enhance gsk - 3β phosphorylation remains to be defined . however , it has been shown that increases in calcium may lead to enhanced gsk - 3β phosphorylation , via activation of the calcium sensitive kinase pyk2 . 35 whether as 2 o 3 acts through a similar mechanism will have to be investigated . nevertheless , the end result of as 2 o 3 - mediated increase in gsk - 3β try - 216 phosphorylation is the increase in cyclin d1 thr - 286 phosphorylation , a key step in its degradation . another recently defined mechanism of regulating cyclin d1 is the ikk system . the ikk complex is the major regulatory component in the nk - κb pathway . it comprises the catalytic subunits ikkα and ikkβ , and a regulatory subunit ikkγ / nemo . 36 interestingly , ikkα has been shown recently to phosphorylate cyclin d1 at thr - 286 , the same site targeted by gsk - 3b . ikkα needs to be activated by phosphorylation at a serine residue 176 ( ser - 176 ) before participating in the regulation of nf - κb by phosphorylating iκb . 38 ikkα ser - 176 phosphorylation is mediated by nk - κb inducing kinase ( nik ). 36 hence , the finding of as 2 o 3 - induced increase in ikk phosphorylation is another important original observation . furthermore , as 2 o 3 - mediated an increase in physical interaction between ikk and cyclin d1 , as shown in immunoprecipitation experiments . finally , an ikk specific inhibitor bms - 345541 37 alleviated as 2 o 3 - induced cyclin d1 down - regulation . taken together , these results indicated that ikk was also an effector of as 2 o 3 treatment . the mechanism by which as 2 o 3 increases ikk phosphorylation is unclear . however , nik is activated by a host of stimuli , including tumor necrosis factor and interleukin - 1 . 38 the potential interaction of as 2 o 3 with these signaling molecules requires future studies . the inventors further showed that as 2 o 3 - mediated cyclin d1 thr - 286 phosphorylation increased its ubiquitination . moreover , the time course of ubiquitination was commensurate with the timing of the biologic functions of as 2 o 3 on the mcl lines . after as 2 o 3 treatment , increased ubiquitination was first detected at 30 minutes and continued to increase . at two hours , significant down - regulation of cyclin d1 was first observed , which was associated with a parallel hypophosphorylation of rb . finally , significant activation of caspase 3 was observed at four hours . these sequence of events were consistent with cyclin d1 down - regulation initiated by thr - 286 phosphorylation . cyclin d1 is a cytosolic and nuclear protein . therefore , polyubiquitination is involved , which targets the protein to degradation in proteasomes . indeed , we showed that inhibition of proteasomes successfully prevented as 2 o 3 - induced down - regulation of cyclin d1 . on the other hand , inhibition of lysosomes , the site of degradation of monoubiquitinated proteins , 39 did not interfere with as 2 o 3 - induced down - regulation of cyclin d1 . these results confirm that as 2 o 3 down - regulated cyclin d1 by promoting its proteasomal degradation . the capability of as 2 o 3 in augmenting proteasomal degradation of cyclin d1 is reminiscent of its action on another fusion oncoprotein pml - rara in apl . as 2 o 3 enhances the conjugation of a ubiquitin - related peptide sumo - 1 to the pml part of the pml - rara protein . 40 this directs pml - rara to nuclear bodies , which are nuclear matrix domains containing 11s proteasome constituents recruited by as 2 o 3 treatment . in this way , as 2 o 3 triggers proteasome - dependent degradation of sumo - conjugated pml - rara . 41 therefore , as 2 o 3 may act together with component of the proteasomal system to effect degradation of target proteins . findings of the current study corroborate with this proposition . the inventors have clearly shown that as 2 o 3 suppresses mcl cell growth by targeting cyclin d1 . furthermore , there are a number of important ramifications arising from this study that will form the lead for further investigations . as 2 o 3 appears to be capable of inducing the phosphorylation of not only gsk - 3β , but also ikk . the issues of whether this is mediated by different mechanisms or a common pathway , and the possibility that as 2 o 3 might mediate phosphorylation of other biologically important molecules , will warrant exploration . finally , cyclin d1 over - expression is pathogenetically important in a vast diversity of cancers . it is important to determine if as 2 o 3 also targets cyclin d1 in these cancers , and is therefore of therapeutic potential . based on these observations , the inventors used oral - as 2 o 3 in the treatment of 14 patients with refractory or relapsed mcl , which over - expressed cyclin d1 . the inventors observed an overall response in 9 patients ( 64 %). four patients achieved complete remission , two patients complete remission unconfirmed , and three patients with partial remissions . these results were very good , given that these patients had refractory or relapsed disease . these clinical observations obtained by the inventors are a direct in vivo proof of the inventors &# 39 ; observations in their experimental system . taken together , the inventors have discovered several novel findings . the inventors have discovered that as 2 o 3 decreased cyclin d1 . the inventors further discovered that the decrease in cyclin d1 was post - transcriptional . the inventors moreover discovered that as 2 o 3 induced gsk - 3β and ikk activation and hence phosphorylation of cyclin d1 . the inventors then showed that phosphorylated cyclin d1 was degraded in the proteasome . finally , the inventors have made the novel observation that oral as 2 o 3 induced a high response rate clinically in patients with refractory or relapsed mcl , a cancer that over - expressed cyclin d1 . the present invention of using oral arsenic trioxide in suppressing cyclin d1 is an important paradigm applicable to the treatment of cancers that are dependent on cyclin d1 for proliferation , survival , metastasis and differentiation . the following delivery systems , which employ a number of routinely used pharmaceutical carriers , are only representative of the many embodiments envisioned for administering the instant compositions . injectable drug delivery systems include solutions , suspensions , gels , microspheres and polymeric injectables , and can comprise excipients such as solubility - altering agents ( e . g ., ethanol , propylene glycol and sucrose ) and polymers ( e . g ., polycaprylactones and plga &# 39 ; s ). implantable systems include rods and discs , and can contain excipients such as plga and polycaprylactone . oral delivery systems include tablets and capsules . these can contain excipients such as binders ( e . g ., hydroxypropylmethylcellulose , polyvinyl pyrilodone , other cellulosic materials and starch ), diluents ( e . g ., lactose and other sugars , starch , dicalcium phosphate and cellulosic materials ), disintegrating agents ( e . g ., starch polymers and cellulosic materials ) and lubricating agents ( e . g ., stearates and talc ). transmucosal delivery systems include patches , tablets , suppositories , pessaries , gels and creams , and can contain excipients such as solubilizers and enhancers ( e . g ., propylene glycol , bile salts and amino acids ), and other vehicles ( e . g ., polyethylene glycol , fatty acid esters and derivatives , and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid ). dermal delivery systems include , for example , aqueous and nonaqueous gels , creams , multiple emulsions , microemulsions , liposomes , ointments , aqueous and nonaqueous solutions , lotions , aerosols , hydrocarbon bases and powders , and can contain excipients such as solubilizers , permeation enhancers ( e . g ., fatty acids , fatty acid esters , fatty alcohols and amino acids ), and hydrophilic polymers ( e . g ., polycarbophil and polyvinylpyrolidone ). in one embodiment , the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer . solutions , suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents ( e . g ., gums , zanthans , cellulosics and sugars ), humectants ( e . g ., sorbitol ), solubilizers ( e . g ., ethanol , water , peg and propylene glycol ), surfactants ( e . g ., sodium lauryl sulfate , spans , tweens , and cetyl pyridine ), preservatives and antioxidants ( e . g ., parabens , vitamins e and c , and ascorbic acid ), anti - caking agents , coating agents , and chelating agents ( e . g ., edta ). example 1 of as 2 o 3 in mcl by targeting cyclin d1 call lines . the mcl lines jeko - 1 and granta - 519 were obtained from german collection of microorganisms and cell cultures ( acc 553 and acc 342 , braunschweig , germany ). jeko - 1 cells were cultured in rpmi 1640 with 20 % fetal bovine serum ( fbs ), and granta - 519 cells in dmem with 10 % fbs ; both with 50 units / ml penicillin and 50 μg / ml streptomycin , at 5 % co 2 . example 2 of as 2 o 3 in mcl by targeting cyclin d1 reagents and antibodies . reagents and antibodies used included cell culture reagents ( invitrogen , carlsbad , calif ., usa ); kinase inhibitors and their inactive analogues ( calbiochem , darmstadt , germany ); antiserum to phospho - gsk3 ( tyrosine 216 , try - 216 ) ( upstate , lake placid , n . y ., usa ); antisera to cyclin d1 , phospho - cyclin d1 ( thr - 286 ), gsk3β , phospho - gsk3β ( tyr - 216 ), iκb kinase ( ikk ) α / β , phospho - ikkα / β ( serine 176 / 180 , ser - 176 / 180 ), rb and phospho - rb ( serine 795 , ser - 795 ), caspase - 3 and β - actin ( cell signaling technology , beverly , mass ., usa ); protein g - agarose ( upstate ); ecl kit ( amersham , piscataway , n . j ., usa ); cell proliferation kit i ( mtt ) ( roche applied science , indianapolis , ind ., usa ); annexin v - fitc kit ( beckman coulter , fullerton , calif ., usa ); and rneasy kit and one - step rt - pcr kit ( qiagen , valencia , calif ., usa ). example 3 of as 2 o 3 in mcl by targeting cyclin d1 cell viability assays . cells were seeded on 96 - well microplates at 2 × 10 4 / well in 100 ml growth medium containing different concentration of as 2 o 3 as indicated at 37 ° c . for 72 hours . mtt labeling reagent ( 10 μl , 5 mg / ml ) ( roche applied science , indianapolis , ind ., usa ) was added to each well at 37 ° c . for 4 hours , followed by 100 μl solubilization at 37 ° c . overnight . solubilized fomarzan crystals were quantified spectophotometrically at 590 nm with a microplate elisa reader . example 4 of as 2 o 3 in mcl by targeting cyclin d1 apoptosis assay . cells were seeded at 1 × 10 6 / ml in different concentrations of as 2 o 3 as indicated at 37 ° c . for 24 hours , harvested , rinsed in ice - cold phosphate buffered saline ( pbs ), and resuspended in 500 □ 1 binding buffer containing annexin v - fitc and propidium iodide ( pi ) ( beckman coulter , fullerton , calif ., usa ) for 20 minutes on ice . the percentages of apoptotic cells ( annexin - v positive , pi negative ) were determined on a flow cytometer ( epics , beckman coulter ) with appropriate color compensation . cell cycle analysis . cells were seeded at 1 × 10 5 / ml in different concentrations of as 2 o 3 as indicated at 37 ° c . for 8 hours , harvested , washed in ice - cold pbs , resuspended in 500 μl pbs , stained with pi for 10 minutes on ice . cell cycle was determined by flow cytometry ( epics , beckman coulter ). example 5 of as 2 o 3 in mcl by targeting cyclin d1 semi - quantitative reverse transcription polymerase chain reaction ( rt - pcr ) for cyclin d1 . cells were seeded at a density of 1 × 10 6 / ml in different concentrations of as 2 o 3 at 37 ° c . for 8 hours , washed with pbs buffer and lysed with rtl buffer . rna was extracted with an rneasy kit , followed by cdna synthesis and a 30 - cycle pcr with a one - step rt - pcr kit with the forward primer 5 ′- ctg gcc atg aac tac ctg ga - 3 ′ and the reverse primer 5 ′- gtc aca ctt gat cac tct gg - 3 ′. cycling conditions were denaturation ( 1 minute at 94 ° c ., first cycle 5 minutes ), annealing ( 2 minutes at 50 ° c .) and extension ( 3 minutes at 72 ° c ., last cycle 10 minutes ), example 6 of as 2 o 3 in mcl by targeting cyclin d1 western blotting analysis . cells were seeded at a density of 1 × 10 6 / ml overnight . where applicable , cells were pre - treated with various inhibitors for 30 minutes , and then incubated with 4 μm as 2 o 3 for different time periods as indicated . cells were lysed in lysis buffer ( 50 mm tris - hcl , 100 mm nacl , 5 mm edta , 40 mm nap 2 o 7 , ph 7 . 5 , 1 % triton x - 100 , 4 μg / ml aprotinin , 1 mm dithiothreitol , 200 μm na 3 vo 4 , 0 . 7 μg / ml pepstatin , 100 μm phenylmethylsulfonyl fluoride , and 2 μg / ml leupeptin ). clarified lysates were resolved on 12 % sds - phenylmethylsulfonyl fluoride and transferred to nitrocellulose membranes . the membranes were blocked with 5 % non - fat milk , washed , incubated with the appropriate antibodies followed by horseradish peroxidase - conjugated secondary antisera . immuno - reactive bands were visualized by chemiluminescence with the ecl kit , detected on x - ray films and quantified by densitometric scanning ( eagle eye ii still video system , stratagene , la jolla , calif ., usa ). example 7 of as 2 o 3 in mcl by targeting cyclin d1 coimmunoprecipitation assays . cells were seeded at 1 × 10 6 / ml overnight treated with 4 μm as 2 o 3 at 37 ° c . for different time periods as indicated , and lysed in lysis buffer . cell lysates were incubated with an anti - cyclin d1 , anti - ubiquitin , anti - calpain 2 or anti - ikkα / β antibodies ( 4 μg / sample ) at 4 ° c . for 1 hour , followed by incubation with 30 μl of protein g - agarose ( 50 % slurry ) at 4 ° c . for another 2 hour . immunoprecipitates were washed four times with 400 μl lysis buffer , resuspended in 50 μl lysis buffer and 10 ml 6 × sample buffer and boiled for 5 minutes . immunoprecipitates were then analysed by western blot analysis . results of example 1 - 7 of as 2 o 3 in mcl by targeting cyclin d1 as 2 o 3 induced dose and time dependent apoptosis in mcl cells . mtt test showed that as 2 o 3 induced a dose - dependent cytotoxicity in jeko - 1 and granta - 519 cells ( fig1 a ). flow cytometric analysis showed that as 2 o 3 treatment led to induction of apoptosis ( fig1 b ). western blot analysis showed that caspase 3 activation was involved in as 2 o 3 - induced apoptosis ( fig1 c ). cyclin d1 was down - regulated in mcl by as 2 o 3 . to determine the molecular mechanisms of as 2 o 3 - induced apoptosis in mcl , the expression of cyclin d1 was examined . western blot analysis showed that as 2 o 3 - induced a time and dose dependent suppression of cyclin d1 in both cell lines . treatment with as 2 o 3 at 4 □ m led to suppression of cyclin d1 , first detectable at 2 hours and almost complete at 8 - 12 hours ( fig2 a ). as 2 o 3 suppression of cyclin d1 was also dose - dependent ( fig2 b ). as 2 o 3 induced down - regulation of cyclin d1 disrupted its signaling . to investigate if cyclin d1 down - regulation is biologically relevant , rb phosphorylation was investigated . as 2 o 3 treatment led to a time dependent decrease in rb phosphorylation , which occurred at a similar time - frame as compared with cyclin d1 down - regulation ( fig3 a and b ). cell cycle analysis by flow cytometry showed that there was an increase in the proportion of apoptotic cells . down - regulation of cyclin d1 by as 2 o 3 was post - transcriptional rt - pcr showed that cyclin - d1 gene transcription was unaffected by as 2 o 3 treatment of up to 8 μm , suggesting that the down - regulation of cyclin d1 was post - transcriptional ( fig4 a ). as 2 o 3 - induced cyclin d1 down - regulation was related to gsk3 □ activation . western blot analysis showed that as 2 o 3 treatment resulted in significant increases in cyclin d1 phosphorylation at thr - 286 , a prerequisite for cyclin d1 degradation ( fig4 b ). cyclin d1 phosphorylation by gsk - 3β requires prior activation of gsk - 3β by phosphorylation at tyr - 216 . as 2 o 3 treatment in fact significantly increased gsk - 3β tyr - 216 phosphorylation , suggesting that gsk - 3β might mediate as 2 o 3 - induced cyclin d1 phosphorylation and hence degradation . to confirm the role of gsk - 3β as a mediator of as 2 o 3 , jeko - 1 cells were pre - incubated with the gsk - 3β inhibitor 6 - bromoindirubin - 3 ′- oxime ( bio ; 10 μm ) before as 2 o 3 treatment . the results showed that bio successfully prevented as 2 o 3 - induced down - regulation of cyclin d1 . collectively , these observations indicated that as 2 o 3 down - regulated cyclin d1 post - transcriptionally , probably by increasing its degradation . as 2 o 3 - induced cyclin d1 down - regulation was also dependent on ikkα / β . to determine if ikk was involved in as 2 o 3 - induced down - regulation of cyclin d1 , ikkα / β phosphorylation at ser - 178 / 180 was examined . as 2 o 3 significantly increased ikkα / β ser - 178 / 180 phosphorylation , which was required for activation of ikkα / β ( fig5 a ). pre - treatment with the ikkα / β inhibitor bms - 345541 ( bms ; 10 μm ) significantly prevented as 2 o 3 - induced cyclin d1 down - regulation , suggesting that ikkα / β was a molecular mediator of as 2 o 3 ( fig5 b ) immunoprecipitation with an anti - ikkα / β antibody showed that cyclin d1 bound ikkα / β . similarly , when cyclin d1 was immunoprecipitated , ikkα / β was also confirmed to co - immunoprecipitate ( fig5 c ). these results confirmed that as 2 o 3 activated ikkα / β , which participated in the down - regulation of cyclin d1 . as 2 o 3 promoted cyclin d1 ubiquitination . to study if as 2 o 3 - induced cyclin d1 down - regulation was mediated via ubiquitination , immunoprecipitation experiments were performed on lystaes from jeko - 1 cells treated with as 2 o 3 . immunoprecipitation with an anti - ubiquitin antibody showed a time - dependent increase in bound cyclin d1 ( fig6 a and b ). similarly , lysates immunoprecipitated with an anti - cyclin d1 antibody also showed a time dependent increase in bound ubiquitin . these results showed that as 2 o 3 promoted cyclin d1 ubiquitination , confirming that as 2 o 3 - induced gsk - 3β and ikkα / β activation was biologically relevant . as 2 o 3 induced cyclin d1 degradation in 26s and 20s proteasomes but not lysosomes . pre - incubation of jeko - 1 cells with the 26s and 20s proteosome inhibitors mg132 ( 30 μm ), bortezimab ( 10 μg / ml ) and lactacystin ( 10 μm ) attenuated as 2 o 3 induced cyclin d1 down - regulation ( fig7 a ). however , pre - incubation with the lysosomal inhibitor ammonium chloride ( nh 4 cl ) had no effect on as 2 o 3 - induced down - regulation of cyclin d1 ( fig7 b ). the results confirmed that as 2 o 3 down - regulated cyclin d1 by promoting its ubiquitination , hence targeting it to the proteosome for degradation . overall model an overall model of the action of as 2 o 3 on mcl is shown in fig8 . example 8 of oral - as 2 o 3 in the clinical treatment of patients with refractory and relapsed mcl that over - expressed cyclin d1 patients , consenting patients with relapsed or refractory b - cell lymphomas , and an ecog performance status of & lt ; 2 were recruited all patients gave informed consent , and the treatment was approved by the institute review board of queen mary hospital . example 9 of oral - as 2 o 3 in the clinical treatment of patients refractory and relapsed mcl that over - expressed cyclin d1 treatment . treatment was initiated with oral - as 2 o 3 ( 10 mg / day for patients below 70 years old with normal renal function ; 5 mg / day for patients over 70 years old , or with impaired renal function ), ascorbic acid ( aa , 1 g / day ) and chlorambucil ( 4 mg / day ) as outpatients until disease response or progression was documented . in patients with bulky disease , debulking with vpp ( vincristine 2 mg / day × 1 , prednisolone 30 mg / day × 14 and procarabzine 50 - 100 mg / day × 14 ) was used . after maximum response was achieved , chlorambucil was taken off and a maintenance regimen of as 2 o 3 ( 5 - 10 mg / day ) and aa ( 1 g / day ) was given for two weeks every 2 months for a planned two years . responses were classified according to standard ncl criteria , 16 and monitored by regular physical examination , marrow and blood assessment , and computerized tomographic scans . results of examples 8 - 9 of oral - as 2 o 3 in the clinical treatment of patients with refractory and relapsed mcl that over - expressed cyclin d1 characteristics of patients with mcl . table 1 shows results of the clinical use of oral - as 2 o 3 in patients with refractory or relapsed mantle cell lymphoma that over - expressed cyclin d1 . the results showed an overall response rate of 64 %. four patients achieved complete remission ( cr ), whereas two patients achieved complete remission unconfirmed . of the fourteen patients treated ( table 1 ), eleven had advanced relapses ( r ) ( r2 , n = 5 ; r3 , n = 4 ; r4 , n = 2 ). three patients treated in r1 had advanced age ( 76 , 77 and 90 years ). all but two patients had received an anthracycline based multi - agent chemotherapy . other previous treatment included rituximab ( n = 8 ), autologous hematopoietic stem cell transplantation ( hsct ) ( n = 3 ), and bortezomib ( n = 1 ). other poor prognostic indicators included marrow infiltration ( n = 11 ) and extensive extranodal involvement ( n = 9 ), so that 12 / 14 ( 86 %) cases had stage iv disease . the median time from initial diagnosis to as 2 o 3 treatment was 33 ( 8 - 85 ) months treatment response . nine patients responded , giving an or rate of 64 %. four patients ( cases 1 - 4 ) achieved cr . two patients ( cases 5 , 6 ) achieved unconfirmed cr ( cru ). they had become asymptomatic without any detectable superficial diseases ( fig9 a ). marrow and peripheral blood involvement was also cleared . however , small residual internal lymph nodes remained . these lymph nodes were negative on gallium scan and had remained static in size . three patients had partial responses ( pr ) with & gt ; 50 % reduction in the size of assessable lymph nodes ( fig9 b and c ). outcome . of the four patients with cr , one had relapsed at 16 months . she achieved a cr3 again with daily as 2 o 3 and resumption of chlorambucil . two patients were still on maintenance as 2 o 3 + aa treatment , while one had completed the planned two years of treatment . of the two patients with cru , one patient had relapsed at 20 months . he achieved cr5 again with as 2 o 3 and chlorambucil therapy . for the three patients with pr , one patient developed progressive disease while on maintenance therapy 12 months later and died of refractory lymphoma . two defaulted treatment and both relapsed . toxicity . significant ( w . h . o grade 3 - 4 ) neutropenia and thrombocytopenia was observed in 7 patients . these patients had previously multiple chemotherapy , or autologous hsct . the neutropenia responded to hematopoietic growth factors . no significant sepsis or bleeding were observed . other side effects included fever ( n = 7 ), herpes zoster reactivation ( n = 3 ), fluid accumulation ( n = 2 ), nausea ( n = 3 ) and headache ( n = 2 ). no significant qt prolongation or arrhythmia was observed . five patients did not report any side effects at all . 2 . sherr c j , mccormick f . the rb and p53 pathways in cancer . cancer cell 2002 ; 2 : 103 - 112 . 3 . guo y , stacey d w , hitomi m . post - transcriptional regulation of cyclin d1 expression during g2 phase . oncogene 2002 ; 21 : 7545 - 56 . 4 . guo y , yang k , harwalkar j , nye j m , mason d r , garrett m d , hitomi m , stacey d w . phosphorylation of cyclin d1 at thr 286 during s phase leads to its proteasomal degradation and allows efficient dna synthesis . oncogene 2005 ; 24 : 2599 - 612 . 5 . diehl j a , zindy f , sherr c j . inhibition of cyclin d1 phosphorylation on threonine - 286 prevents its rapid degradation via the ubiquitin - proteasome pathway . genes dev 1997 ; 11 : 957 - 72 . 6 . diehl j a , cheng m , roussel m f , sherr c j . glycogen synthase kinase - 3beta regulates cyclin d1 proteolysis and subcellular localization . genes dev 1998 ; 12 : 3499 - 511 . 7 . alt j r , cleveland j l , hannink m , diehl j a . phosphorylation - dependent regulation of cyclin d1 nuclear export and cyclin d1 - dependent cellular transformation . genes dev 2000 ; 14 : 3102 - 14 . 8 . kwak y t , li r . becerra c r , tripathy d , frenkel e p , verma u n , ikappab kinase alpha regulates subcellular distribution and turnover of cyclin d1 by phosphorylation . j biol chem 2005 ; 280 : 33945 - 52 . 9 . swerdlow s h , berger f , isaacson p i , muller - hermelink h k , nathwani b n , piris m a , harris n l . mantle cell lymphoma . in jaffe e s , harris n l , styein h , vardiman j w ( eds ). world health organization classification of tumours . pathology and genetics of tumours of hematopoietic and lymphoid tissues . lyon , france , iarc press , 2001 , 168 - 170 . 10 . the non - hodgkin &# 39 ; s lymphoma classification project . a clinical evaluation of the international lymphoma study group classification of non - hodgkin &# 39 ; s lymphoma . blood 1997 ; 89 : 3909 - 3918 . 11 . tsujimoto y , yunis j , onorato - showe l , erikson j , nowell p c , croce c m . molecular cloning of the chromosomal breakpoint of b - cell lymphomas and leukemias with the t ( 11 ; 14 ) chromosome translocation . science 1984 ; 224 : 1403 - 6 . 12 . tsujimoto y , jaffe e , cossman j , gorham j , nowell p c , croce c m . clustering of breakpoints on chromosome 11 in human b - cell neoplasms with the t ( 11 ; 14 ) chromosome translocation . nature 1985 ; 315 : 340 - 3 . 13 . witzig t e . current treatment approaches for mantle - cell lymphoma . j clin oncol 2005 ; 23 : 6409 - 14 . 14 . lenz g , dreyling m , hoster e , wormann b , duhrsen u , metzner b , eimermacher h , neubauer a , wandt h , steinhauer h , martin s , heidemann e , aldaoud a , parwaresch r , hasford j , unterhalt m , hiddemann w . immunochemotherapy with rituximab and cyclophosphamide , doxorubicin , vincristine , and prednisone significantly improves response and time to treatment failure , but not long - term outcome in patients with previously untreated mantle cell lymphoma : results of a prospective randomized trial of the german low grade lymphoma study group ( glsg ). j clin oncol 2005 ; 23 : 1984 - 92 . 15 . forstpointner r , dreyling m , repp r , hermann s , hanel a , metzner b , pott c , hartmann f , rothmann f , rohrberg r , bock h p , wandt h , unterhalt m , hiddemann w ; german low - grade lymphoma study group . the addition of rituximab to a combination of fludarabine , cyclophosphamide , mitoxantrone ( fcm ) significantly increases the response rate and prolongs survival as compared with fcm alone in patients with relapsed and refractory follicular and mantle cell lymphomas : results of a prospective randomized study of the german low - grade lymphoma study group . blood 2004 ; 104 : 3064 - 71 . 16 . romaguera j e , fayad l , rodriguez m a , broglio k r , hagemeister f b , pro b , mclaughlin p , younes a , samaniego f , goy a , sarris a h , dang n h , wang m , beasley v , medeiros l j , katz r l , gagneja h , samuels b i , smith t l , cabanillas f f . high rate of durable remissions after treatment of newly diagnosed aggressive mantle - 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