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DUKE UNIVERSITY MEDICAL CENTER vv~u~n~rr~ ~o~ ~ .... Dr. Arthur D. Eisenberg ~soc~ate Re,.batch iDirector :ReSea~ - USA, In( ,, , ............. ~; , : !: ; ,=~n~oS,ed=p!ease find a Preliminary Application for a r~search' proposal entit!ed iUol=~Uia:~ ,~iath=ogenesis of leu:kemia~in sub,m:ittihg t0 =the ~d~l '",for," ,~ob~:~, ' :,i~=;r,'::,:i=,f~di.gi,=,, : :1 ~=i~;m==,,,',reque'~ting:i f~ndi~ fo,r,:,' Philip M. Rosoff. M.D. Associate Professor of ,Pediatrics , ~ediatri¢ Hematology-On(~ology Naldl C=oii~ 27710. Te~=l= (~1~) 684-.:~401 - FAX 019) 68 I-'/9~0

,Dr' 'Arthur I;D;. Eisenberg : ,~0ci~te ::iResea:rch Director ..~,,~b ~;un~i' for:Tobacco Research-USA. Inc. York, NY 10022 Deari Dr. Eisenberg: ' ' " ...... ' 'February 7, 1997 Enclosed please find a Preliminary Application for a research proposal entitled 'IVlolecular pathogenesis of leukemia.in Down Syndrome" that I am submitting to the Council for Tobacco Research for. consideration for funding. I am requesting fur ling for the three year duration of the $100,000. If you have any questions~,:~. you. ~ ..... ': ~ -...--.-.,~irv~mlv, i .... i::, :,~ :,!:~ ~ '~,, ~, ,:~ : ,;, : ,~ the first year's annual d!=redt do not hesitate to contact!me..':i:~:Th , Hematology.!Onc6iO~=: ;,:=, :: ,~ :: =~ ;: ,: ;; !i=i 'i :: =

MOLECULAR PATHOGENESIS OF LEUKEMIA IN DOWN SYNDROME Background: Cnic-en cc-n w;th Dowr" Sy"c~rome ITriscmy 2!: b, ave a 20-fold c:re~ "~-: :' :e,eo:,q~ ieu~emia cver their iifetmes. Inthe firs*, three years of ti;e.,t~,~ :&-t -.~.:4. r~sK for cievelcl:ing acute megakaryobJastic ieuKemia (AMKL). This alimc~ :,::cbr~.' i..,~wirlg a pericd cf myelodysplasia arc a syndrome uniquetc 5'..~r,~,::'--e ,:C.S) known as transient myeioproliferative disorder (TMD). :i:~::;qp ~etal i:fe and the diagnosis can be made antenatally. Up ~tili~ deve ~-F. AMKL within 3-36 months. It has been demonstrate,d th~ Illltl .AMK,. tf-a~ develops iater arises from the same clo~e. DS c le~kem!.s, independent of a prior history of TMD, are qorma} co~nterparls. This may be due to their inc Since many chemotherapeutic agents induce a ir~.creased sensitivity may be due 1o an inherent death ~see below). TMD resembles another malignant diso[der }s kr~own to have a high rale of spontaneous the neuroblasts. Whether TMD disappears 7here is a ~a~e group of patients wilhout clini been noted to have TMD in the neonatal pert( leukemic ce~ls, but are extreme somatic mosl being lrisomic. In a series of 14 patients, non~ 35-40%. These observations suggest that sufficient to predispose palients to develop somatic cell trisomy 21 is required for progres AI} ol the features constituting DS chromosome 21, specifically a sho~ section Down regionL A numbe[ of genes have been ~n !t)e palhogenesis of leukemia in DS, inclu( These genes encode transcription factors. e~ther ETS-2 or ERG-1 in rodent fibroblasts investigators have speculated that these pathogenesis of AMKL in Down Syndrome. i' ~ene~ated, called Ts65dn. These animals skills, and neuronal organization in the central~ those observed in DS patient. These animals hematc~o~esis or immune dysfunction as~ TM[~ o,nly occurs at or near bi~h, su~ ~:-i : .actcr that is over-expressed and s~ :~is~my :.'~ :ells could be more sensitive to th "-ematrp.:),etic hormone. This factor could ei 7~y ~nat i: s present at high levels in prenata eve~l ,~ utero, then fall to lower levels after b cells ~tl e TMD clone) would die, except for t} been ~[rll i that to l, may a sign a routine mode!, ; ,of, :, ; ,, in system that not been examined with D8 (M. that them may ibei", possible that them for

ievels< t0 ngr- M:-:.nv "~'-.;:'::!:g,e'~ :e :~.:s : -: ..... ~- ~ ..... e : ~,--~ ,-.: :~re ~e:e~)e~' completely factc, r :~d~p~u:~:]erl 'r ~:t ~, ..... ,..~ ~_ ":' , . : This project was .... l::~..~.~.d n ~a!,e i9~95 ~p:::,:; m~ .::.::re ~ .~ previous work was ;n t~e ~e~, ,., ~,g,,,<~. Irar:,s;:,::t[;::r" "~ 7 .:;:m[~" "",~,~, l~" ..... ~ to change the d~:~ect~o':: o~ m~ research .... ~'b .~ea ,,/~:~c:h '~a~ m:"~ pr:,:eqt~a:~ relevance. Therelc,.re, 1h£- - .... ~ ' limited. Much of ou~ tir~:e ~s been sp~f #eneratinq the rea~enls ~,r be used [,n experiments proposeg in the ;le:~t sect[:)n. We t,ave h:i[~at~d a .n[:~rca~ p~toco~ 1c:. coi~ecl blood and bone marrow specimens from children enro~ ~{) ~,,, :~r Mut.-D)s~ip~na~ Down Syndrome Clinic. We haw studied the expression o~ both ets-2 and ~rg. 1 in DS ce}~ lines and 2N cells and have sk, ow::, 1hat 1hey am ov~r~e~pms~-~d at i.gX n lh~ }.srme~. We haw:, preliminaG evidence su(Ggest}ng that ove~exK, m~sion ,{~t e:theL.~l ~,t;u..~* ~{~ ~:~g-7 ~. hematopoietic cells can decrease 1heir dependence upu~ growff~ lactors 1or survival anc~ allow lhem Io "escape" from apoptosis at g~owlh 1actor w~thdrawal. We can also show that over-expression of lhese genes leads 1o increased pre~iie~al~on ui fibroblasts ~n a manner propo~ional 1o the amount of proleins expressed. Fir~ally~ we t~ave also begun t analyze lhe mechanisms by wMch lhese 9e~., may iranslorrn hemalopo~etic cells. Stem cell factor (SCF) binds 1o its receptor~ c-kit which is highly expressed ~n earty hematopioetic progenilors and some myeloid leukemias, particularly AMKL. c-kit is a lyrosine kinase membrane receptor which can be irreversibly activaled by ceflain mutations~ leading to Iranslormation. Over-expression ot c~kit could make ce~ls more sensitive to SCr, The c-kit plomoler has al least seven ets-binding sites. We are expressing these genes in I;OS cells lo look lor Iransactivation ol a c-kit promoler construct by ets-2 arid/or erg-1. We now have a full length phospho~lation mulanl o! ~ts-2 (V72A) which we believe will act as dominanl-negalive in vivo, We have this mutanl~ along with wild type ets~2 and erg-l and lhe ets-domain of both ets-2 and erg-1 expressed in a variety of ~ector~, ~nctuding relroviral recto a dexamethasone4nducible promoter construe!= myc. and f i AG-lagged vectors, and expressed as GST-fusion proteins. We have generaled ~nti~(human)~ets.-2 monoclonal antibodies that are reaclive with the dilferent domains ol lhe prolein, and in the process of making s~m~ar anli.-~r.q. ~ ~nt~bodies. general. An understan{fintl oi the ni, olec:~l;:~r '" ~"-{- ~' pal o9~.~.o,, oi ~he i~creused ~sk developing transient leukemi~:~ ~l~d ac~te ileuk~:~:~[~a '.~f,, <;},~di+~., w[~th DS uou~d uff~:r ~.~',~r abnormal children witt DS "IbiS" g{~al~, .4 lb ;s rt~se~r,ch [.;-a)eci are I- slL~::~ these in depth both clinia{lly ar,l [~iochem~cally ~)v ,abcratcrv ~s urtq,Jeiv stJ[ted for l~:ls wE. rk because of my clinica~ p.as:t~:)q and :RDs m,, access :c bat,.er't SamE' e: AMKL has never beer, deterG qed iq DS ~e w,q c:l,ecl b~ood samgles ~r.c~ all pat.ents

who are born at Duke Hospital or who are referred to the Down Syndrome C!;nio at D~uke. We will expand tile study to a wider geographic area w~thin a year with collaboration with other regional physicians via the D$ Clinic. If warranted, further tests will includ~ an examination of bone marrow aspirates. The results from this study should {live a Irue incidenc~ of these disorders as well as giving us the opportunity to develop a Iongitu~!inal database on these patients. Moreover, these data should complement those obtained from the Pediatric Ontology Group Protocol #9481 which is a national laboratory sludy collecting and analyzing bone marrow and peripheral blood from D8 pat!enls with confirmed diagnoses of TMD, myelodysplasia and AMKL, in which we are participating. 2. What is the expression of ets-2, erg-i in normal and pathological specimens from children with DS? We will investigate the amount of protein, specific mRNA, and the gene copy number for ets-2, erg-1 on peripheral blood and bone marrow from patients with !! DS. We have already seen that some transformed DS cells express significantly more ets-2 =and erg-1 protein than normal DS cells. We will investigate the possible reasons for this at the molecular level: the purpose of this study will be to examine a large number of cl nical samples to see if this is an isolated or more widespread phenomenon. We will also test these cells for c-kit expression. 3. What is the role of apoptosis in TMD and AMKL in DS? We determine the percen :age of apoptotic cells in clinical samples from out own longitudinal study at Duke as well ~ on the specimens obtained from the Pediatric Oncology Group national study. With:the I~tter, we will be able to analyze samples from patients initially diagnosed with TMD .as well as that percentage who then develop myelodysplasia and eventually AMKL. 4. What is the sensitivity to hematopoletic growth factors in DS cells and cells from Ts65dn mice? Bone marrow from patients with DS and Ts65dn mice will be placed in short term cultures and their sensitivity to IL-3, SCF, and GM-CSF will be tested. Dose-resP0nse and survival curves for each factor alone and in combination with others will be generated. Similar survival curves will be done with true pathological specimens obtained from the ~..103 cell bank. 5. What is the normal and abnormal hematology of the Ts65dn mouse? Recently, these animals have also been noted to have red cell macrocytosis like patients with DS. Wp will continue and expand upon these preliminary results doing experiments simila~ to those described for our longitudinal study of infants and children with DS. 6. What is the expression of ets-2 and erg-1 proteins and mRNAs in these ammals? Peripheral blood leukocytes (pooled from several animals), bone marrow, primary i' and immodalized fibroblasts, and placenta will be examined for protein and mRNA expression. 7. What is the "transformability" of ceils from Ts65dn mice compared with nqrma/ /ittermates? We will assess the comparative ease with which Ts65dn cells car~ be transformed by recombinant retroviruses containing either proto-oncogenes (ets-2,~ and erg-1) or an oncogenic form of ras (vail2). Primary cells, including fibroblasts,'I perlp~eral blood leukocytes and bone marrow cells from Ts65dn mice or their heterozygous littermates wiII be infected with these retroviruses and G418-reslstant colonies IsOlated. Future studies will include examining placental tissues for factors that Influence! hematopoiesis. 3 50544043

, , '= ~i =, 'Associate Professor of Ped~atd~ : ,~ ~ ,; ;~ .... ' "~ ~' ~ 'i~ i!' = : DEGREE INST~uTIDN: ~,: AND LO'{, ~ .,~N : ',:'~f~P;EC~:e): ,: Y~R(S) : Fi~O, OF . ":; ' i[ B.A. i 1 974 BiologytPsychology~ New York: Universi~ .;. Case Western Resewe Univprsil.,' ~i: . M.D. i 1 978 Medicine School of Medicine ~ ~ , ~,, ,, , ,,, , RES~RCH AND PROFESS~ON~ ~xPE~IEH~E: Co~cl~d].g W=~ :preset posi~on, li~ Inichro.ologi~ order, previo~ e~lo~ent, '~rie~ce, and hon~. IndUe present m'embemhip o!, ~7 ~=Fede~ G~vemme~ pubi~ adv~s~ com~aa. List, in chmnot~ic~ o~er, ~s c~plete r~etenc~ to ~1 pub~icatlons du~=~g the' p~t ~ree years ~ to ~eprese~tive ~er publicaEons pennant to ~is ~plica~on ~=~e I st of publ¢~ns in ~e I~t ~ree ~ exceeds. ~ ~ges, se]~ the m~ pe~ent pu~lca~ons. DO NOT ~CE~O PAGES. P0~t-Doctoral PositionS)t: :: ;=: ~ '. ' ; 1978-1 981 Clinical #ellow in ~lped:,~td~ and Hematolo~-Oncology . : 1 981-1 98 2 Postdoctoral Fellow, D;ept. Cell. & Developmental Bmology,~ Hazard Un~vers~ 1 982-1 985 Postdoctoral Fellow !~ept. of Bi~hem. & Mol~ular Biology, Hawed Un~ersi~ Facul~ PoSitions : : ) ) . 983-1 985 Instructor, ; ii i " . Dept. of P~dlatncs, Harvard Medical School. 985-1 98 9 Assistant Professor, D~l)tS. of Pediatrics &:,Physiology, Tulfts Medical School. 9 8 9 - 1 9'9 5 Assoc. 'Prof., Depts. 0fi Physiology & Pediatrics, Tufts Medical School. 992-1 995 Assocmate Professor, Dept. of Medmcmne, Tufts Msd=cal School. 9 9 5 -Associate ProfeSsor & ~Chief :Biv. Pediatric Hemato!ogy-Oncolog) Dept. of Pediatrics, Duke University. 1. Alpha Omega Alpha Medical H~neqSociety, 1977 .... 5' Scholar:of the Leukemia ~'J~,~!' jfl:~merica 1990 199":=:: 6; Stohlmann Scholar of the Leu;~emi~Socie~ of Amedca; 1995-1996. Memberships I; ,=; :i!, l!,' 1. Americ~n Society for~ Biochemist~ I,&! Molecular Biology, 1989- present. 2. Amencan Soc|ety of immunOlqg=st,% 1989 - present; 3. Amencan Associabon for the Adv~i)cement of Science, 1979 - present. 4. Society: for Pediatric'Research. ~ £f1~8 - present. 5. American Society of Hemat010.~y~l~,91 - present. :. ' 6. American Society of 'Pediatric,~ -~Heil ~l~tology-Oncology, 1996 - present. 7. American Society of: Clinical ~c.01~gy, 1997 - present. ; Associate Editor, The Journal PUBLICATIONS. (representative 1, Rosoff. P.M. aqd . have opposite effects,on phospi-~; Biol. Chem. 260: 9209~9215~ 2. Oettgen, H.CJ,'. Terho~s receptor complex induces a membr 1989 1993. , (1985) "Upopolysaccharlde and phorbol esters Induce differerttlation but turnover and Ca2+ mobilization in 70Z/3 pre-B iymphocytes." J. Cantley, L.C.~ and Rosoff, P.M, (1985) :'Stimul~tion of the :iT3-T cell inflcc~° Cell 40: 583-590~ .=~ (Form Page 6) t=age ~ :i 50544044

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C~19525-24 ?~cne Indicate d~e total annual funds available to you this year for all resean:h projects under yore supe; visi0r~. $ 136,979 ENDL-NG OR PLANNED Titb oflh cject SOUrCeS (give grant ..nmnbers) 3~cie~, cf A~rioa Total Value "mual of Grant mt (direct costs) ~o You 614,N37 2C0,0021 Ave:. A Avail; ~ble 19i,71 18),00 ::-_: .':::,:.-- .e::-. ~7; ,<:"'. tr:: ::ira of thls proposal. Total Duration (give inclusive dates) and March cf