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Lnciosed please find ~.~ Preiirnina~7 Ai:.,pi:~c~ibn I(.~ a resear<h proposal ~r;tilt(d 'Mo!ecula~ pati]ogenesis of leukemia in Down Syndrome:: that i art, subrnitling tc th~ Councilfo~ 7obacco Reseamh !e~ consideration !o~ fundinU. I am ~equesti~g lu~lding for 1he lhree yea~ du~alion ol the project with 1he lirsl year~s ~:~nnual direct cost o~ St00,000. II you hav~. a~]y qLteslions, please do no1 hesilale to conlact me. -lhank ]70U. - ' Sincere!v

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factor independent. Indeed, it has also been found tI~t ' I~v~/s of ~CF are significantly/ higher in the fetus and neonate than in later infancy, childhood an~l aclulthood v~here they fall t o levels < 10 ngfml. Many hematopoietic cell lines, bothitranSformed and not, are dependent upon exogenous growth or survival factors: withdrawal. .. l~ads to apoptotic cell death. In this model, then, the cell population that survives to ,emerge later as AMKL would be either completely factor independent or effectively so in vivo This project was started in late 1995 upon:.:, my ~move to Duke University. My previous work was in the area of signal transduction !in T ,~iymphocytes. However, I wished to change the direction of my research to an ' ~ . al~ea which had more potential clinical relevance. Therefore, the amount of data we ha~e accumulated to date is necessarily limited. Much of our time has been spent generat;!ng the reagents to be used in the experiments proposed in the next section. We= have. =initiated a clinical protocol to collect blood and bone marrow specimens from children .enrolled in our Multi-Disciplinary Down Syndrome Clinic. We have studied the expression of iboth'ets-2 and erg-1 in DS cell lines and 2N cells and have shown that they are over-exp!iessed' at 1.5X in the former. We have preliminary evidence suggesting that over-expressi!on of either/or ets-2 and erg-1 in hematopoietic cells can decrease their dependence upon growth factors for survival and allow them to "escape" from apoptosis at growth i'factor:~ withdrawal. We can also show that over-expression of these genes leads to ~increased ;proliferation of fibroblasts in a manner proportional to the amount of proteins' expressed. Finally, we have also begun to analyze the mechanisms by which these genes may.I, t'ransform hematopoietic cells. Stem cell factor (SCF) binds to its receptor, c-kit, which is highly expressed in early hematopioetio progenitors and some myeloid leukemlas, particularly AMKL. c-kit is a tyrosine kinase membrane receptor which can be irrerversibly .activated by certain mutations, leading to transformation. Over-expression of c-kit could make cells more sensitive to SCF. The c-kit ptomoter has at least seven ets-binding sites. We are expressing these genes in COS cells to look for transactivation of a c-kit promoter constiuct by ets-2 and/or erg-1. We now have a full length phosphorylation mutant of ets-2 (T72A) which we believe will act as a dominant-negative in vivo. We have this mutant,' along with wild type ets-2 and erg-l, and the ets-domain of both ets-2 and erg-1 expressed..! in a variety of vectors, including a retroviral vecto, a dexamethasone-inducible promoter construct, myc- and FLAG-tagged vectors, and expressed as GST-fusion proteins. We have generated anti-(human)-ets-2 monoclonal antibodies that are reactive with the different :domains of the protein, and are in the process of making similar anti-erg-I antib0dies.iI I believe that DS offers a unique molecular model in which to study leukemogenesis in general. An understanding of the molecular paihogenesis of the increased risk to developing transient leukemia and acute leukemia in children with DS could offer major insights into molecular carcinogenesis. However, there iS a paucity of data about the natural history of these disorders, their true incidence, and the expression of these and other Down Region chromosome 21-encoded genes~: in both hematologically normal and abnormal children with DS. The goals of this research: project are to study these diseases in depth both clinically and biochemically. My !aboratory,~is uniquely suited for this work because of my clinical position and thus my access t0~ patient samples. Research Proposal; 1. Cfinical-patholog[cafstudies!. A :,'true incidence rate for TMD and AMKL has never been determined in DS. We will collect blood sampIes from all patients

who are born at Duke Hosp£tal or who are referred to the Down Syndrome We will expand the study to a wider geographic area w£thin a year with collaboration with other regional physicians via the DS O!in~.o. ff warranted, further tests wUl include an examination of bone marrow aspirates. The results from this study should give a true incidence of these disorders as wel; as giving us the opportunity to develop a ~ongitudlna~ database on these patients. ~oreover, these da~a should complement those obtained from the Pediatric Ontology G~oUp Protocol #g4B1 which ~s a nationa~ ~aborato~y study colleo4ing and analyzing bone marrow and periphera~ b~ood from DS patients with confirmed diagnoses of TMD, myelodysplasla 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 an~i 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 clinical 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 percentage of apoptotic cells in clinical samples from out own longitudinal study at Duke as well as on the specimens obtained from the Pediatric Oncology Group national study. With the latter, 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 hematopoietic 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-response 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 FOGi cell bank. . ' i 5. What is the normal and abnormal hematology of the Ts65dn mouse? Recently, these animals have also. been noted to'have red cell macrocyt0sis like patients with DS. We will continue and expand ~pon these preliminary results do!ng experiments similar 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 animals? Peripheral blood leukocytes (pooled from several animals), bone marrow, primary and immortalized fibroblasts, and placenta will" be examined for protein and mRNA expression. 7. What is the "transformab!lity" of cells from Ts65dn mice compared with normal littermates? We will assess the comparative ease with which Ts65dn cells can be transformed by recombinant retroviruses containing either proto-oncogenes (ets-2 and erg-l) or an oncogenic form of ras (vail2). Primary ceils, including fibroblasts, peripheral blood leukocytes and bone marrow cells from littermates will be infected with ~these retroviruses Future studies will include examining placental hematopoiesis. 3 50544034 Ts65:dn mice or their heterozygous and i G418-resistant colonies isolated. tissues for factors that influende

BIO( Rosoff, Philip Ma~ ' ,1 ,A~O~ ate Professor of Pediatr{cs 'NST~UT )NAr =DL LOCATION )-m(ffapp,~,,~ye) Y~R(s) FIELD OF STUDY New York University m=: I ' BLXT]m I ~ 974 ) Biology/Psycho ogy Case Western Resew, University ) m: M~mD";' m 1978 Medicine REp,~RCH ~ND PROFESS~ONAL~EXPERIENCE: Concluding w~ present pO~};~, list. In chm.alog~ order, previous employment, e~erienca. ana nonom, include present membemHip on any F~e~ Government public advlsoqt comml~ee. Ust, in chronological order, ~e t[~es, al a~ors, ~d complete references to all pu~ca~ons'durlng the past ~ree y~rs ~d ~'reprb~ve ead[er publica~ons ~d~nent to th~ ~p cation. If ~ st of publ~ca~ons In ~e last ~ree ypars e~eeds ~,o pages, select ~,~ most pe~nent p~bafions. DO NOT ~CEED ~O PAGES. post-Doctoral Positions , ~ : : ~ ,= ~ ~, 1978-1981 Clini~lFellow ~ Pediatrics ~d Hematol0gy-oh~fogy 1 9 81-1 9 8 2 Postdo=oral F~ilow, Dept. Cell. & Developmebt~l~iology, Haward Universi~ 1"9 8 2-1 9 8 5 Postdo=oral Fellow, Dept. of Die,hem. & aoleo~l~r Biology, Hazard Universi~ 1983-1 985 Instruct, }r, Dept:. of Pediatrics, Harvard Medical Sdhool. 1985-1 989 Assista, ~t Prgfqssor, Depts. of PeDiatrics & Physio!~gy, Tufts Medical School. =rof.,! Dlepts. of Physiology i& Pedlatrics,'."l'Lri'~s Medical School. 1989-1995 Assoc. I 1 992-1 995 Associ~ te Professor, Dept. of Medicine, Tufts M.#dical School. 1 9 95-Associate Professor & 'Chief, Div. PediatriC. HematdlogyL~6cology, Dept. of Pediatrics, Duke University. , Honors '= • Alpha Omega Alpha IMediqaJ.Honor Society, 1977. ' 2. Award of Dept. of Ped.iatrips; :Case western Reserve School lb~i!iMedicine, 5/78. 3. David Abraham Postdoctoral=Fellowshlp, Dana~Farbe( CanC~r' institute, 1983-84 4. Pew SchoJa, r Award, 11987~-1D91. i i = ':!: !'i: " 5. Scholar of the Leukemia SoCiety of America, 1~90-199& 6. Stohlmann Scholar ~f the' Leukemia Society o! America, 19~i996. / Memberships / ! : i i 1.. American Society for Biochemistry & Molecular! Biology, 1989 ! oresent 2. American Society ot: I" 'mmurlelogists, 1989 - pre;sent. : : i' !,',L " 3. American Association for It~ Advancement of Science, ,1979 -;:present. 4. Society for Pediatric Research, 1988 - present ;, 5. American Society of Hem'atlology, 1991 present. 6. American Society of Pediatric Hematology-Oni-iology, i:!996 -: present. 7. American Society of Cliniqal~'Oncology, 1997 - l.;;resenti ' : ' :i" Associate Ed£tor, The JournpIiof Immunology, ~89 - li,99&i ,=,:, ,,!,,,'i= PUBLICATIONS (representativ from total of 40). ' 1. Ros0ff, P.M. and C&r ey, L.C. (1985) and phorbol esters induce differentiation but have opposite effects on 'linositoI turnover and mobilization in 70Z/3 pre-B lymphccytes." J. Biol. Chem. 260: 9209. I =,' :,. ' 2. Oettgen, B.C., C.,, Cantley,,L.C., ~nd (1985) "Stimulation of the T3-T cell receptor compIex induces a m ~e Cell 40: 583-590. PHS 398 (Per. &*£5) I:

_anH .... !}9~ 'SIT'_ a::c:. :; :he 73-, :e:~ :e:e:::-ass: :ate: C.a2= ~mances the act:v:v o~ t~e Na- ~- e~.:;:ar~e: !.r a e~kerr!c !~umar T ce! ":e. J. Biol. Chert - 4£53- ~ 4359. .4. Rosoff, ~.M.. Burakoff, S.E~.. and Greensteir,. J. ::195% 'The rcie of the L374 moies.Jle i': mitog~ an.:igen-initiated signa; transduction.~ Cell 49: 8~5-853. 5, Rqsoff, P.M.. Walker, R., and Winberry, L. (1987) "Pertussis toxin stimulates seccnd rues: :.~roducticn in human T lymphocyles." J, Immuno1139: 2419-2423. Roscff, P.M., Savage, N., and Dinarello, C.A. (1988) 'Interleukin-1 stimu!ates diacylg ::roduction in T lymphocytes by a novel mechanism." Cell 54: 73-81. .7. Rosofl P.M., Hall, C., Gramates, S., and Terlecky, S, R. (1988) "DIDS (4,4'-diisothiccyanatost 2,2'-disul{onic acid) inhibits the CD3-T cell antigen receplor-stimulaled Ca2+ transporter in human T J. Biol. Chem. 263: 19535-19540. 8. Corey, S.J. and Rosoff P.M. (1989) "Granulocyte-macrophage colony stimulating factor (G~ primes neutrophils by modulating the activity of a pertussis toxin-sensitive G protein". J. Biol. 264: 14165-14171. 9. Rosoff, P.M. (1990) "The Interleukin-1 receptor and lransmembrane signalling" in S{ }mmunol. (G. Klaus, ed.) 2: 129-138. 1 0. Rogers, T.R., Corey, S.J. and Rosoff, P.M. (1990) "Identification of a 43-Kilodalton membrane as the pertussis toxin receptor in human T lymphocytes." J. Irnmunol. 145: 678-683. 1 1. Corey, S.J. and Rosofl P.M. (1991) "Unsaturated fatty acids and lipoxygenase products r, phagocytic NADPH activity by non-detergent membrane interactions." J. Lab. Olin. Meal. 118: 343-351. 12. Prasad, K.R. and Ro~otf, P.M. (1992) "Characterization of the energy-dependent, mating activated Ca2+ influx in Saccharomyces cerevisiae." Cell Calcium13: 615-626. 1 3. Rosofl, P.M. and Mohan, C. (1992) "Unidirectional, heterologous desensitization of the pertussi ~eceptor by the CD3-TCR complex." J. Immunol. 149; 3191-3199. 1 4. O'Riordan, C. and Rosoti, P.M. {1993) "Reconstitution of a T cell receptor-stimulated plasma me[ calcium transporter: lack of dependence on inositol phosphates." Cell Calcium 14:119-133. 15. Maschek, B.J., Zhang, W., Rosolf, P.M., and Reiser, H. (1993) "Modulation of the intracellular and inositol lrisphosphate concentrations in routine T lymphocyles by the glycosylphosphatidylir anchored protein sgp-60." J. Immunol.150: 3198-3206. 1 6. Rosoft, P.M. (1995) "Brief report: Congenital bone marrow failure with myelodysplasia in siblir Ped. Hematol/Oncol. 17: 56-60. 17. Zhang, X-M., Berland, R. and Rosoff, P.M. (1995) "Differential regulation of accessory mit signaiing receptors by the T cell antigen receptor." ~olec. Immunol. 32: 323-332. 18. Ro.~otl, P.M. (1995) "Myelodysplasia and UDP-galactose-4-epimerase deficiency." J. Ped~ 605-608. 1 9. Wong, W.S., Simon, D.I., Rosoft, P.M., Rao, N.K. and Chapman, H.A. (1996) "Mechanisms of p toxin-induced myelomonocytic cell adhesion: role of MAC-1 (CD11b/CD18) and urokinase receptor ((: Immunology 88: 90-97. 20. Wong, W,S. and Rosotl, P.M. (1996) "Pharmacology of pertussis loxin B-oligomer". Canad Physiol. Pharmacol. 74: 559-564. 2 1. Warwick, A., Hayer, K. and Rosoff. P.M. (1997) "Cystic fibrosis and homozygous sickle cell disea: 'patient from the Dominican Republic: report cf a case.'= Ped. Pulm.: in press. influx 260: n ~nd ~enger ycerol ilbene- :cells." I-CSF) 3hern. In. in. ~roteln gulate factor- s toxin nbrane Ca2 + ~osit:ol- igs" J. ogenic 127: tussis ;D87)". ian : J. ~e In a

support (dh'e~ cols'~, Tide of Projec~ 4) Peddatric Cncolo~ Group Studies i CA1552.~2/~ Identify and describe any overlap of this application None 'i ~ Date of" Termination of Grant Indicate the total annuat funds availableto you projects under your supervisi.on. Title of Project l~leoular Pathogenesis of Leukemia iu Do)m S~ Mo!eo~ Pathogenesis of Leukemia in I~m ~ (give grant numbe, rs) Leukemia Sooiety of , Identify and describe any overlap of this Aim I o£ the NIH grant and the l aims Dimes grant, overlap with the aim. of

The Couacfl For Tobacco Research - U.S~4-, Inc. 1997 subrai~ Full Appli~tiom. .~o 5054403S=