US 20070209082A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0209082 A1 Lih et al. (43) Pub. Date: Sep. 6, 2007 (54) TXR1 AND ENHANCED TAXANE Publication Classification SENSTIVITY BASED ON THE MODULATION OF A PATHWAY MEDIATED (51) Int. Cl. THEREBY AOIK 67/027 (2006.01) CI2O I/68 (2006.01) (76) Inventors: Chih Jian Lih, Palo Alto, CA (US); A6IR 48/00 (2006.01) Stanley N. Cohen, Stanford, CA (US) A6II 3L/337 (2006.01) CI2P 2/06 (2006.01) C07K I4/705 (2006.01) Correspondence Address: C07K 6/30 (2006.01) BOZICEVIC, FIELD & FRANCIS LLP (52) U.S. Cl. .............................. 800/14: 514/449; 514/44; 1900 UNIVERSITY AVENUE 435/6: 435/69. 1; 435/325; 435/320.1; SUTE 200 530/350; 530/388.8; 536/23.5 EAST PALO ALTO, CA 94.303 (US) (57) ABSTRACT (21) Appl. No.: 11/357,728 Methods and compositions for enhancing taxane sensitivity are provided. Aspects of the Subject methods include admin (22) Filed: Feb. 16, 2006 istering to a subject a tXrl pathway modulatory agent in Related U.S. Application Data conjunction with a taxane. Also provided are tXrl polypep tides and nucleic acids encoding the same. The Subject (60) Provisional application No. 60/654,343, filed on Feb. methods and compositions find use in a variety of different 17, 2005. applications. Patent Application Publication Sep. 6, 2007 Sheet 1 of 7 US 2007/0209082 A1 Fig 1. A. 5 dTR is s dTR 3 dTR 3 tr downstream u gene crp Tc RP B. 120 e-M2182tTA 60 40 M2182TA M2182TATc Cone 18 Cone 18+Tc CHO M2182tTA Clone 18 -Tc +c -Tc +Tc B-tubulin (25X, 154bp) Extes (25X, 22Obp) Osas sessie etPisaray be it (25x,B -actin 12Obp) Patent Application Publication Sep. 6, 2007 Sheet 2 of 7 US 2007/0209082 A1 Fig. 2 A. GSV D. M2182tTA-- Cone-r- 18 integration S. -TC +Tc - c. --Tc -a-tubulin 52121713 Chro Tosome 21254 - - Txt 1 12q13 Pre-intune Seut B. E. MWNPNAGOPGENPPPNGCPGGSNPAHPPPINPPFPPGPCPPPPGAPHG NPAFPPGGPPHPvPQPGYPGCQPLGPYPPPYPPPAPGIPPVNPLAPGMVG PAVIVDKKMOKKMKKAHKKMHKHQKHHKYHKHGKHSSSSSSSSSSDSD C. M2182tTA ClOne 18 anti-TxT txr1 (1.2kb) b-actin txr1 (20X 156bp) Patent Application Publication Sep. 6, 2007 Sheet 3 of 7 US 2007/0209082 A1 Fig. 3 Clone 18 M2182tTA E. No taxo 2.5M taxo F. 508 0.6 g 0.4 > 2 0.2 on S1 SI2 Control C. G. g -le-Clone 18 control s -e-M2182TAvcontrol s --Core 18S -a-M2182TASI1 -o-Clone 18S2 o -o-M2182tTAVS2 d O S o r 2 s s s s as o a O 100 taxol (nM) D a & H. s e eS of (-tubulin o-tubulin Tx 1 Tx1 Patent Application Publication Sep. 6, 2007 Sheet 4 of 7 US 2007/0209082 A1 Fig. 4 A. B. taxol (nM) 4. 5 10 Cone R1 -TC +TC X C. tit.1.3.1.-1 100 8) 6 OnM 2.5M 3M 4nM SnM 10nM 120 too OnM 2.5M 3M 4M SnM 10nM Patent Application Publication Sep. 6, 2007 Sheet 5 of 7 US 2007/0209082 A1 Fig.5 A. A382A Chris -ic ce 18- ckne 3 ic C. M2182ttA core r2 he attaplest TXRA WS vs ws WS -- -- N28A a M8A T. cyre 8 wic M28TA . tc. le -- - +c 1 2 3 1 2 3 2 3 1 2 3 Ma82TA cine Ra heatTAlpStixRA D. 2. s tr1 g -- trff TSP-1 sansiliityS; Q G B. 3. M2182TA Clone18 -TC +TC -Tc +TC . W. Cockkinos contellation coefficient r1 vs TSP-1 -0.86 tor ws taxo -0.83 TSP-1 vs taxo 0.67 Patent Application Publication Sep. 6, 2007 Sheet 6 of 7 US 2007/0209082 A1 80 60 Control AD ET VB VC EpoB x B. D. taxol. OnM SM 1.4 t. Tan. -------- TSP-1 Quvrn OS ... e. itsP1 is uom TSP1 w >< 1.2 7.5ugm, O So 8 > th- is 0.8 - X 06 2 0.8 TSP. O 804 3 0.4 Ougm ><>< 0.2 D.2 SZNZOO taxonM taxoSM 0. Control AD ET VB VC EpoB E. taxo 10nM Control taxo 10nM TSP-17.5ug/ml TSP-17.5ug/ml Control STSP-1 ... taxol: onM 1M 2nM 3nM 4nM 5nM in an ISP. Patent Application Publication Sep. 6, 2007 Sheet 7 of 7 US 2007/0209082 A1 Fig 7. Clone 18 M2182tTA o to a o o to p o y CD47 ; 20 0. - AaT-6 to 150pm --ABT-5 to room orpae T-12sum : 20 s 1 so 4N1K 250M US 2007/0209082 A1 Sep. 6, 2007 TXR1 AND ENHANCED TAXANE SENSTIVITY sents the meant-SD from eight replicates: (FIG. 1C) Tc effect BASED ON THE MODULATION OF A PATHWAY on taxol resistance in clone 18. Equal numbers of M2182tTA MEDIATED THEREBY and clone 18 cells were seeded and treated with the indicated concentration of taxane in the presence (+Tc) or absence CROSS-REFERENCE TO RELATED (-Tc) of tetracycline (1 lug/ml) for colony forming assays. APPLICATIONS Cell colonies were stained by 0.5% crystal violet. (FIG. 1D) Tc effect on taxol uptake in clone 18. Equal numbers of the 0001 Pursuant to 35 U.S.C. S 119 (e), this application indicated cell lines were seeded, incubated with H. taxol. claims priority to the filing date of U.S. Provisional Patent washed, and Subjected to Scintillation counting for measur Application Ser. No. 60/654,343 filed on Feb. 17, 2005; the ing the rate of uptake. Chinese hamster ovary (CHO) cells, disclosure of which application is herein incorporated by which are known to contain high efflux activity were used as reference. controls (Parekh & Simpkins, Biochem Pharmacol (1996) 51:301–311). Each histogram represents the meaniSD of BACKGROUND four independent experiments. (FIG. 1E) Gene expression of 0002 Taxane is a family of major chemotherapeutic B-tubulin isoforms. Poly(A) RNAs extracted from indicated agents that have anti-neoplastic effects against a wide spec cell lines were used as templates to quantitatively analyze trum of human cancers. Despite a dramatic response of expression of B-tubulins I and III by RT-PCR. The cycle Susceptible tumors to initial treatment with taxanes, the number and product size are indicated. Subsequent development of resistance to these drugs has 0006 FIGS. 2A-2F. Cloning, Gene Expression, and proved to be a major limitation to long-term use of these Localization of tXr1 (FIG. 2A) Schematic diagram of GSV agents as anticancer drugs. Two well-studied mechanisms integration site and tXrl gene structure. GSV provirus inte associated with such resistance are: (1) mutations in ATP grated (inverse triangle, arrowhead indicates the direction of dependant B-glycoproteins that lead to exclusion of taxanes transcription from the Tc-regulated promoter) at chromo and a variety of other drugs from cells (i.e. transporter Some locus 12q13 (horizontal line). The genomic nucleotide related multidrug resistance; MDR-1); and (2) mutations coordinate for the integration site and coordinates for the that affect the binding of taxanes to microtubules. However, start and end of tXrl transcripts are indicated below the line it has long been apparent that still other undefined mecha in bold type. Four exons of tXrl gene are shown as Solid nisms are also implicated in taxane resistance. boxes and the cDNA nucleotide coordinates for the start and end of each exon are italicized. The codons initiating and SUMMARY terminating translation (start and stop, respectively) are 0003 Methods and compositions for enhancing taxane indicated.(FIG. 2B) TXrl protein sequence deduced from sensitivity are provided. Aspects of the subject methods cDNA sequence. The peptide used as antigen for raising include administering to a Subject a tXrl pathway modula antibodies is underlined. (FIG. 2C) Effect of Tc on tXr1 tory agent in conjunction with a taxane. Also provided are transcript in clone 18. Northern blot (top panel), poly(A) tXrl polypeptides and nucleic acids encoding the same. The RNA extracted from indicated cells/conditions were frac Subject methods and compositions find use in a variety of tionated by electrophoresis, transferred onto filter, and different applications. probed with radioactively labeled tXr1 and B-actin cDNA fragments. The size of the tXrl transcript is indicated. The BRIEF DESCRIPTION OF THE FIGURES same RNAs were used as template for RT-PCR analysis (bottom panel), the cycle number and product size of the 0004 The file of this patent contains at least one drawing RT-PCR product are indicated. (FIG. 2D) Effect of Tc on executed in color. Copies of this patent with color draw Txrl protein in clone 18. Cell lysates extracted from indi ing(s) will be provided by the Patent and Trademark Office cated cells/conditions were Subjected to immunoblotting upon request and payment of the necessary fee. analysis and probed with anti-TXrl and anti-C-tubulin anti 0005 FIGS. 1A to 1E. Identification and Characterization bodies. (FIG. 2E) Intracellular localization of Txrl. HeLa of Taxol Resistant Cell Clones. (FIG. 1A) Structure of cells were immunofluorescence stained with preimmune integrated provirus derived from retroviral gene search serum (left, upper) or anti-Txrl polyclonal antibodies (left, vector (GSV). 3'dLTR or 5'dLTR (open boxes), designate lower). Chinese hamster ovary cells transfected with pCMV defective retroviral long terminal repeats lacking promoter EGFPN1 (right, upper) or pEGFPTXrl (right, lower) were and enhancer sequences required for virion production; SA, stained with DAPI (blue). Fluorescent images were obtained splicing acceptor site from adenovirus; neo, reporter gene by confocal microscopy. (FIG. 2F) Multiple tissue Northern encodes resistance to G418 (arrow in box indicates the blot. A filter (Clontech) containing RNA extracted from the sense direction of transcription; the broken arrow at the top indicated tissues was probed with radioactively labeled tXr1 indicates the location and 5' to 3’ direction of the primer used and B-actin cDNA fragments.