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(12) Patent Application Publication (10) Pub. No.: US 2012/0070450 A1 Ishikawa Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2012/0070450 A1 Ishikawa Et Al US 20120070450A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0070450 A1 Ishikawa et al. (43) Pub. Date: Mar. 22, 2012 (54) LEUKEMA STEM CELLMARKERS Publication Classification (51) Int. Cl. A 6LX 39/395 (2006.01) (75) Inventors: Fumihiko Ishikawa, Kanagawa CI2O I/68 (2006.01) (JP): Osamu Ohara, Kanagawa GOIN 2L/64 (2006.01) (JP); Yoriko Saito, Kanagawa (JP); A6IP35/02 (2006.01) Hiroshi Kitamura, Kanagawa (JP); C40B 30/04 (2006.01) Atsushi Hijikata, Kanagawa (JP); A63L/7088 (2006.01) Hidetoshi Ozawa, Kanagawa (JP); C07K 6/8 (2006.01) Leonard D. Shultz, Bar Harbor, C7H 2L/00 (2006.01) A6II 35/12 (2006.01) ME (US) CI2N 5/078 (2010.01) (52) U.S. Cl. .................. 424/173.1; 424/178.1; 424/93.7: (73) Assignee: RIKEN, Wako-shi (JP) 435/6.14; 435/723; 435/375; 506/9: 514/44 A: 530/389.6; 530/391.7:536/24.5 (57) ABSTRACT (21) Appl. No.: 13/258,993 The invention provides a test method for predicting the initial onset or a recurrence of acute myeloid leukemia (AML) com PCT Fled: prising (1) measuring the expression level of human leukemic (22) Mar. 24, 2010 stem cell (LSC) marker genes in a biological sample collected from a Subject for a transcription product or translation prod uct of the gene as an analyte and (2) comparing the expression (86) PCT NO.: PCT/UP2010/0551.31 level with a reference value; an LSC-targeting therapeutic agent for AML capable of Suppressing the expression of a S371 (c)(1), gene selected from among LSC marker genes or a Substance (2), (4) Date: Dec. 7, 2011 capable of suppressing the activity of a translation product of the gene; a method for producing a sample containing hematopoietic cells for autologous transplantation or alloge (30) Foreign Application Priority Data neic transplantation for AML patients comprising obtaining an LSC-purged sample with at least 1 kind of LSC marker as Mar. 24, 2009 (JP) ................................. 2009-072400 an index; and a method of preventing or treating AML. Patent Application Publication US 2012/0070450 A1 I’50IJI Patent Application Publication Mar. 22, 2012 Sheet 2 of 7 US 2012/0070450 A1 AK5 HCST CD3D CTSC GPR84 DOK2 A.OX5AP CACNB4 PDE9A PDK RG BK HOMER3 cN NCF4 PRKC) RNASE2 CD33 LGALS1 CD93 HCK ITGB2 TNFSF3B PXN g ... CD97 CEC2A FYB NEK6 TYROBP WNN1 CCL5 TNF FCGR2A PTH2R WT RAB20 Patent Application Publication Mar. 22, 2012 Sheet 3 of 7 US 2012/0070450 A1 xwu win. ; : 3 were Relativeymywrapinput cell ro, Relativesmritraruvu all fic, --r -...' sowrt--" " “. : rimstarrierrwroulture'r rary Relative cello, Fikista cil ris, Felite ceilio, (Y) 3 3 : its." - %-wr-will "rrow; c: r : . --. Fielä it air-3, IHHIWWMMMIWWM-al-----------r. g 3 retroitwirelyi wurup. Fixtis till it, s . EaE. ut g lit.& m rewrist Relatiya till ff), Relative all no. Rashtiye aire, Relative cello, Patent Application Publication Mar. 22, 2012 Sheet 4 of 7 US 2012/0070450 A1 : s g ir " . : 3. E t g . r t s al E. D k s e asser f "r g E. : 3. i. Patent Application Publication Mar. 22, 2012 Sheet 6 of 7 US 2012/0070450 A1 N y a) O () : ...) 3. Patent Application Publication Mar. 22, 2012 Sheet 7 of 7 US 2012/0070450 A1 . US 2012/0070450 A1 Mar. 22, 2012 LEUKEMLA STEM CELLMARKERS hematopoiesis: are leukemias a stem cell disorder or a reacquisition of stem cell characteristics? Proc Natl Acad TECHNICAL FIELD Sci USA 100 Suppl 1, 11842-11849 (2003). 0001. The present invention relates to leukemic stem cell 0006 non-patent document 2: Hope, K.J., Jin, L. & Dick, markers and the field of treatment of acute myeloid leukemia. J. E. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal BACKGROUND ART capacity. Nat Immunol 5, 738-743 (2004). 0002 Acute myeloid leukemia (AML) is the most com 0007 non-patent document 3: Jordan, C. T. & Guzman, mon/highly frequent (onset rate) adult leukemia, character M. L. Mechanisms controlling pathogenesis and Survival ized by the clonal expansion of immature myeloblasts initi of leukemic stem cells. Oncogene 23, 7178-7187 (2004). ating from rare leukemic stem cells (LSCs) (non-patent 0008 non-patent document 4: Lapidot, T. et al. A cell documents 1-3). The functional and molecular characteristics initiating human acute myeloid leukaemia after transplan of human LSCs are largely undetermined. Although conven tation into SCID mice. Nature 367, 645-648 (1994). tional chemotherapeutic agents can temporarily remit AML. 0009 non-patent document 5: Ishikawa, F. et al. Chemo recurrence later is the difficult problem that prevents us from therapy-resistant human AML stem cells home to and helping patients. For the development of an effective thera engraft within the bone marrow endosteal region. Nature peutic agent or treatment method, elucidation of the recur Biotechnol 25:1315-1321 (2007). rence mechanism by clarifying the leukemia features 0010 non-patent document 6: Clarke, M. F. et al. Cancer unknown to date is strongly desired. stem cells perspectives on current status and future direc 0003. A recent study demonstrated that a certain ratio of tions: AACR Workshop on cancer stem cells. Cancer Res leukemias and cancers consists of a heterogenous cell frac 66,9339-9344 (2006). tion and is not configured with a homogenous cell population capable of clonal proliferation. Lapidot and Dick identified SUMMARY OF THE INVENTION Such heterogeneity in acute myeloid leukemia (AML) and Problems to Be Solved by the Invention reported that CD34+CD38-cells are transplanted selectively in CB17-scid and NOD/SCID mice (Non-patent Document 0011 A problem to be solved is to find a molecular target 4). that is specific for human leukemic stem cells (LSCs) and 0004. The present inventors have succeeded in the devel provide a therapeutic means that will lead to radical treatment opment of an animal model capable of reproducing features of acute myeloid leukemia (AML) and the like. of human, rather than mouse, AML, particularly AML of individual patients, rather than a cell line, and permitting Means of Solving the Problems long-term assessment (Non-patent Document 5, Patent 0012. The present inventors found sets of genes differen Application PCT/JP2008/068892). The present inventors fur tially expressed between LSCs and non-stem cells, and pro ther identified using a neonatal NOD/SCID/IL2rg KO mouse posed the possibility that these genes serve as therapeutic model, which is one of the most sensitive human stem cell targets for AML (Ishikawa F. et al., Nature Biotechnol assays, that CD34+CD38-AML cells meet all criteria for 25:1315-1321, 2007 and PCT/JP2008/068892), but were cancer stem cells recommended by the American Association unable to rule out the possibility that the genes are at the same for Cancer Research (Non-patent Document 6). Specifically, time differentially expressed in normal hematopoietic stem CD34+CD38-AML cells self-renew, produce non-stem leu cells (HSCs) as well. Hence, a therapeutic agent and thera kemia cells, and have the exclusive capability of causing peutic method for AML with low prevalence of adverse reac AML in living organisms. By repeating primary human AML tions cannot be realized unless not only a comparison is made in NOD/SCID/IL2rg KO mice, the present inventors searched between LSCs and non-stem cells, but also a set of genes that for the mechanism behind the chemotherapy resistance and are differentially expressed between LSCs and HSCs are recurrences, which pose the most important problem in the identified as targets. The present inventors succeeded in reality of this disease, and identified the following two essen developing a mouse model enabling reproduction of human tial features of human AML stem cells. First, AML stem cells AML (mice generated by transplanting a fraction containing are present predominantly in the endosteal region of the bone leukemic stem cells derived from a human AML patient to marrow; when human AML transplantation recipient mice NOD/SCID/IL2rg" mice), transplanting a small number of were treated with chemotherapeutic agents, the great major bone marrow cells derived from an AML patient, and recon ity of chemotherapy-resistant AML cells were found in osteo structing the pathology of AML in the animal model. The blast niches. Second, AML stem cells (not CD34+CD38+and present inventors then prepared LSCs derived from an AML CD34-AML cells) are stationary and hence exhibit resistance patient and those from an AML transplantation recipient to cell cycle-dependent chemotherapeutic agents. These his mouse, as well as bone marrow samples and cord blood tological experiments and cell cycle analyses agree with the samples (HSCs are contained) derived from healthy donors, clinical evidence that a large number of AML patients achieve conducted a comprehensive analysis, and have developed the remission via chemotherapy induction but eventually experi present invention. ence recurrences. To develop a novel therapeutic strategy 0013. Accordingly, the present invention provides the fol designed to exterminate LSCs seems to be an exact step lowing. toward overcoming recurrences of AML. 1 A test method for predicting the initial onset or a recur Prior Art References rence of acute myeloid leukemia, comprising (1) a step of measuring the expression level of leukemic stem Non-Patent Documents cell marker genes in a biological sample collected from a 0005 non-patent document 1: Passegue, E., Jamieson, C. Subject for a transcription product or translation product of H., Ailles, L. E. & Weissman, I. L. Normal and leukemic the genes as an analyte, and US 2012/0070450 A1 Mar. 22, 2012 naling-related genes consisting of AK5, ARHGAP18. FYB, PDE9A, PDK1, PRKAR1A, PRKCD, PXK, RAB20, HCK, LPXN, PDE9A, PDK1, PRKCD, RAB20, RAB8A and RAB8A, RABIF, RASGRP3, RGS18 and S100A11; RABIF; transcription factors consisting of WT1 and HLX; transcription factor genes consisting of WT1, MYC and and other genes consisting of CYBB, CTSC and NCF4.
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