Diagnostic and therapeutic targets for leukemia

#The present invention relates to methods of identifying candidate compounds for the treatment of leukemia and diagnostic methods based on histone methylation.

#Higher-order chromatin structures are of profound importance in gene regulation and epigenetic inheritance (Wu and Grunstein (2000) Trends Biochem. Sci. 25:619-623). Post-translational modifications of core histones influence the establishment and maintenance of higher-order chromatin structures. The unstructured tails of certain core histones are extensively modified by acetylation, methylation, phosphorylation, ribosylation and ubiquitination. A “histone code” hypothesis, linking histone modifications to chromatin structures, has been the focus of intensive recent studies (Strahl and Allis (2000) Mol. Cell. Biol. 22:1298-1306; Turner (2000) Bioessays 22:836-845). Histone methylation has emerged as a major form of histone modification. (Strahl and Allis (2000) Mol. Cell. Biol. 22:1298-1306; Zhang and Reinberg (2001) Genes Dev. 15:2343-2360). In particular, a large family of SET domain-containing histone methyltransferases (HMTases) has been identified (Lachner and Jenuwein (2002) Curr. Opin. Cell Biol. 14:286-298). SET domain proteins have been shown to methylate various N-terminal lysine residues of histone H3 and H4. Histone lysine methylation has been associated with diverse biological processes ranging from transcriptional regulation to the faithful transmission of chromosomes during cell division (Grewal and Elgin (2002) Curr. Opin. Genet. Dev. 12:178-187).

#Further, lysine methylation catalyzed by SET domain containing proteins has been linked to cancer (Schneider, et al. (2002) Trends Biochem. Sci. 27:396-402). For example, the H3-K4 methyltransferase MLL is frequently translocated in leukemia (Ayton and Cleary (2001) Oncogene 20:5695-5707; Milne, et al. (2002) Mol. Cell 10:1107-1117; Nakamura, et al. (2002) Mol. Cell 10:1119-1128) and the H3-K27 methyltransferase EZH2 is overexpressed in a number of tumors and its expression level correlates with the invasiveness of these tumors (Bracken, et al. (2003) EMBO J. 22:5323-5335; Kleer, et al. (2003) Proc. Natl. Acad. Sci. USA 100:11606-11611; Varambally, et al. (2002) Nature 419:624-9).

#FIGS. 1A-F. Knockdown of CALM-AF10 in U937 cells impairs their proliferation and leukemogenesis in vitro and in vivo. a, Top panel depicts the fusion protein and the target region for knockdown. The two arrows indicate the primer position used for RT-PCR. Bottom panel is RT-PCR results evaluating the knockdown efficiency. β-actin serves as a control. V: vector control, KD: Knockdown. b, c, Effects of CALM-AF10 knockdown on cell proliferation in RPMI medium (b) and methylcellulose (c). Only colonies having over 50 cells were counted. The numbers below the pictures in c represent colony numbers with standard deviation. d-f, Effects of CALM-AF10 knockdown in vivo. d, Knockdown CALM-AF10 extends survival time of transplanted mice when compared with those receiving transplantation of parental U937 cells. e, H&E staining demonstrating that mice transplanted with CALM-AF10 knockdown cells (bottom panels) have less infiltration of leukemic cells in spleen, kidney and pancreas when compared with mice transplanted with control U937 cells (top panels). Arrows indicate infiltration of transplanted U937 cells. Original magnification: ×40 for spleen, ×20 for others. f, FACS analysis of proliferation in bone marrow and spleen of transplanted U937 hCD45 positive cells. CALM-AF10 knockdown affects U937 cell proliferation in both bone marrow and spleen.

#FIGS. 2A-F. Expression of CALM-AF10 is sufficient to cause mouse bone marrow cell transformation and hDOT1L plays an important role in this process. a, Schematic representation of the retroviral transduction procedures. b, Expression confirmation of the transduced genes by RT-PCR. U937 cells serve as a positive control. GAPDH serves as a control for equal input RNA in RT-PCR. c, Serial colony plating assay of bone marrow cells transduced by wild-type and mutant CALM-AF10 with vector control. Presented is the average colony numbers with standard deviations of three independent experiments. The p value of the third round colony numbers between the wild-type and the OM-LZ deletion mutant is presented. d, Growth curve of the transduced cells in mFTOC culture. e, Immunophenotyping by FACS of CALM-AF10 transformed cells. The cell surface markers used in the analysis are indicated. f, Diagram of the 2nd transduction procedure (left panel), and the effect of wild-type and catalytic-deficient hDOT1L on colony formation of CALM-AF10 transformed cells (right panel).

#FIGS. 3A-B. Hoxa5 is involved in leukemic transformation by CALM-AF10. a, Left panel, RT-PCR analysis of the expression of late Hoxa genes and Bmi-1 in wild-type and CALM-AF10 knockdown U937 cells. Right panel, RT-PCR analysis of expression of late Hoxa genes and Bmi-1 in mouse bone marrow cells derived from second round colonies, and transduced by wild-type and OM-LZ deletion CALM-AF10 mutants. Lack of detectable signal for Hoxa9 is not due to primer failure as the same primers detected Hoxa9 expression in MLL-ENL transduced cells (lane 9). b, Hoxa5 knockout attenuates the transformation capability of CALM-AF10, but has no effect on that of MLL-AF10. Serial colony plating assays were performed as that in FIG. 2a. Presented is the average colony numbers with standard deviations of three independent experiments. Due to embryonic lethality of Hoxa5 knockout in C57B6 strain, bone marrow cells used in the assays were derived from Hoxa5 knockout and wild-type littermates of MF1 strain (Jeannotte et al., (1993) Genes Dev 7, 2085-96).

#Chromatin structure is important in gene regulation and epigenetic inherence. It is known that post-translational modifications of histones are involved in the establishment and maintenance of higher-order chromatin structure; further, it has been reported that the tails of certain core histones are modified by acetylation, methylation, phosphorylation, ribosylation and ubiquitination. The present invention is based, in part, on the first demonstration that (1) CALM-AF10 fusion appears to be both necessary and sufficient to mediate leukemogenesis in vitro and in vivo; (2) that hDOT1L and its H3-K79 methyltransferase activity are implicated in CALM-AF10 mediated leukemic transformation; and (3) that the Hoxa5 gene is involved in CALM-AF10 mediated transformation. While not wishing to be limited to any theory of the invention, the inventors also provide an experimental model where hDOT1L retains nuclear localization of CALM-AF10 through protein-protein interaction. CALM-AF10 in association with hDOT1L is then recruited to the Hoxa5 promoter to mediate H3-K79 methylation, which in turn leads to Hoxa5 upregulation and leukemogenesis.

#The present invention will now be described with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention.

#Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.