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WO1994026930A1 - Procedes pour depister et traiter des leucemies resultant d'anomalies chromosomiques de la region all-1 - Google Patents

Procedes pour depister et traiter des leucemies resultant d'anomalies chromosomiques de la region all-1 Download PDF

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WO1994026930A1
WO1994026930A1 PCT/US1994/004496 US9404496W WO9426930A1 WO 1994026930 A1 WO1994026930 A1 WO 1994026930A1 US 9404496 W US9404496 W US 9404496W WO 9426930 A1 WO9426930 A1 WO 9426930A1
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Carlo Croce
Eli Canaani
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Thomas Jefferson University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the field of methods for diagnosis and treatment of human leukemias wherein hematopoietic cells of patients have translocations in a small region of chromosome 11 designated as ALL-l. Diagnostics and therapeutics based on nucleic acid and amino acid sequences are 10 provided.
  • Chromosome translocations have been shown to play an important role in the pathogenesis of human leukemias and lymphomas by either activating cellular protooncogenes or by leading to the formation of chimeric genes capable of transforming hematopoietic cells.
  • Erikson et al .
  • C L chronic myelogenous leukemia
  • Translocations t (1,-19) , t(15;17) , and t(6;9) are other examples of gene fusions, involving in the first two cases transcription factors (Nourse et al . , Cell 1990, 60, 535-545; Kamps et al . , Cell 1990, 60, 547-555; Kakizuka et al .
  • the alternative molecular consequence of translocations is deregulation of protooncogenes by their juxtapositioning to an enhancer or promoter which is active in the type of cell from which the tumor arises.
  • the immunoglobulin (Ig) and T cell receptor (TCR) enhancers participate in at least 15 different translocations associated with Burkitt lymphoma, chronic ly phocytic leukemia, follicular lymphoma, mantle cell lymphoma, and acute T or B cell leukemia.
  • Ig immunoglobulin
  • TCR T cell receptor
  • Chromosomal region llq23 has been shown to be involved in different chromosomal translocations in human acute leukemias of different hematopoietic lineages. Ilq23 chromosome abnormalities have been reported in acute lymphoblastic leukemia and in acute nonlymphoblastic leukemia
  • Chromosome 11 band q23 is frequently rearranged in acute lymphocytic (ALL) , in acute myelomonocytic (AMMOL) , acute monocytic (AMOL) and acute myeloid (AML) leukemias, mostly in reciprocal exchanges with various translocation partners.
  • ALL acute lymphocytic
  • AMMOL acute myelomonocytic
  • AMOL acute monocytic
  • AML acute myeloid leukemias
  • Reciprocal translocation between llq23 and chromosomal regions 9p22, 6q27, lp21, 2p21, lOpll, 17q25 and 19pl3 are found in 5-6% of AML.
  • Heim and Mitelman, supra are found in 5-6% of AML.
  • interstitial deletions in llq23 have been detected both in ALL and AML.
  • Reciprocal translocations between chromosome region llq23 and chromosomal regions 9p22, 6q27, lp21, 2p21, lOpll, 17p25 and 19pl3 are found in 5-6% of ANLL.
  • t(4;ll) chromosome translocation In clinical terms, rearrangements of llq23, in particular the t(4;ll) chromosome translocation, have some distinct features.
  • the patients are often quite young; t(4;ll) accounts for the vast majority of cytogenetically abnormal ALLs in infants.
  • the leukemic cells show both B-cell and myeloid marker (Stong et al . Blood 1986, 67, 391-397) and the disease is consequently considered "biphenotypic.
  • the leukemic cells have a CD10-/CD19+ early B cell precursor phenotype and most of them express a myeloid associated antigen (CD15) ; Pui et al . , Blood 1991, 77, 440-447. Myelomonocytic and biphenotypic leukemias carrying the t(4;ll) aberration have also been reported; Nagasaka et al . , Blood 1983, 61, 1174-1181.
  • the cDNA sequence of the ALL-l gene on chromosome 11 is provided.
  • a partial sequence of the AF-4 gene is also provided in the context of the sequences of two reciprocal endproducts of a translocation.
  • Amino acid sequences corresponding to the cDNA sequences of the entire ALL-l gene and the partial sequence of the AF-4 gene, and sequences relating to chimeric genes formed by chromosome translocations with chromosome 4, 9 and 19, respectively, are provided.
  • Probes are provided for detecting chromosome abnormalities involving the ALL-l gene on chromosome 11, including probes for detecting chimeric genes generated by translocations.
  • Monoclonal antibodies for diagnosis and treatment and antisense oligonucleotides for treatment of acute leukemias are also described.
  • Figure 1 is a drawing depicting a physical map of YAC B22B, which has been described in Rowley et al. , Proc. Natl . Acad. Sci . USA 1990, 87, 9358-9362.
  • ura and trp correspond to the termini of the vector.
  • a 40 kb segment located towards the ura end and lacking No I and MluI sites is not included in the map.
  • Pulse field analysis indicates two or three Sfil sites located to the left of cosmid 43.
  • Figure 2 is a photograph showing the results of Southern blot analysis of tumor D ⁇ As . Blots were hybridized to the radiolabeled 0.7 kb Ddel fragment derived from the terminus of cosmid 53. Aliquots of 10 ⁇ g were analyzed.
  • Figure 3 is a drawing showing mapping of tumor breakpoints .
  • the internal N ⁇ tl fragment of YAC is shown in the same orientation as in Figure 1.
  • the dotted line represents a region not cloned in the cosmids. Restriction sites within this region are deduced from the size of the relevant germline fragments detected in genomic Southern blots using the indicated probe. Additional EcoRV and Xbal sites are not shown. Some of the samples were not analyzed with BamHI . Lines below the map correspond to the smallest genomic fragments found rearranged.
  • the breakpoint cluster region is believed to span approximately the region encompassed by the two nearest BamHI sites flanking the arrow; more specifically, the breakpoint cluster region is believed to span exons 6-12 illustrated in Figure 10.
  • Figure 4 is a photograph showing the results of Northern blot analysis of RNA from cell lines and a primary leukemia using pooled probes. 10-20 ⁇ g aliquots of total RNA were analyzed on a formaldehyde gel. Following hybridization, blots were washed in a solution containing 0.1% SSC and 0.1% SOS at 700. RNAs were obtained from: a) K562 cells; b) the glioblastoma T98G cell line; c) the SupB pre B ALL cell line; d) the MV4;11 cell line; and e) a patient with t(9;ll) .
  • Figure 5 is a photograph showing the results of Southern blot analysis of DNAs from primary tumors and cell lines with llq23 abnormalities using a modified 0.5 kb Ddel probe.
  • the germline Ba.mHI and Xbal fragments are of 9 and 12 kb, respectively.
  • Figure 6 is a photograph showing the results of Northern blot analysis of RNAs from cell lines using a 1.5 kb EcoRI probe generated from cosmid 20. Lanes included SK DHL (a) ; KC122 (b) ; MV 4;11 (c) ; T98G (d) ; All-1 (e) ; Bl (f) ; K562 (g) ; Jurkat (h) ; GM607 (i) ; 697 (j) ; RS4;11 (k) ; GM1500 (1) ; LNCaPFGC (m) ; PC3 (n) . 28S and 18S indicate migration of ribosomal RNA.
  • Figure 7 shows physical maps of ALL-l cD ⁇ A and gene.
  • Figure 8 shows nucleotide sequence and predicted amino acid sequence of ALL-l cD ⁇ A.
  • Figure 9 depicts homology between ALL-l and Drosophila trithorax (D. Trx) proteins (top and center) , and the structure of ALL-l zinc finger-like domains (bottom) . Bars indicate identical residues. One dot and two dots indicate first and second degree conservative differences, respectively.
  • Figure 10 A-C shows exon-intron structure of ALL-l breakpoint cluster region Figure 10A and partial sequence of the two reciprocal ALL-l/AF-4 fused transcripts (Figure 10B and Figure IOC) .
  • exons containing the zinc finger- like domains (8-12) are represented by cross-hatched boxes.
  • t(4;ll) breakpoints shown (arrowheads in Figure 10A) , included are those of the MV4;11 (MV) , RS4;11 (RS) , and Bl (Bl) cell lines.
  • CL. and I.V. represent leukemic cells with t(4,-ll) from two patients.
  • B, R, G, X, H correspond to sites for the enzymes BamHI, .EcoRI, Bglll, Xbal, and Hindlll, respectively.
  • small and large letters represent introns and exons, respectively. Cytosine in position 4141 of ALL-l sequence ( Figure 2) is replaced by thymidine in clone 25, resulting in alteration of Leucine into Phenylalanine ( Figure IOC) .
  • Figure 11 A-E shows the non ALL-l sequences within the fused RNAs unique to cells with t(4;ll) chromosome translocations (Figure 11A-C) which originate from chromosome 4 ( Figure 11D and HE) .
  • ALL-l cD ⁇ A probe ( Figure HA) , to non ALL-l sequences from cD ⁇ A clone 16 ( Figure 11B) , and to non ALL-l sequences from cD ⁇ A clone 25 ( Figure 11C) .
  • ALL-l is a Philadelphia-chromosome positive cell line (B cell leukemia) lacking Hq23 aberrations (Erikson et al . , Proc Natl . Acad . Sci . USA 1986, 83, 1807- 1811) .
  • K562 originated from chronic myelogenous leukemia (Lozzio, CB and Lozzio, BB, Blood 1975, 45, 321-324) .
  • SKDHL is a B cell lymphoma cell line (Saito et al . , Proc . Natl . Acad . Sci . USA 1983, 80, 7476-7480) .
  • the second and third probes were also used in hybridization to Southern blots ( Figure 11D and HE, respectively) with D ⁇ As from Chinese hamster ovary (CHO cells and CHO cells containing chromosome 4 (CHO/4) .
  • "Fused 1" and "fused 2" correspond to the altered ALL-l RNAs of 14 kb and 12.7 kb, respectively.
  • Figure 12A-C depicts the genomic analysis of the t(6:ll) (q27:q23) chromosome translocation.
  • FIG. 12B Chromosome 6-specific probe XRO .5 detects DNA rearrangement in the bone marrow from a patient, whose karyotype showed Hq23 deletion;
  • Figure 12C Sequence of the t(6;ll) breakpoint region. Cen and Tel denote the direction of the telomeres and centromeres of the two chromosomes . Open vertical boxes represent defined exons. Restriction sites: B, BamHI, H., HindHI, G, Bglll; Rm, EcoRI and X, Xbal.
  • Figure 13A-C shows the cloning and sequencing of AF-6 cDNA and of ALL-l/AG-6 fusion transcript.
  • Figure 13A AF-6 cDNA clones. Dashed lines indicate different sequences possibly representing alternative non-coding exons. Restriction sites: A, Apal; B, BamHI,-, H, HindHI and S, Sad .
  • Figure 13B Predicted amino acid sequence of AF-6 cDNA coding region. Arrow indicates the RNA fusion point.
  • Figure 14 shows a comparison of the GLGF repeat within the AF-6 protein to GLGF repeats of other patients.
  • GLGF repeats are the third GLGF in human ZO-1 (ZO-1 3) ; the second GLGF in rat PSD95 (PSD95 2) , and the third GLGF in Drosophila large disc tumor suppressor gene (dlq3) .
  • Bold amino acids are consensus amino acids conserved among the four proteins.
  • Figure 15 depicts a Northern analysis of AF-6 RNA in human cell lines. 5-10 ⁇ g of polyadenylated RNA were analyzed on agarose gel containing formaldehyde. RNAs were obtained from the lines KC122, K562, B-l, MV4;11, SKDHL, T98G, 293 (a-g, respectively) .
  • Figure 16A and 16B shows genomic analysis of the t(ll,-17) chromosome translocation.
  • Figure 16A Physical map of the genomic junction of patient GUS [der (17)] and a map of the corresponding normal region (chr. Hq23) . Numbered open boxes in the top line represent ALL-l exons. Darkened segment of der (17) correspond to chromosome 17 sequences, and open box therein represents an exon. Fragment Rl .7 was used as a probe for the genomic Southern analysis as well as for cDNA screening. Cen and Tel show directions of the centromeres and telomeres, respectively.
  • R EcoRI; H, HindHI; B, BamHI; G, Bglll, X, Xbal.
  • Figure 16B Southern genomic analysis of a DNA from patient GE with AML and t(ll;17) , and a normal DNA (lanes b and a, respectively) . DNAs were digested with EcoRV and hybridized with the Rl.7 probe. Germline fragment is 18 kb.
  • Figure 17A-C shows cloning and sequencing of AF-17 cDNA and of the junction within ALL-l/AF-17 fusion transcript.
  • Figure 17A Physical map of AF-17 cDNA clones. Restriction sites: S, Sad; H, HindHI; H2, Hindi. Initiation (ATG) and termination (TAA) are shown by arrows.
  • Figure 17B Predicted amino acid sequence of AF-17 protein. Cysteines within the cysteine-rich region at the N-terminus are underlined. Also underlined is the leucine zipper at positions 729-764. Arrow indicates point of fusion with the ALL-l protein.
  • Figure 17C All-l/AF-17 RNA junction cloned from the leukemic cells of patient GUS.
  • Figure 18A and 18B depicts ho ology between the AF-17 protein and the human Brl40 (peregrin) protein.
  • Figure 18A Alignment of AF-17 and Brl40 cysteine-rich domains. Bars indicate identical residues; one dot and two dots indicate first and second degree conservative differences, respectively.
  • Figure 18B Potential zinc fingers within the cysteine-rich domain of AF-17.
  • Figure 19 shows Northern analysis of AF-17 RNA in human cell lines. 5-10 ⁇ g of polyadenylated RNA were analyzed on agarose gel containing formaldehyde . RNAs were obtained from the cell lines KCl-122, MV4;11, ALL-l, GM-607, B 1, 380, PC3, GM 1500, K562, T93G, 679 (a to j, respectively) .
  • Figure 20 depicts landmarks, common motifs and homologous sequences within the partner proteins AF-4, AF-9, ENL, AF-6 and AF-17, and within the ALL-l protein. Arrows indicate fusion points between ALL-l and the partner proteins. Striped regions in AF-9 and ENL indicate domains of highest homology between the two proteins. NTS, nuclear targeting sequence, LZ, leucine zipper, MTase, methyl transferase.
  • Figure 21A and 21B shows use of the B859 probe in detecting ALL-l abnormalities.
  • Figure 21A The B859 probe and the breakpoint cluster region of the ALL-l gene (BCRHq23) . Numbered boxes are the exons of the ALL-l gene. Thin lines display the subclones used for sequencing. Cen. and Tel. denote the centromere and telomere.
  • Figure 21B Southern analysis of the ALL-l gene rearrangements in patients with acute leukemia. Patient's DNA samples were digested with BamHI and probed with the B859 probe. Numbers in each lane correspond to the case numbers in Table 2.
  • Figure 22 shows the nucleotide sequence of the breakpoint cluster region within the ALL-l gene. The predicted amino acid sequences of each exon are shown under the corresponding nucleotide sequences. A consensus sequence for topoisomerase II recognition site is underlined.
  • Figure 23 is a schematic representation of the exons, Alu repeats, and the breakpoints in the breakpoint cluster region in the ALL-l gene. Filled boxes are exons. Alu repeats are shown as open boxes. Arrows point to the positions of the breakpoints with their corresponding case numbers presented in Table 2. Hatched box represents a 130 bp novel repetitive sequence .
  • Figure 24a and 24b shows Southern analysis of ALL-l gene rearrangements in adult AML patients without cytogenetic evidence of Hq23 translocations.
  • the label above each lane corresponds to a unique patient identification number taken from (Caligiuri et al . , Cancer Res . 1994 54, 370-373) .
  • Patients nos . 23 and 24 had trisomy 11 as a sole cytogenetic abnormality whereas patient no. 1 had a normal karyotype. Arrows indicate rearranged bands. N, normal control.
  • Figure 24a Blots examined with the B859 probe.
  • B859 is a cDNA probe (Caligiuri et al . , Cancer Res .
  • Figure 25a-c shows the structure of partial duplication of the ALL-l gene.
  • Figure 25a Restriction enzyme maps of lambda clones ( ⁇ 23 and ⁇ 24) corresponding to rearranged BamHI fragments from two AML patients with trisomy 11. Boxes represent ALL-l exon positions determined by subcloning and partial DNA sequence analysis. The junction point of the duplication is indicated by the juncture of the black and shaded bars. Position of the SASl probe is shown. B, BamHI; R, EcoRI; H, HindHI; X, Xbal.
  • Figure 25b Proposed structure of the partially duplicated ALL-l gene contains a direct tandem duplication spanning exons 2-6.
  • Figure 25c DNA sequence across the junction points of clones ⁇ 23 and ⁇ 24 are aligned with sequences from introns 1 and 6 of the ALL-l gene. ⁇ 24 has a 2 bp N-segment. Heptamer-like signal sequences (Akira et al . , Science 1987 238, 1134-1138) near the junction points in both clones are underlined. Nonamer-like signal sequences are not present.
  • Figure 26a and b shows RNA-PCR analysis of trisomy 11 patient samples.
  • Figure 26a Agarose gel of RNA-PCR products
  • the ALL-l gene located at human chromosome 11 band q23 is rearranged in acute leukemias with interstitial deletions or reciprocal translocations between this region and chromosomes 1, 2, 4, 6, 9, 10, 15, 17 or 19.
  • the gene spans approximately 100 kb of DNA and contains at least 21 exons. It encodes a protein of approximately 4,000 amino acids containing three regions with homology to sequences within the Drosophila trithorax gene including cysteine-rich regions which can be folded into six zinc finger-like domains.
  • the breakpoint cluster region within ALL-l spans approximately 8 kb and encompasses several small exons (including exons 5-12) , most of which begin in the same phase of the open reading frame .
  • the t(4;ll) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-l and from a gene on chromosome 4.
  • This gene on chromosome 4 is termed "AF-4" while the chimeric gene resulting from the t(4;ll) translocation is termed "ALL-l/AF-4. " It is believed that the Hq23 abnormality of translocation with 4q21 gives rise to one or two specific oncogenic fusion proteins.
  • the t(9;ll) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-l and from a gene on chromosome 9.
  • This gene on chromosome 9 is termed "AF-9” while the chimeric gene resulting from the t(9;ll) translocation is termed "ALL-l/AF-9. " It is believed that the Hq23 abnormality of translocation with 9p22 gives rise to one or two specific oncogenic fusion proteins.
  • the t(ll;19) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-l and from a gene on chromosome 19. This gene on chromosome 19 is termed "ENL” while the chimeric gene resulting from the t(ll;19) translocation is termed "ALL-1/ENL.” It is believed that the t(ll;19) translocation gives rise to one or two specific oncogenic fusion proteins.
  • the gene on chromosome 17 is termed AF-17 and the chimeric gene resulting from the t (11:17) translocation is termed ALL-l/AF-17.
  • a DNA fragment which detects DNA rearrangements by Southern analysis in the majority of patients with t(4;ll) , t(9;ll) and t(ll;19) chromosomal aberrations has been cloned from chromosome 11.
  • This locus is referred to as ALL-l for acute lymphocytic leukemia, although the same locus is also involved in acute myelomonocytic, myelogenous and monocytic leukemias carrying translocations involving Hq23.
  • DNAs and RNAs were extracted from cell lines and primary tumors by conventional methods. Southern and Northern analysis were performed as described in Shtivelman et al . , Na ture 1985, 315, 550-554) .
  • cosmids were, digested with a variety of restriction enzymes, and analyzed by Southern blotting for fragments which do not react with radiolabeled total human DNA. End fragments of cosmids were identified by hybridizing cosmids' digests to radiolabeled oligonucleotides corresponding to the recognition sequences for T7 and T3 RNA polymerases . If the end fragments contained human repeats, they were isolated, digested with frequent cutters and analyzed as described above. The 0.7 kb Ddel probe was thus obtained from a terminal 3.5 kb Ec ⁇ RV fragment of cosmid 53.
  • the insert was partially digested by a combined application of dam methylase and the restriction endonuclease Mbol, Hoheisel et al . , Nuc . Acid Res . 1989, 17, 9571-9582. Both enzymes act on the sequence GATC, but Mbol is unable to cut the methylated form. More than a hundred cosmid clones, detected with a probe for human repetitive sequences, were obtained. The cosmids were mapped by screening for those with sites for NotI and Mlul enzymes, and for those hybridizing to CD3, trp and ura probes. Some cosmids were established using unique (repeat free) probes obtained from termini of cosmids.
  • a 0.7 kb Ddel fragment derived from the terminus of cosmid 53 detected rearranged fragments in tumor D ⁇ As digested with EcoRV, Xbal, or Bair-HI . Examples of these analyses are shown in Figure 2.
  • the leukemic cells from patients A.G., E.C, A.L., B.H., I.B., G.F., P.P., and V.S. contained novel EcoRV or Xbal fragments of various sizes. This probe detected rearrangements in 6/7, 4/5, and 3/4 patients with the t(4;ll) , t(9;ll) and t(ll;19) translocations, respectively.
  • pooled unique fragments from cosmid 20 were labeled, together with the terminal fragment of cosmid 53, and were used to probe RNAs from cell lines and patients with or without Hq23 translocations (Figure 4) .
  • the pooled probe detected 5 kb and 10 kb RNA species in the K562, glioblastoma T986 and Sup B cell lines (lanes a, b, c) . It also hybridized with a 5 kb RNA from patients with t(4;ll) , t(9;ll) , and t(ll;19) ( Figure 4, lanes d, e,) . In another patient with t(4;ll) the probe detected the 10 kb RNA species alone.
  • tumor DNAs from the leukemic cells of patients with t (6,-H) (q27;q23) , t (1;11) (p34 ;q23) , t (10;11) (pll-15;q23) and del (11) (q23) were digested with BamHI, Xbal, EcoRV and HindHI enzymes and subjected to Southern analysis using the modified 0.5 kb Ddel fragment as a probe.
  • This probe was obtained from the 0.7 kb Ddel probe by digestion with Alul, which ultimately improved performance by removing a 0.24 kb internal fragment that had caused a higher background in Southern analyses .
  • the internal fragment and the two end fragments were electrophoresed to isolate the two terminal fragments, which were then ligated to form a 0.5 kb fragment which was cloned into a plasmid vector.
  • Results of Southern blotting are shown in Figure 5. Rearranged fragments were found in the DNAs of patients with t(6;ll) , t(l;ll) and t(10,-ll) (lanes a, d, e, respectively) and in two patients
  • segments of cosmid 20 found fully or partially free of repetitive sequences were examined as probes to polyadenylated RNAs obtained from a variety of hematopoietic and nonhematopoietic cell lines.
  • Three ALL cell lines, MV 4;11, RS 4;11 and Bl containing the t(4,-ll) chromosome translocation were included in the analysis. These three cell lines had rearrangements at the breakpoint cluster region, as shown in Figure 5, lanes b and c.
  • a 1.5 kb EcoRI DNA segment generated from cosmid 20 was used as a probe and identified a 12.5 kb RNA in all cell lines ( Figure 6) .
  • the breakpoint in the RC-K8 cell line containing the t(ll;14) (q23;q32) is apparently telomeric to the locus discussed here.
  • other unidentified loci on chromosome 11 could be involved.
  • the ALL-l locus might also be affected in these patients, but this may occur at a different site.
  • RNA species of similar size have recently been reported by others. For example, Ziemin-van der Poel et al . , Proc . Natl . Acad. Sci . USA
  • (#1) which is derived from the same general location, detects predominantly the 12.5 kb species. While the instant probe detects 11 kb transcript solely in leukemic cells with the t(4,-ll) chromosome translocation, the Ziemin-van der Poel et al . study identifies an 11 kb mRNA in the RS4;11 cell line, as well as in small amounts in all cells tested. The results show, however, a clear qualitative alteration in expression of a region adjacent to the breakpoint cluster region on chromosome 11 in cells with the t(4;ll) chromosome translocation.
  • telomeric to the cluster region detected two additional transcripts of 11.5 and 11 kb in the RS 4;11 cell line, as well as in all hematopoietic and nonhematopoietic cells tested (Ziemin-van der Poel et al . , Proc . Natl . Acad. Sci . USA 1991, 88, 10735-10739) .
  • Translocation junction fragments were cloned from leukemic cells with t(4;ll) and showed clustering of the breakpoints in an area of 7-8 kb on chromosome 4.
  • Sequencing analysis indicated heptamer and nonamer-like sequences, associated with rearrangements of immunoglobulin and T cell receptor genes, near the breakpoints. These sequences suggested a direct involvement of the VDJ recombinase in the Hq23 translocations.
  • RNAs transcribed from the der (11) and der (4) chromosomes were analyzed using segments of cloned DNAs as probes. Probes from both sides of the region identified a major transcript of 15-16 kb (previously estimated as 12.5 kb) (Cimino et al . , Cancer Research 1991, 51, 6712- 6714; Cimino et al . , Cancer Research 1992, 52, 3811-3813) in cells with or without Hq23 abnormalities. The gene coding for these RNAs was termed ALL-l. Leukemic cells with the t(4;ll) chromosome translocation contained, in addition to the normal species, shorter RNAs transcribed from the der (11) and der (4) chromosomes .
  • ALL-l RNA was extended to clone and sequence ALL-l RNA, to further characterize the ALL-l gene, and to identify chimeric transcripts produced in cells with the t(4,-ll) chromosome translocation. Structure of the ALL-l gene and cDNA
  • a human fibroblast cDNA library and a K562 cDNA library were screened (Chu et al . , EMBO J. 1990, 9, 985-993; Shtivelman et al . , Nature 1985, 315, 550-554) . Positive clones were used as probes for further screening.
  • clones mg 11.1, cl4, and gc3 were obtained from a genomic DNA library made in the EMBL-3 vector (Stratagene) . Restriction enzyme mapping of the cDNA and genomic clones and analysis of the hybridization pattern of cDNA fragments to genomic DNA indicated that the ALL-l gene is composed of a minimum of 21 exons, some of them (6-12) very small (shorter than 150 bp) . The first intron was found to be the largest, spanning approximately 35 kb of DNA.
  • the nucleotide sequence of ALL-l cDNA was determined using an automatic sequencer (ABI) .
  • the sequence revealed a single long open reading frame predicting a protein of approximately 4,000 amino acids with molecular weight of approximately 400,000 Daltons ( Figure 8) .
  • GenBank and SWISS data bases were screened by the FASTA program.
  • Nucleotides 9353-9696 were found to be nearly identical to an anonymous sequence (EST00626) cloned from human fetal brain cDNA library (Adams et al. , Nature 1992, 355, 632- 634) .
  • the third region of homology constitutes the extreme C-terminus of the two proteins; both species end in an identical sequence.
  • the first homology region is cysteine-rich and contains sequence motifs analogous to four zinc finger domains (3-6) within the trithorax gene (Mazo et al . , supra) .
  • the second region of homology is also cysteine-rich and corresponds to zinc fingers 7 and 8 of the Drosophila gene.
  • the human putative zinc finger structures are shown at the bottom of Figure 9.
  • the multiple conserved cysteines and histidines at the 3' end of the motifs allow two or three arrangements of the putative fingers.
  • the structure of these cysteine-rich domains appears to be unique to the trithorax and ALL-l genes.
  • RNAs resulting from the t(4;ll) chromosome translocations Clustering of t(4;ll) breakpoints has previously been found within a small segment of the ALL-l locus (Cimino et al . , Cancer Research 1991, 51, 6712-6714; Cimino et al . , Cancer Research 1992, 52, 3811-3813) . This region includes 7 coding exons (6-12) containing 74, 132, 114, 147, 96, 121, and 123 bp respectively. Exons 8-12 contain four zinc finger motifs. Exons 7-11 all begin in the first nucleotide of a codon.
  • t(4;ll) translocations should result in fusion of the ALL-l gene to the gene aforementioned and, consequently, in production of two reciprocal chimeric RNAs .
  • a cD ⁇ A library was constructed from R ⁇ A extracted from the RS4;11 leukemic cell line established from a patient with the t(4;ll) chromosome translocation (Stong, RG, and Kersey, JH, Blood 1985, 66, 439-443) .
  • This RS4;H cD ⁇ A library was constructed by treating polyadenylated RNA with 1 mM methyl mercury for 10 minutes at room temperature, followed by neutralization with 10 mM mercaptoethanol and alcohol precipitation.
  • cD ⁇ A was prepared by using the Time Saver kit (Pharmacia) and was cloned into the lambda ZAP II vector (Stratagene) .
  • the library (2 X 10 6 clones) was screened with a probe composed of exons 3-13. Twenty positive clones were purified and mapped. Two clones varied from normal ALL-l cDNA and were further analyzed by sequencing.
  • Clone 16 contained normal ALL-l sequences 3' to the beginning of exon 9. 5' to this position, ALL-l information was substituted with a new DNA fragment composed of an open reading frame (ORF) that joins in phase the rest of ALL-l ORF ( Figure 10B) .
  • Clone 25 had a reciprocal configuration in which exon 7 of ALL-l is linked to a new DNA segment containing an open reading frame. Here again, the two ORFs are joined in phase ( Figure IOC) .
  • RNAs specific to cells with the t(4;ll) chromosome translocation Two such transcripts were identified: a 14 kb RNA (previously estimated as 11.5 kb) containing 3' ALL-l sequences and a 12.7 kb RNA (previously estimated as 11 kb) hybridizing to 5' ALL-l probe. These RNAs were transcribed from chromosome derivatives 4 and 11, respectively.
  • RNAs from cell lines with or without the t(4;ll) chromosome translocation As a control, the RNAs were first hybridized to 3' ALL-l cDNA probe which detected the major normal transcript of 15-16 kb
  • Clone 16 probe identified a 9.5 kb RNA in all cells examined and a 14 kb transcript in RS4;H, MV4;H and B-l cells ( Figure HB) . It was concluded that clone 16 originated from the 14 kb altered ALL-l transcript and that the non-ALL-1 sequence within this RNA is expressed in human cells as a 9.5 kb transcript, which corresponds to the normal AF-4 transcript on a non-rearranged chromosome 4.
  • clone 25 originated from the second altered 12.7 kb ALL-l
  • ALL-l gene Cloning and sequence analysis of the ALL-l gene indicates that it encodes an unusually large protein of 4,000 amino acids with a mass of approximately 400 kD .
  • the striking feature of the protein is its homology to the Drosophila trithorax gene. The homology is reflected in three ways. First, the transcripts and proteins have a similar size; the Drosophila gene is transcribed into a 15 kb RNA encoding a protein of 3759 amino acids (Mozer, BA, and David, IB, Proc . Na tl . Acad. Sci . USA 1989, 86, 3738-3742; Mazo et al . , Proc . Natl . Acad . Sci . USA 1990, 87, 2112-2116) .
  • the trithorax gene in Drosophila acts to maintain spatially-restricted expression patterns of the Antennapedia and Bithorax complexes during fruit fly development (Ingham, PW, Cold Spring Harbor Symp . Quant . Biol . 1985, 50, 201-208) .
  • Trithorax activates transcription of multiple genes of the two complexes and, as such, counteracts the activity of Polycomb group genes which act as repressors of transcription for the same genes (McKeon, J and Brock, HW, Roux' s Arch . Dev. Biol . 1991, 199, 387-396) .
  • antibodies to the protein react with specific regions of the chromatin in the salivary glands of Drosophila.
  • ALL-l gene is a transcription factor and that it is involved in regulation of genes controlling human development and/or differentiation. While expression of ALL-l during embryonic development has not yet been investigated, the isolation of ALL-l sequences from a human fetal cDNA library indicates transcription of the gene during fetal development. Previous studies (Cimino et al . , Cancer Research 1992, 52, 3811-3813) demonstrated ALL-l RNA in a variety of hematopoietic cell lines, as well as in tumors originating from precursors of epithelial and glial cells.
  • t(4;ll) chromosome translocation cleaves the ALL-l gene within the coding region and results in fusion of the open reading frames of ALL-l and a gene on chromosome 4 (termed AF-4) in phase.
  • the breakpoints on chromosome 11 cluster in a region containing several small exons, 5 of them (exons 7-11) begin in the first letter of a codon. Splicing from the same exon on chromosome 4, adjacent to the breakpoint in RS4;H, to each one of the five exons on chromosome 11 will retain the two open reading frames fused in phase.
  • Two chimeric proteins from the 12.7 and 14 kb R ⁇ As are predicted for cells with the t(4;ll) chromosome translocation.
  • the lack of information about the normal AF-4 protein precludes at this time the determination if it is also a transcription factor that exchanges functional domains with ALL-l to give a chimeric transcription factor. This occurs in the t(l;19) and t(15,-17) chromosome translocations (Kamps et al . , Cell 1990, 60, 547-555; ⁇ ourse et al . , Cell 1990, 60, 535-545; Kakizuka et al., Cell 1991, 66, 663-674; de The et al .
  • the present invention provides methods of diagnosis for human leukemia by providing a tissue sample from a person suspected of having acute lymphocytic, myelomonocytic, monocytic or myelogenous leukemia, and determining if there are breakpoints on chromosome 11 in the ALL-l locus.
  • the sequence of the ALL-l cDNA can be used to generate probes to detect chromosome abnormalities in the ALL-l breakpoint cluster region. These probes may be generated from both the sense and antisense strands of double-stranded DNA.
  • ALL-l probe refers to both genomic and cDNA probes derived from the ALL-l gene.
  • genomic probes capable of detecting chromosomal translocations involving the ALL-l breakpoint cluster region span sequences from at least 10 kb centromeric to at least 10 kb telomeric to the breakpoint cluster region, which has been shown to span at least exons 6-9, and may span exons 5-12 of the ALL-l gene. It is believed that cDNA probes capable of detecting chromosomal translocations involving the ALL-l breakpoint cluster region span sequences ranging from 2 kb centromeric to 2 kb telomeric to the breakpoint cluster region.
  • preferred embodiments of the present invention for detecting chromosomal abnormalities involving ALL-l provide genomic and cDNA probes spanning the chromosome 11 regions described above. cDNA probes are more preferred, and probes comprising the exons included in the breakpoint cluster region are most preferred.
  • Part or all of the ALL-l cDNA sequence may be used to create a probe capable of detecting aberrant transcripts resulting from chromosome 11 translocations.
  • the EcoRI probe for example, was derived from a genomic clone but its location lies within an exon.
  • preferred embodiments of the present invention for detecting aberrant transcripts provide cDNA probes spanning the ALL-l gene.
  • ALL-l/AF-4 sequences provided in SEQ ID NO: 23 and SEQ ID NO:24 can be used to create probes to detect t(4;ll) chromosome abnormalities and aberrant transcripts corresponding to t(4,-ll) translocations. Additional sequences (see below) include those specific for the ALL-l/AF-6, ALL-l/AF-9 and ALL- l/AF-17 chimeric genes. Also included in the invention and described below are specific ALL-l probes capable of detecting chromosomal abnormalities in the ALL-l gene irrespective of the nature of the fusion partner gene.
  • PCR Polymerase Chain Reaction
  • restriction fragment length analysis oligonucleotide hybridization using, for example, Southern and Northern blotting and in si tu hybridization.
  • PCR technology is practiced routinely by those having ordinary skill in the art and its uses in diagnostics are well known and accepted. Methods for practicing PCR technology are disclosed in PCR Protocols : A Guide to Methods and Applications, Innis, M.A. et al .
  • PCR technology allows for the rapid generation of multiple copies of DNA sequences by providing 5' and 3' primers that hybridize to sequences present in a DNA molecule, and further providing free nucleotides and an enzyme which fills in the complementary bases to the DNA sequence between the primers with the free nucleotides to produce a complementary strand of DNA.
  • the enzyme will fill in the complementary sequences between probes only if both the 5' primer and 3' primer hybridize to DNA sequences on the same strand of DNA.
  • one of the two probes can be generated from the ALL-l cDNA and one probe from the AF-4 gene.
  • RNA is isolated from hematopoietic cells of a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia, and cDNA is generated from the mRNA. If the cDNA of the chimeric ALL-l/AF-4 gene is present, both primers will hybridize to the cDNA and the intervening sequence will be amplified.
  • the PCR technology therefore provides a straightforward and reliable method of detecting the chimeric gene.
  • the preferred primers for PCR are selected, one from a portion of SEQ ID NO: 1, corresponding to the ALL-l cDNA, and one from a portion of either SEQ ID NO: 19 or SEQ ID NO: 22, corresponding to AF-4 gene sequences.
  • the sequences chosen from SEQ ID NO: 1 comprise at least a portion of SEQ ID NO: 20, which corresponds to exon 9, or SEQ ID NO: 21, which corresponds to exon 7.
  • diagnostic kits can be assembled which are useful to practice oligonucleotide hybridization methods of distinguishing chromosome 11 abnormalities from non-rearranged chromosomes 11.
  • diagnostic kits comprise a labelled oligonucleotide which hybridizes, for example, to the chimeric transcript that results from t(4;ll) translocations but which does not hybridize to nucleic acid transcripts not associated with aberrations.
  • diagnostic kits of the present invention comprise, for example, a labelled probe that includes ALL-l and AF-4 sequences which make up the chimeric transcript associated with t(4;ll) translocations.
  • probes comprise oligonucleotides having at least a portion of the sequence of the ALL-l/AF-4 gene of SEQ ID NO: 23 or SEQ ID NO: 24.
  • labelled probes of the oligonucleotide diagnostic kits according to the present invention are labelled with a radionucleotide.
  • the oligonucleotide hybridization-based diagnostic kits according to the invention preferably comprise DNA samples that represent positive and negative controls.
  • a positive control DNA sample is one that comprises a nucleic acid molecule which has a nucleotide sequence that is fully complementary to the probes of the kit such that the probes will hybridize to the molecule under assay conditions.
  • a negative control DNA sample is one that comprises at least one nucleic acid molecule, the nucleotide sequence of which is partially complementary to the sequences of the probe of the kit. Under assay conditions, the probe will not hybridize to the negative control DNA sample.
  • Probes useful as diagnostics can be used not only to diagnose the onset of illness in a patient, but may also be used to assess the status of a patient who may or may not be in remission. It is believed that emergence of a patient from remission is characterized by the presence of cells containing chromosome abnormalities. Thus, patients believed to be in remission may be monitored using the probes of the invention to determine their status regarding progression or remission from disease. Use of such probes will thus provide a highly sensitive assay the results of which may be used by physicians in their overall assessment and management of the patient's illness .
  • Antisense oligonucleotides which hybridize to at least a portion of an aberrant transcript resulting from chromosome 11 abnormalities involving the ALL-l gene are also contemplated by the present invention.
  • the oligonucleotide may match the target region exactly or may contain several mismatches.
  • molecules which bind competitively to RNA coded by, for example, the chimeric ALL-l/AF-4 gene, for example are envisioned for therapeutics.
  • Preferred embodiments include antisense oligonucleotides capable of binding to at least a portion of SEQ ID NO: 23 and SEQ ID NO: 24.
  • Preferred embodiments of the present invention include antisense oligonucleotides capable of binding to a region of the ALL-l/AF-4 mRNA corresponding to the ALL-l sequences which encode a peptide having homology with the Drosophila trithorax protein and antisense oligonucleotides capable of binding to a region of the mRNA encoding a zinc finger-like domain in the ALL-l protein.
  • oligonucleotide sequences shorter than 15 bases may be less specific in hybridizing to the target and may be more easily destroyed by enzymatic degradation. Hence, oligonucleotides having at least 15 nucleotides are preferred. Sequences longer than 21 nucleotides may be somewhat less effective in interfering with ALL-l expression because of decreased uptake by the target cell. Therefore, oligonucleotides of 15-21 nucleotides are most preferred.
  • oligonucleotide as used herein includes both ribonucleotides and deoxyribonucleotides, and includes molecules which may be long enough to be termed “polynucleotides . " Oligodeoxyribonucleotides are preferred since oligoribonucleotides are more susceptible to enzymatic attack by ribonucleotides than deoxyribonucleotides. It will also be understood that the bases, sugars or internucleotide linkages may be chemically modified by methods known in the art. Modifications may be made, for example, to improve stability and/or lipid solubility.
  • oligonucleotides of the present invention may be synthesized by any of the known chemical oligonucleotide synthesis methods. See for example, Gait, M.J., ed. (1984) , Oligonucleotide Synthesis (IRL, Oxford) .
  • antisense oligonucleotides hybridizable with any portion of these sequences may be prepared by the synthetic methods known by those skilled in the art . It is generally preferred to apply the therapeutic agent in accordance with this invention internally such as intravenously, transdermally or intramuscularly. Other forms of administration such as topically or interlesionally may also be useful. Inclusion in suppositories is presently believed to be likely to be highly useful. Use of pharmacologically acceptable carriers is also preferred for some embodiments .
  • the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • a pharmaceutical carrier such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • the liquid vehicles and excipients are conventional and commercially available. Illustrative thereof are distilled water, physiological saline, aqueous solution of dextrose, and the like.
  • the antisense oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques. For example, oligonucleotides have been successfully encapsulated in unilameller liposomes. Reconstituted Sendai virus envelopes have been successfully used to deliver RNA and DNA to cells (Arad et al . , Biochem . Biophy. Acta . 1986, 859, 88-94) .
  • the antisense oligonucleotides may be administered in an amount effective to result in extracellular concentrations approximating in vi tro concentrations described below.
  • the actual dosage administered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health and sex of the patient, the route of administration, and other factors.
  • the daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 1,000 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required.
  • the antisense oligonucleotides may be administered in amounts effective to kill leukemic cells while maintaining the viability of normal hematologic cells. Such amounts may vary depending on the nature and extent of the leukemia, the particular oligonucleotide utilized, the relative sensitivity of the leukemia to the oligonucleotide, and other factors. Concentrations from about 10 to 100 ⁇ g/ml per 10 5 cells may be employed, preferably from about 40 to about 60 ⁇ g/ml per 10 5 cells . Supplemental dosing of the same or lesser amounts of oligonucleotide are advantageous to optimize the treatment.
  • dosages from about 2 to about 20 mg antisense per ml of marrow may be effectively utilized, preferably from about 8 to 12 mg/ml. Greater or lesser amounts of oligonucleotide may be employed.
  • the present invention is also directed to monoclonal antibodies capable of binding to chimeric ALL-l/AF proteins including ALL-l/AF-4, ALL-l/AF-6, ALL-l/AF-9 and ALL-l/AF-17, and includes monoclonal antibodies capable of binding to a region of the protein having homology with the Drosophila trithorax protein and monoclonal antibodies capable of binding to a zinc finger-like domain.
  • Such monoclonal antibodies are useful as diagnostic and therapeutic agents for leukemias characterized by t(4;ll) , (t(6;ll) , t(9;ll) and t(H;17) translocations.
  • the present invention encompasses immunoassays for detecting at least portions of either the ALL- l/AF-4, ALL-l/AF-6, ALL-l/AF-9 and ALL-l/AF-17 proteins.
  • the instant invention contemplates diagnostic kits comprising a monoclonal antibody to at least a portion of the ALL-l fusion proteins listed above in combination with conventional diagnostic kit components.
  • the present invention is also directed to pharmaceutical compositions comprising monoclonal antibodies and a suitable pharmaceutical carrier, which are well known in the pharmaceutical art, and are described, for example, in
  • Polyclonal antibodies to the instant polypeptides are also within the ambit of the invention.
  • Such polyclonal antibodies may be produced using standard techniques, for example, by immunizing a rabbit or a rat with a protein or peptide of the invention, removing serum from the rabbit, and harvesting the resultant polyclonal antibodies from the serum.
  • the polyclonal antibodies may be used as an IgG fraction or may be further purified in varying degrees. Procedures for preparing, harvesting and purifying polyclonal antibodies are well known in the art, and are described, for example, in Methods in Immunology: A Laboratory Text for Instruction and Research, Garvey et al . , Ed., W.A. Benjamin, Reading MA, 1977, 3rd ed. , chapter 22, 24-30.
  • Example 1 provides further data for designing methods of diagnosing and treating acute lymphoblastic or nonlymphoblastic leukemia, particularly those involving a chimeric gene in t(4;ll) translocations.
  • the information provided in example 1 includes complete cDNA sequences encoding AF-4. These sequences may be used design probes of at least 15 nucleotides which are capable of identifying chromosome abnormalities within the ALL-l gene of chromosome 11. Examples of such probes comprise, an oligonucleotide sequence or derivatives thereof comprising at least a portion of SEQ ID NO:25 or SEQ ID NO:27. The procedures for using such probes are described above.
  • Example 2 Provide further data for designing methods of diagnosing and treating acute lymphoblastic or nonlymphoblastic leukemia, particularly those involving a chimeric gene in t(9;ll) translocations.
  • the information provided in example 2 may be used design probes of at least 15 nucleotides which is capable of identifying chromosome abnormalities within the ALL-l gene of chromosome 11. Examples of such probes may comprise at least a portion of SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34. Further, probes capable of identifying chromosome abnormalities within the AF-9 gene of chromosome 9 may be designed.
  • probes comprise an oligonucleotide sequence or derivatives thereof comprising at least a portion of SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34. The procedures for using such probes are described above.
  • the experiments reported in Examples 3 and 4 describe the cloning and sequencing of ALL-l/AF-6 and ALL-l/AF-17 genes, respectively.
  • the experiments reported in Example 5 describe a probe capable of detecting abnormalities in the ALL-l region irrespective of the nature of the fusion gene, and the experiments reported in Example 6 describe duplications of the ALL-l region in cells of some patients with leukemia.
  • the invention must be construed to include each of these genes, their products and probes derived therefrom as being useful for the diagnosis and treatment of patients with these types of leukemias.
  • each example must be construed to include the other named fusion genes as being useful in the methods and compositins of the invention.
  • a method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia involving a chimeric gene in t(9;ll) translocations may be performed by first providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia; then isolating RNA from the sample followed by generating cDNA from said RNA and amplifying a chimeric gene sequence in said cD ⁇ A which is generated by said translocation using a set of PCR primers if said chimeric gene is present such that detecting the presence of amplified DNA indicates the tissue sample is derived from an individual suffering from lymphoblastic or nonlymphoblastic leukemia involving a chimeric gene in t(9;ll) translocations.
  • the method may be performed using sets of primers which can be used to amplify a chimeric gene generated by the translocation.
  • primers can be designed, for example, using the sequence information in SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34.
  • primers include SEQ ID NO:39 and SEQ ID NO:40; SEQ ID NO:41 and SEQ ID NO:42; and SEQ ID NO:43 and SEQ ID NO:44.
  • Monoclonal antibody capable of binding to at least a portion of for example, the chimeric ALL-l/AF-9 protein may be produced by standard techniques.
  • Examples of such a monoclonal antibodies, which can bind specifically to at least a portion of the amino acid sequences encoded by SEQ ID NO: 9, SEQ ID NO:11 or SEQ ID NO:13, may be produced using peptides which comprise at least a portion of SEQ ID NO: 9, SEQ ID NO:11 or SEQ ID NO:13.
  • tissue sample containing hematopoietic cells from a person suspected of having acute lymphocytic or nonlymphoblastic leukemia is examined to detect the ALL-l/AF-9 chimeric protein or a portion of the chimeric ALL-l/AF-9 protein.
  • a monoclonal antibody capable of binding to at least a portion of the chimeric ALL-l/AF-9 protein is used.
  • the present invention provides antisense oligonucleotides capable of binding to at least a portion of the chimeric ALL-l/AF-9 mRNA.
  • antisense oligonucleotides include those capable of binding to at least a portion of SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34.
  • Method of treating acute lymphoblastic or nonlymphoblastic leukemia comprise administering an antisense oligonucleotide capable of binding to at least a portion of the chimeric ALL-l/AF-9 mRNA or, alternatively, administering a monoclonal antibody capable of binding to at least a portion of the chimeric ALL-l/AF-9 protein.
  • the formulation and administration of therapeutics are outlined above.
  • a third cDNA clone, k 1.1 represents another RNA variant probably resulting from alternative splicing; an in frame termination codon is present in this clone immediately 3' to the divergence point.
  • AF-4 encodes 2 or more proteins varying at their termini.
  • AF-4 contains an unusually long 3' untranslated region of 5 3kb. This region includes multiple AATAAA sequences located 20 nucleotides 5' of the poly A, as well as in several upstream positions; it also contains several stretches of T.
  • AF-4 protein sequence was searched for homology to other proteins and for the presence of motifs.
  • the sequence AKKRK at positions 811-815 matched the consensus nuclear targeting sequence - (RKTA) KK (RQNTSG) K- (Gomez-Marquez and Segada, 1988) .
  • AF-4 was relatively rich in serine (16%) and proline (11%) compared to the average frequency of these amino acids (7.1% and 4.6%, respectively) .
  • the protein encloses a nuclear targeting sequence
  • AF-9 protein is serine rich (20%) and includes a remarkable uninterrupted stretch of 42 serines at positions 149-190; it also contains proline at a frequency of 7% which is above the average frequency of 4.1%.
  • a homology search showed, unexpectedly, that the predicted protein shared high similarity with the ENL protein SEQ ID NO:31.
  • the latter is located on chromosome 19 and is fused to the ALL-1/HRX gene in t(ll;19) chromosome translocations.
  • the two proteins show 56% identity and 68% similarly.
  • the homology is highest within the 140 amino acids at the N terminus where the proteins are 82% identical, and 92% similar, and within the 67 amino acids at the C terminus where the corresponding values are 82% and 91%.
  • the der 11 chromosome was due to a break in the other ALL-l DNA strands within the intron flanked by exons 6 and 7, and to a breakage of the second AF-9 DNA strand within an intron located 5' of the AF-9 exon mentioned above.
  • RNA 11 is transcribed into an RNA corresponding to cDNA clone E2.
  • a BamHI-Stul cDNA probe detected some normal genomic fragments, which were also detected by the 0.4 kb Hindlll-Avall probe-derived from the genomic junction cloned from DNA of patient CO.
  • the AF-9 protein is linked at position 375 to the ALL-l moiety, while in patient FI the junction point is at amino acids 444 or 477 of AF-9.
  • the reading frames of the two genes are joined in phase.
  • Hq23 abnormalities are the multitude of chromosome partners participating in translocations with the ALL-l locus.
  • This promiscuity in partners for rearrangement and fusion could suggest that the only critical event in all these different translocations is the separation of a DNA binding domain (either the zinc fingers or the AT hooks in the ALL-l gene) from a positive or negative regulatory element, and that the proteins encoded by the partner genes solely provide initiation or termination codons .
  • High molecular weight DNAs from patients with t(9;ll) chromosome translocation were partially digested with Mbol enzyme and cloned into the EMBL-3 phage vector (Promega) .
  • the libraries were plated into the host bacteria CES200 (Wyman et al . , 1986) .
  • the libraries were screened with an ALL-l specific probe (Cimino et al . , 1992) and positive clones were mapped with restriction enzymes.
  • cytoplasmic RNA was extracted by standard techniques (Berger & Chirgwin, 1989) and polyadenylated RNA purified on an oligo dT column.
  • cDNA was prepared using the Timesaver kit of Pharmacia and cloned into the lambda ZAPII vector (Stratagene) . Construction of cDNA libraries from K562 or fibroblasts RNA was described (Shtivelman et al . , 1985; Chu et al . , 1990) .
  • AF-4 cDNA clones kl.l, kl.2, kll and kl2 originated from the K562 library and the clones kcl 6, kcl 10, and kcl 12 were cloned from the KC122 library.
  • AF-9 cDNA clones v4 and v7 were obtained from the fibroblasts library, and k 16 was cloned from the K562 library.
  • RNA from a patient FI Two micrograms of RNA from a patient FI were reverse transcribed in a reaction utilizing the AF-9 oligonucleotide TCCTCAGGATGTTCCAGATGT (SEQ ID NO:39) or the ALL-l oligonucleotide GGCTCACAACAGACTTGGCAA (SEQ ID NO:40) as primers.
  • the cDNAs were amplified with Taq 1 polymerase
  • the AF-9/ALL-1 RNA function of patient C() was obtained in a similar way using the ALL-l primer CAGCGAACACACTTGGTACAG (SEQ ID NO:43) for synthesis of cDNA and the same primer together with the AF-9 primer CAACGTTACCGCCATTTGAT (SEQ ID NO:44) for PCR amplification.
  • ALL-l primer CAGCGAACACACTTGGTACAG SEQ ID NO:43
  • AF-9 primer CAACGTTACCGCCATTTGAT SEQ ID NO:44
  • AML(M4) Her karyotype was 46XX, t(6;ll) (q27,-q23) in 20/20 of bone marrow cells.
  • Patient Ed was a male diagnosed as AML(M5) with a karyotype of 46 XY del(llq23) .
  • the cell lines used for RNA analysis included K562 and KC122 (erythroid and myeloid acute phase of chronic myeloid leukemia) (Lozzio et al . , Blood 1975 45, 321-324; and Kubonishi et al . , Int . J.
  • the rearranged genomic fragments of ALL-l patients 01 and Ed were cloned into the EMBL-3 phage vector (Promega) after partial digestion of the DNAs with the Mbol enzyme and size selection. Phage libraries were screened using a 0.86 kb Bam HI fragment derived from ALL-l cDNA and spanning exons 5-11. Normal genomic library was constructed in a similar way from normal white blood cell DNA. cDNA library was constructed utilizing a kit from Pharmacia. Cytoplasmic poly A-selected RNA was prepared from KC122 cells.
  • RNA samples were reverse transcribed utilizing the AF-6 oligonucleotide 5' ATC TGA ATT CTC CGC TGA CAT GCA CTT CAT AG 3' [SEQ ID NO:79] .
  • the cDNA was amplified using the same AF-6 primer together with the All-1 primer 5' ATC TGA ATT CTC CGC TGA CAT GCA CTT CAT AG 3' [SEQ ID NO: 80] .
  • Both primers contained cloning sites at their 5' termini.
  • the amplified products were cloned into the SK plasmid vector and sequenced.
  • cDNAs and genomic DNAs were excised from the phage vectors and recloned into the SK plasmid vector. Sequencing was performed using the ABI automatic sequencer. Sequence was analyzed using the FASTA, TFASTA and motifs programs.
  • a rearranged ALL-l segment was cloned from the genomic DNA of leukemic cells of patient 01. Mapping of this segment indicated that it originated from the der (6) chromosome (Fig.
  • DNA of the patient Ed with AML and the del(llq23) aberration contained a rearranged BamHI fragment of 12 kb (Fig. 12B) .
  • XRO-5 probe hybridized to human DNA within Chinese hamster cell hybrids containing human chromosome 6. This indicated that the cloned DNA spanned a breakpoint cluster region and that a cytogenetic pattern of del(llq23) could correspond to a t(6;ll) translocation.
  • AF-6 encodes a protein of 1612 amino acids.
  • cDNA clone K10 we find two additional amino acids - glutamic acid at position 101, and a lysine in position 139; both are probably due to alteration in splicing similar to those which we previously detected in ALL-l (Nakamura et al . , Proc . Natl . Acad . Sci . USA 1993 90, 4631- 4635; and Ma et al . , Proc . Natl . Acad . Sci . USA 1993 90, 6350- 6354) .
  • RT-PCR reactions on RNAs from patients 01 and Ed using ALL-l and AF-6 primers flanking the expected junction region.
  • AF-6 protein residue 745-925, shows 23.2% identity over 181 amino acids with the C-terminus of yeast myosin-1 isoform (Johnston et al . , J. Cell Biol . 1991 113, 539-551) .
  • AF-6 protein also shows high similarity, though low identity, (66% similarity plus identity) over amino acids 1000-1594 to amino acids 1400-1980 of the myosin heavy chain from Dictyostelium discoideum (Warrick et al., Proc . Natl . Acad. Sci . USA 1986 83, 9433-9437) .
  • this region is part of the tail domain which assumes, due to a high helical potential, a rod structure.
  • a striking homology was detected in the polypeptide spanning amino acids 997-1080.
  • a series of amino acids in this domain are conserved (Fig. 14) in three other proteins - in the human tight junction protein ZO-1 (Willott et al. , Proc .
  • RNAs extracted from several cell lines Fig. 15
  • An 8 kb transcript was detected in cell lines of myeloid (a) , erythroid (b) , lymphoid (c-e) , glia (f) and epithelial (g) origin.
  • myeloid a
  • erythroid b
  • lymphoid c-e
  • glia f
  • epithelial g
  • t(6,-ll) (q27;q23) translocation is one of the most frequent translocations involving Hq23. Cloning of the AF-6 gene involved in this abnormality would enable now the use of
  • AF-6 One of the main reasons for cloning AF-6 was to see if it is related to the partner genes AF-4, AF-9, and ENL. Among these, AF-9 and ENL are highly related. However, AF-6 showed no sequence homology to any of the three partner genes. Short domains rich in prolines, serines and acidic amino acids were the only motifs shared by the four genes. The C-terminus AF-6 showed homology to the tail domain of myosin-1 isoform from yeast and myosin heavy chain from Dictvostelium discoideum; this domain presumably confers the rod structure to the myosin protein.
  • AF-6 displays a remarkable homology to the GLGF repeat found in the ZO-1, PSD- 95 and dig proteins from human, rat, and Drosophila respectively.
  • the first and the third proteins are presumably homologous and are thought to play a role in signal transduction on the cytoplasmic surface of intracellular junctions (Willott et al . , Proc . Natl . Acad. Sci . USA 1993 90, 7834-7838; Woods et al . , Cell 1991 66, 451-464) .
  • the second protein localizes to synaptic junctions and is thought to be involved in synaptic signalling or organization (Willott et al., Proc . Natl . Acad .
  • AF-6 cytoplasmic or associated with membranes.
  • the presence of this domain in AF-6 raises the possibility that AF- 6 is not a nuclear protein. Indeed, unlike AF-4, AF-9 and ENL, AF-6 does not contain a nuclear targeting sequence.
  • AML patients GUS and GE showed the chromosome translocation t(ll;17) (q23;q21) in their leukemic cells.
  • the cell lines used for RNA analysis included K562 and KC1-22 (erythroid and myeloid acute phase of chronic myeloid leukemia) , MV4:H and B-l (ALLs with the 4:11 translocation) , 380, ALL-l, 697, GM607, (ALLs) , GM1500 (EBV transformed lymphoblastoid cell line) , T98G (glioblastoma) , PC3 (prostate carcinoma) , (Prasad et al . , Cancer 1993 53, 5624-5628; Licht et al., Na ture 1990, 346, 76-79)
  • the junction fragment of patient GUS was cloned from a library prepared from a partial digest of genomic DNA clones into the EMBL-3 phage vector.
  • the library was screened with a 0.86 kb BamHI cDNA probe spanning ALL-l exons 5-11.
  • cDNA libraries were prepared from ALL-l and KCl-22 cytoplasmic RNAs utilizing a kit manufactured by Pharmacia, and the lambda ZAPII vector of Stratagene. RT-PCR reaction was performed as described (Nakamura et al . , Proc . Na tl . Acad. Sci .
  • DNA from patient GUS with AML and t(ll;17) was partially digested with Mbol enzyme, and following size selection was cloned into the EMBL-3 phage vector.
  • the library was screened with a cDNA probe spanning the breakpoint cluster region.
  • a clone composed of a rearranged ALL-l segment was identified among positive clones. Comparison between the physical maps of this clone and the corresponding normal ALL-l DNA (Fig. 16A) indicated that ALL-l sequences upstream of exon 6 were substituted with new DNA; the latter was subsequently found to be derived from chromosome 17.
  • a 1.7 kb EcoRI fragment (R1.7) was found to be devoid of repetitive sequences. This fragment was used as a probe to analyze by the Southern technique DNA from a second patient (GE) with AML and the t(ll;17) aberration. In that DNA we detected an 11.6 kb rearranged EcoRV fragment (Fig. 16B, lane b) . This indicated that in both patients the breaks occurred in the same region on chromosome 17. Fragment Rl .7 was next used as a probe on cDNA libraries derived from RNAs of the cell lines KC1-22 and ALL-l.
  • AF-17 cDNA contains an open reading frame spanning 3279 nucleotides. The first ATG shows a good fit to a Kozak consensus sequence and is preceded by an in-frame termination codon. The predicted protein spans 1093 amino acids. It contains relatively high concentrations of serines, glycines, alanines, leucines and prolines (15%, 11%, 10%, 10%, 10%, respectively) often concentrated in short stretches. In addition, it has a glutamine-rich region (41%) between amino acids 935 and 984 (Fig. 17B) .
  • the same region shows high concentration of hydrophobic amino acids, in particular leucines.
  • domains rich in alanines Liicht et al . , Nature 1990, 346, 76-79] , glycines (Shi et al., Cell 1991 67, 377-388) , glutamines and prolines (Madden et al . , Science 1991 253, 1550-1553) were implicated in transcriptional repression.
  • regions with high concentration of serines and prolines (Gill et al . , Proc . Natl . Acad . Sci . USA 1993 91, 192-196) or glutamines intercalated with hydrophobic amino acids (Theill et al . , Nature 1989 342, 945-948) were found to be involved in transcriptional activation.
  • AF-17 showed 48% identity and 67% similarity to a region within the protein Brl40, previously named peregrin (Accession No. M91585) .
  • This domain is cysteine-rich in both proteins and can be arranged into three zinc fingers according to the consensus C - X 2 - C - X 10 _ 13 C- X 2 _ 4 - C (Fig. 18B) .
  • Related consensus sequences are present in the adenovirus E1A protein and in the steroid receptor superfamily.
  • the human Brl40 protein has a second cysteine- rich domain and is located in the nucleus; the function of this protein is unknown.
  • Inspection of AF-17 predicted protein sequence revealed a leucine zipper dimerization motif between amino acids 729 and 764 (Fig. 17B) . Unlike many leucine zippers, the one in AF-17 is not preceded by a basic region.
  • ALL-l/AF-17 fused gene is transcribed into a chimeric RNA
  • cDNA and genomic DNA sequence information to design primers for amplification by RT-PCR of a putative ALL-l/AF-17 RNA junction from the leukemic cells of patient GUS.
  • An amplification product was indeed found to contain the RNA junction (Fig. 17C) .
  • the open reading frames of the two genes were found to be linked in phase.
  • the t(ll;17) abnormality results in production of an RNA encoding a chimeric ALL-l/AF-17 protein.
  • the cloning and sequence analysis of the partner genes which recombine with ALL-l in Hq23 translocations provides information and reagents which can be used in the diagnosis, prognosis and monitoring of human acute leukemias.
  • this cloning enables construction of biologically active molecules, and might provide insights into the mechanism of leukemogenesis .
  • the most notable feature of AF-17 protein is the leucine zipper protein dimerization motif. Following the t(ll;17) chromosome translocation, this motif will be included in the ALL-l/AF-17 chimeric protein which is presumed to be the critical product of the aberration.
  • AF-17 Since the leucine zipper of AF-17 is not preceded by a basic region required for interaction with DNA, and because leucine zippers are found not only in transcription factors but also in other proteins with diverse functions, it is concurrently not clear whether AF-17 is a transcription factor.
  • AF-17 is the fifth partner gene involved in Hq23 abnormalities to be cloned and characterized. Schematic representation of the proteins encoded by these genes and by ALL-l is shown in Figure 20. Inspection of the sequences within the segments of the partner proteins (right side of the arrows) linked to ALL-l sequences (left side of the fusion point within the top scheme) in the chimeric proteins thought to be critical for leukemogenesis, does not reveal a common motif. AF-9 and ENL are the only partner genes which share sequence homology (Nakamura et al . , Proc . Natl . Acad . Sci . USA 1993 90, 4631-4635) .
  • AF-9 and ENL proteins contain nuclear targeting sequences and are probably nuclear proteins .
  • the AF-6 polypeptide linked to the N-terminus of ALL-l contains the GLGF motif (Prasad et al . , Cancer 1993 53, 5624-5628) whose function is not known, as well as short regions very rich in acidic amino acids, basic amino acids or prolines.
  • the GLGF motif is found in cytoplasmic or membrane-associated proteins and this suggests that AF-6 is not located in the nucleus.
  • AF-4 polypeptide within the ALL-l/AF-4 protein includes several segments with high concentration of serines or prolines
  • the AF-4 protein includes a nuclear targeting sequence and therefore is probably associated with the nucleus. Finally, each of the normal five partner genes is expressed in all cell lines analyzed, both of hematopoietic and non hematopoietic lineages.
  • AF-4, AF-6, and AF-17 polypeptides linked to the ⁇ - terminus of ALL-l each contain stretches of one or more of those amino acids, the analogous polypeptide of AF-9 as well as its homologous C-terminal region in E ⁇ L are devoid of these amino acids.
  • the AF-6 protein is probably located in the cytoplasm or the membrane of the cell, and therefore does not play a role in transcriptional regulation. Considering the above we find it less likely that the partner polypeptides of AF-6, AF-9 and E ⁇ L contribute domains involved in direct activation or repression of transcription.
  • Inactivation could occur by nonproductive binding to the promoter of the normal target (s) for ALL-l or by dimerization of the chimeric protein to the normal protein and sequestering the latter either to a complex with other proteins or into another cellular compartment.
  • the partner polypeptides could best play a role in the elimination of the normal protein activity through dimerization. They could make the dimer nonfunctional by virtue of their presence within, or by sequestering it through interaction with other cellular proteins.
  • the leucine zipper dimerization motif in AF-17 and the GLGF motif in AF-6 could represent protein-protein interaction domains of partner polypeptides.
  • Sequencing reactions were performed by using an automatic sequencer (ABI) . Sequences were reassembled and analyzed in the Genetic Computer Group system. Alu sequences were analyzed by the Pythia service.
  • ABSI automatic sequencer
  • FIG. 21 shows DNA rearrangements detected by B859 probe in some of the various Hq23 aberrations studied in this report.
  • a phage clone, mgll.l which spans the breakpoint cluster region in the ALL-l gene (Gu et al . , Cell 1992 71, 701- 708) , was subcloned into plasmids for sequencing. The complete sequence of the 8342 bp BamHI fragment is presented in Fig. 22. The exons included in this region are shown.
  • the AF4 probe (Cimino et al . , Cancer Res . 1992 52, 3811-3813 and Gu et al . , Proc . Na tl . Acad. Sci . USA 1992 89, 10464-10468) , a modified Ddel fragment, spans nucleotides 3071 to 3261 and 3502 to 3754 ( Fig . 22 ) .
  • the 8342 bp sequence was first screened for Alu repeats. Eight Alu repeats were identified and their positions are indicated in Table 1. The orientation of these Alu repeats is the same as that of the ALL-l gene. Classification of these Alu repeats was based on recently published diagnostic criteria (Milosavljevic et al . , J " . ⁇ ol . Evol . 1991 32, 105-121) . After the ALL-l exons and Alu repeats were precisely identified, the rest of sequence was searched for other homologous sequence (s) in GenBank.
  • a 130 bp fragment encompassing nucleotides 7429 to 7559 in intron 9, shows around 80 percent sequence identity to genomic sequences in several genes such as TRE17, ApoA4, Factor VIII c subunit, Factor IX, a nuclear gene for mitochondrial ATP synthase c subunit, and G6PD gene (GenBank accessions: X63596, M14642, M88636, K02402, X69907, and Z29527, respectively) . These similar sequences were located in 5' regulatory regions, or in 3' segments, or in introns, suggesting that they may represent a group of repetitive elements with low frequency in the genome.
  • the DNA rearrangements in the ALL-l gene involved in acute leukemia can be detected by a single probe, B859. Digestion with BamHI is normally sufficient for the analysis. However, if only one or no rearranged fragments are detected, the sample DNA should be digested by other restriction enzymes such as HindHI, and probed with B859.
  • the germline sequence of the partner chromosome at the breakpoint should have been Alu. However, this is not the case in any of the five translocations. Nevertheless, the high concentration of the Alu sequences within the region, in particular, within the area spanned by exons 6 and 7, suggested a possible role for the Alu in the translocations. This indirect role might be destabilization of the region so as to make it more prone to breaks.
  • topoisomerase II recognition sites were found in three out of ten cases when three mismatches were allowed in the consensus sequence. Thus, in the majority of the de novo All-1 rearrangements topoisomerase II recognition sites are not present at the breakpoints, and the enzyme is probably not involved. It will be necessary to sequence the breakpoint in secondary leukemias to determine whether in these cases topoisomerase II recognition sites are consistently associated with the breakpoints.
  • i Gu et al., Proc . Natl . Acad. Sci . USA 1992 89, 10464- 10468 ii: Prasad et al . , Cancer Res . 1993 53, 5581-5585 iii: Nakamura, et al . , Proc . Natl . Acad . Sci . USA 1993 90, 4631-4635
  • Genomic DNA was extracted from bone marrow aspirates by a standard procedure (Gustincich et al . , BioTechniques 1991, 11, 298-301) . Approximately 8 ⁇ g of genomic DNA was digested to completion with BamHI or HindHI. Restriction enzyme digests were separated by electrophoresis on 0.7% agarose gels and blotted onto positively charged nylon membranes. Southern blotting, probe radiolabeling, and hybridization were performed by standard techniques. A single blot was prepared. After probing with SASl, the blot was stripped, then probed again with B859.
  • Clones corresponding to the rearranged ALL-l BamHI fragments were isolated from bacteriophage XEMBL3 libraries made from size-fractionated BamHI digests of patient DNA. Recombinants were identified in phage libraries by filter hybridization using the B859 probe. Construction of libraries, screening, phage purification, and restriction enzyme mapping were done by standard techniques . Subclones were constructed in the pBluescript II plasmid vector. DNA sequence of selected portions of subclones was determined by cycle sequencing using an Applied Biosystems 373A DNA sequencer. Programs from Genetics Computer Group (GCG) system (Devereux et al . , Nucl . Acids Res . 1984, 12, 387-395) were used for data analysis.
  • GCG Genetics Computer Group
  • RNAzolTM Biotecx Laboratories
  • RT Reverse transcriptase
  • RNA-PCR amplification was performed with rTth DNA polymerase.
  • Nested PCR amplification was performed with Taq DNA polymerase.
  • Oligonucleotide primers were used without further purification.
  • Primers are 3.1c (AGGAGAGAGTTTACCTGCTC) [SEQ ID NO: 83] from exon 3, 5.3 (GGAAGTCAAGCAAGCAGGTC) [SEQ ID NO:84] from exon 5, 6.1 (GTCCAGAGCAGAGCAAACAG) [SEQ ID NO:85] from exon 6, and 3.2c (ACACAGATGGATCTGAGAGG) [SEQ ID NO:86] from exon 3.
  • Primers used in reactions are as follows: 1) RT reaction - 3.1c, 2) RNA-PCR amplication - 5.3/3.1C, 3) nested PCR amplification - 6.1/3.2c. RT reaction was performed for 15 minutes at 57°C using 500 ng RNA.
  • RNA-PCR amplification was performed for 35 cycles (95 C, 1 minutes; 53°C, 1 minutes; 72°C, 1 minute) .
  • Nested PCR amplification was performed using 0.5 ⁇ l of the RNA- PCR product for 30 cycles (95°C, 1 minute; 60°C 1 minute; 72°C, 1 minute) .
  • PCR products were analyzed by 2% agarose gel electrophoresis .
  • Figure 24 shows Southern blot rearrangements in the
  • AML cytogenetic evidence of Hq23 translocations.
  • the rearrangements were detected with a cDNA probe (B859) (Gu et al . , Cell 1992 71, 701-708 and Caligiuri et al . , Cancer Res . 1994 54, 370-373) which spans the ALL-l breakpoint cluster region.
  • Two of these patients (no ⁇ . 23 and 24) had trisomy 11 as a sole cytogenetic abnormality whereas one patient (no. 1) had a normal karyotype (Caligiuri et al . , Cancer Res . 1994 54, 370-373) .
  • a single rearranged ALL-l band is seen for each patient in both BamHI and HindHI restriction enzyme digests.
  • Clones corresponding to the rearranged BamHI fragments from the two trisomy 11 patients were isolated and characterized. Each clone begins and ends with a portion of ALL-l exon 5 delineated by the BamHI cloning site within this exon (Fig. 25A) . The 5'- 3' order of ALL-l exons within each clone is 5-6-2-3-4-5. This novel exon structure indicates that the ALL-l rearrangement in each patient is the result of a direct tandem duplication of a portion of the ALL-l gene (Fig. 25B) . The junction point of this duplication fuses the 5' portion of intron 6 to the 3' portion of intron 1. The precise junction points for the two clones are different. DNA sequence across the junctions (Fig.
  • 25C shows a 1 bp N-segment in one clone ( ⁇ 24) and heptamer- like signal sequences (Akira et al . , Science 1987 238, 1134- 1138) near the junction points in both clones.
  • RNA-PCR was performed using oligonucleotide primers specific for the ALL-l duplication. Discrete bands of the predicted size were detected for the two patients with trisomy 11 (Fig. 26A) . Sequence analysis of nested PCR products (Fig. 26B) shows an in-frame fusion of exon 6 with exon 2.
  • the partial ALL-l duplication creates a novel type of fusion protein in which a truncated polypeptide chain encoded by ALL-l exons 1-6 is fused near the amino-terminus of the native ALL-l protein.
  • the partially duplicated protein may be involved in cellular transformation, as postulated for other ALL-l fusions (Cimino et al. , Cancer Res . 1991 51, 6712-6714; Gu et al., Cell 1992 71, 701-708; Tkachuk et al . , Cell 1992 71, 691-700; Morrissey et al . , Blood 1993 81, 1124-1131; Nakamura et al., Proc. Natl . Acad.
  • ALL-l amino- terminal domains from their normal protein environments is the critical structural alteration leading to ALL-l associated leukemogenesis. Because the ALL-l gene is fused with itself, it follows that partner genes from other chromosomes are not necessary for involvement of ALL-l in leukemia.
  • Trisomy 11 is a rare recurrent finding in AML, estimated to occur at a frequency of about 0.7% (CALGB AML cytogenetic data base) . Trisomy of other chromosomes is reported frequently in hematologic malignancy, sometimes in association with disease progression (Heim et al .
  • TCT TCA AAA AGG ACA GAT GCA ACC ATT GCT AAG CAA CTC TTA CAG 945 Ser Ser Lys Arg Thr Asp Ala Thr lie Ala Lys Gin Leu Leu Gin 305 310 315
  • AGC AGA AGG TAT TCA GTG TCG GAG AGA AGT TTT GGA TCT AGA ACG 1440 Ser Arg Arg Tyr Ser Val Ser Glu Arg Ser Phe Gly Ser Arg Thr 470 475 480
  • AGG TGG ACT TCT TTA AAG CAT TCT AGG TCA GAG CCA CAA TAC TTT 1710 Arg Trp Thr Ser Leu Lys His Ser Arg Ser Glu Pro Gin Tyr Phe 560 565 570
  • AGT AGA GAG AGA GAC CGG GAG AGA GAA AAG GAG AAT AAG CGG GAG 2475 Ser Arg Glu Arg Asp Arg Glu Arg Glu Lys Glu Asn Lys Arg Glu 815 820 825
  • AAA 3060 lie Lys His Val Cys Arg Arg Ala Ala Val Ala Leu Gly Arg Lys 1010 1015 1020
  • Trp Glu Arg Glu Lys lie Leu Ser Ser Met Gly Asn Asp Asp 1040 1045 1050
  • ATC CCT GTA AAA CAA AAA CCA AAA GAA AAG GAA AAA CCA CCT CCG 3915 lie Pro Val Lys Gin Lys Pro Lys Glu Lys Glu Lys Pro Pro Pro 1295 1300 1305
  • ATA ACA CCC AGG GTG GTT TGC TTT CTC TGT GCC
  • AGT GGG CAT 4140 lie Thr Pro Arg Val Val Cys Phe Leu Cys Ala Ser Ser Gly His 1370 1375 1380
  • AAG CGA GGC AAG AGA TCA GCT GAA GGA CAG GTG GAT GGG GCC GAT 7830 Lys Arg Gly Lys Arg Ser Ala Glu Gly Gin Val Asp Gly Ala Asp 2600 2605 2610
  • GGT AAA GAA AAT GGA ACA GAG AAC TTA AAG ATT GAT AGA CCT GAA 8100 Gly Lys Glu Asn Gly Thr Glu Asn Leu Lys He Asp Arg Pro Glu 2690 2695 2700
  • CAG CAT GTC AGT TCT GTA ACC CAA AAC CAA AAA CAA TAT GAT ACA 180 Gin His Val Ser Ser Val Thr Gin Asn Gin Lys Gin Tyr Asp Thr 50 55 60
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE
  • AAG CCC ACG GCT TAT GTC CGG CCC ATG GAT GGT CAA GAT CAG GCC CCT 1332 Lys Pro Thr Ala Tyr Val Arg Pro Met Asp Gly Gin Asp Gin Ala Pro 290 295 300
  • GAG CCC CCA CAA AGG CAA ACC GTT GGA ACC AAA CAA CCC AAA AAA CCT 2244 Glu Pro Pro Gin Arg Gin Thr Val Gly Thr Lys Gin Pro Lys Lys Pro 595 600 605
  • GCCACAGCAC ATGGGGTGCC ACCTCGAGGT CTGCACAGGA GGACTTGGCG CTGCCATTTC 4850
  • CAGAGTAAGA CAAATGCAAA GCATCTTAAG GAGTGAAAAT AGAGTCTAAA TCTTGCCTTT 6830

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Abstract

Sont décrits des procédés pour le diagnostic et le traitement de leucémies humaines impliquant des points de cassure sur le chromosome 11 dans le locus ALL-1. Cette région ALL-1 des points de cassure, une région d'approximativement 8 kb sur le chromosome 11, est également décrite. La région ALL-1 est impliquée dans des translocations observées dans les leucémies aiguës lymphocytaires, myélomonocytaires, monocytaires et myélogènes. Sont également décrites des sondes qui identifient les aberrations chromosomiques intéressant la région ALL-1 des points de cassure sur le chromosome 11. Sont d'autre part décrites des séquences d'ADNc du gène ALL-1 sur le chromosome 11, du gène AF-9 sur le chromosome 9, du gène AF-4 sur le chromosome 4, du gène AF-6 sur le chromosome 6 et du gène AF-17 sur le chromosome 17 ainsi que les séquences aminoacides correspondantes. Sont décrites des sondes permettant de détecter des anomalies chromosomiques intéressant ces gènes. Sont aussi décrits des gènes chimères impliqués dans ces translocations. Sont par ailleurs décrits des anticorps monoclonaux permettant le diagnostic et le traitement, ainsi que des oligonucléotides antisens permettant le traitement des leucémies aiguës.
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US6040140A (en) * 1991-12-11 2000-03-21 Thomas Jefferson University Methods for screening and treating leukemias resulting from all-1 region chromosome abnormalities
US5567586A (en) * 1995-05-18 1996-10-22 Thomas Jefferson University Methods of indentifying solid tumors with chromosome abnormalities in the ALL-1 region
US5723579A (en) * 1996-02-02 1998-03-03 Bayer Corporation Fibrinogen binding peptides
WO1998024928A2 (fr) * 1996-12-06 1998-06-11 Niels Pallisgaard Detection d'anomalies chromosomiques
WO1998024928A3 (fr) * 1996-12-06 1999-03-25 Niels Pallisgaard Detection d'anomalies chromosomiques
US6180760B1 (en) * 1997-09-22 2001-01-30 Japan Science And Technology Corp. Actin filament-binding protein “l-Afadin”
EP0905239A3 (fr) * 1997-09-22 2001-02-07 Japan Science and Technology Corporation Gène codant Afadin-1
EP0905239A2 (fr) * 1997-09-22 1999-03-31 Japan Science and Technology Corporation Gène codant Afadin-1
WO2003057240A2 (fr) * 2002-01-09 2003-07-17 Smithkline Beecham P.L.C. Nouvelle utilisation
WO2003057240A3 (fr) * 2002-01-09 2003-09-25 Smithkline Beecham Plc Nouvelle utilisation
US8124351B2 (en) 2005-11-18 2012-02-28 Board Of Regents, The University Of Texas System Quantification of fusion proteins and their activity from chromosomal translocation
US8349560B2 (en) 2007-06-15 2013-01-08 The Ohio State University Research Method for diagnosing acute lymphomic leukemia (ALL) using miR-222
US9085804B2 (en) 2007-08-03 2015-07-21 The Ohio State University Research Foundation Ultraconserved regions encoding ncRNAs
US8911998B2 (en) 2007-10-26 2014-12-16 The Ohio State University Methods for identifying fragile histidine triad (FHIT) interaction and uses thereof
US9125923B2 (en) 2008-06-11 2015-09-08 The Ohio State University Use of MiR-26 family as a predictive marker for hepatocellular carcinoma and responsiveness to therapy
US8916533B2 (en) 2009-11-23 2014-12-23 The Ohio State University Materials and methods useful for affecting tumor cell growth, migration and invasion
US8946187B2 (en) 2010-11-12 2015-02-03 The Ohio State University Materials and methods related to microRNA-21, mismatch repair, and colorectal cancer
US10758619B2 (en) 2010-11-15 2020-09-01 The Ohio State University Controlled release mucoadhesive systems
US11679157B2 (en) 2010-11-15 2023-06-20 The Ohio State University Controlled release mucoadhesive systems
US8664192B2 (en) 2011-03-07 2014-03-04 The Ohio State University Mutator activity induced by microRNA-155 (miR-155) links inflammation and cancer
US9249468B2 (en) 2011-10-14 2016-02-02 The Ohio State University Methods and materials related to ovarian cancer
US9481885B2 (en) 2011-12-13 2016-11-01 Ohio State Innovation Foundation Methods and compositions related to miR-21 and miR-29a, exosome inhibition, and cancer metastasis
US8859202B2 (en) 2012-01-20 2014-10-14 The Ohio State University Breast cancer biomarker signatures for invasiveness and prognosis
US9434995B2 (en) 2012-01-20 2016-09-06 The Ohio State University Breast cancer biomarker signatures for invasiveness and prognosis
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