Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis Running Head: Inducible murine model of CML-like disease Scientific Section Heading: Neoplasia

In order to develop murine models of leukemogenesis, a series of transgenic mice expressing BCR-ABL in different hematopoietic cell subsets were generated. Here we describe targeted expression of P210 BCRABL in stem and progenitor cells of murine bone marrow using the tet-off system. The transactivator protein tTA was placed under the control of the murine stem cell leukemia gene (SCL) 3' enhancer. Induction of BCR-ABL resulted in neutrophilia and leukocytosis, and the mice became moribund within 29 to 122 days. Necropsy of sick mice demonstrated splenomegaly, myeloid bone marrow hyperplasia, and extramedullary myeloid cell infiltration of multiple organs. BCR-ABL mRNA and protein were detectable in the affected organs. FACS analysis demonstrated a significant increase of mature and immature myeloid cells in bone marrow and spleen together with increased bilineal B220+/Mac-1+ cells in the bone marrow. tTA mRNA was expressed in FACS-sorted hematopoietic stem cells which were expanded 26-fold after BCR-ABL induction. 31% of the animals demonstrated a biphasic phenotype, consisting of neutrophilia and subsequent B-cell lymphoblastic disease, reminiscient of blast crisis. In summary, this phenotype recapitulates many characteristics of human chronic myeloid leukemia (CML) and may help elucidate basic leukemogenic mechanisms in CML stem cells during disease initiation and progression. only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Koschmieder et al, Page 4 Introduction Chronic myeloid leukemia (CML) was the first human malignancy shown to be associated with a specific genetic lesion, the Philadelphia chromosome, an abnormality first described as a shortened chromosome 22 and subsequently identified as a chromosomal translocation t(9;22), that fuses parts of the breakpoint cluster region BCR with the gene encoding the tyrosine kinase ABL. The BCR-ABL protein is a constitutively active tyrosine kinase that is associated with different forms of leukemia, and animal models as well as clinical trials with the ABL kinase inhibitor imatinib have implicated BCR-ABL as the cause of the leukemia.. The most common form, p210 BCR-ABL is found in more than 90% of patients with CML, a malignant disorder of hematopoietic stem cells (HSC). Previous studies have demonstrated that the phenotype of p210 BCR-ABL transgenic mice is dependent on the expression pattern and cell type specificity of the regulatory elements used to drive expression of the oncogene. We developed a series of transgenic mice in which expression of the BCR-ABL fusion protein in different subsets of hematopoietic progenitor cells is adjustable by the administration of tetracycline. These models demonstrated that BCRABL alone is sufficient to induce leukemia and that its continued expression is required for maintenance of the leukemic phenotype. The regulation of the tetracycline-controlled transactivator (tTA) by the mammary tumor virus long terminal repeat (MMTV-LTR) resulted in the rapid and exclusive development of a pre-B cell leukemia. This phenotype was consistent with high expression of MMTV-LTR in early B cells. We reasoned that promoter or enhancer elements derived from HSC-specific genes should result in a more accurate model of the development of CML. Previously, regulatory elements derived from the Ly-6E.1 gene and the H2K gene have been used to target adult HSCs. However, the expression of the Ly-6E1 gene is dependent on the genetic background of the mice, and the H2K gene is expressed in most tissues, which makes these elements less desirable. We isolated and characterized regulatory elements of the human CD34 gene locus that are capable of expressing human CD34 in HSC and subsequently modified the only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Koschmieder et al, Page 5 expression cassette to allow for inducible expression of a heterologous transgene in mice. These animals were bred to a line of mice that carries p210 BCR-ABL under the control of a tetracycline-responsive element (TRE). Double-transgenic mice consistently developed a myeloproliferative syndrome with preferential expansion of the megakaryocytic lineage causing thrombocytosis, a feature also seen in CML patients. However, the expression pattern of tTA in these transgenic mice with highest expression in megakaryocyte-erythrocyte progenitor cells (MEP) was slightly different from that of human CD34 expressed from an unmodified human CD34 PAC clone. The SCL gene encodes a basic helix-loop-helix transcription factor expressed in hematopoietic cells, endothelial cells, the central nervous system, and the embryonic skeleton. Within the hematopoietic system, SCL is normally expressed in erythroid cells, mast cells, megakaryocytes, and multipotent progenitor cells. Studies with null-mutants have shown that SCL is a critical regulator of hematopoiesis. Based on these results it was predicted that normal SCL-expressing progenitor cells should be multipotent, and a knock-in strategy has indeed confirmed this hypothesis. Detailed analysis of enhancer elements within the murine SCL gene has identified a fragment within the 3’-region of the gene, which restricted expression of a heterologous LacZ transgene within adult murine bone marrow to hematopoietic stem and progenitor cells and megakaryocytes. We have modified this construct to allow inducible expression of transgenes by placing tTA expression under the control of this 3' enhancer fragment. Here we describe that induction of BCR-ABL expression invariably resulted in neutrophilia and subsequent splenomegaly as well as organ invasion by myeloid cells, thus recapitulating major features of CML in patients. only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Koschmieder et al, Page 6 Materials and Methods Generation of the SCLtTA construct and of transgenic mice The transgenic construct (Fig. 1A) consisting of the SV40 minimal promoter, an intervening sequence (intron), the tTA gene, and the 5.5 kb Bgl II fragment from the murine 3'SCL enhancer was generated by inserting the rabbit beta-globin intron 2 into the SfiI site in the SV40 minimal promoter contained in pGL2. The tTA sequence with poly adenylation (polyA) site was isolated by digestion of pUHD 15-1 with Hind III/Eco RI and then ligated into the Sal I digested SV40/intron plasmid. This fragment was released by Hind II-blunted/Nhe I digestion and ligated into the multiple cloning site 5 ́ of the 5.5 kb murine SCL 3’-enhancer in pBluescript KSdigested with Bgl II-blunted/Xba I (Fig. 1A). The transgenic construct was released from the vector backbone by digestion with Xho I, and the fragment was purified using QiaEx II following the manufacturer's instructions (Qiagen, Valencia, CA). The DNA was resuspended in microinjection buffer (1 mM Tris/HCl pH 8.0, 0.1 mM EDTA) and injected into the pronucleus of BDF mice. Nine different founder animals were identified of which 6 transgenic lines were subsequently established. In order to test for expression of a functional transactivator protein, SCLtTA mice were bred with Tet-O-cre transresponder mice. SCLtTA/BCR-ABL double-transgenic mice were generated by cross breeding of male SCLtTA mice with female TRE-BCR-ABL mice (founder line 2) in the presence of tetracycline administered with the drinking water (0.5g/L) to prevent expression of the transgene during embryogenesis. Genotyping Transgenic mice were identified by either Southern blot analysis (for the identification of founder animals) or PCR with tTA gene-specific primers. The upstream primer (5'-AGA AGA GAT GAG GTG TGA GGA T – 3’) and downstream primer (5'-GGA TCC ACA AGA ATGAGCATGTA-3’) amplified a 405 base pair only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Koschmieder et al, Page 7 fragment within the tTA gene. SuperMix polymerase (GIBCO, BRL, Grand Island, NY) was used with amplification conditions of 35 cycles at 94° C for 40 sec, 65° C for 30 sec, and 72° C for 30 sec. Southern Blot analysis of EcoRI-restricted genomic DNA was performed using positively charged nylon membranes (Biotrans +, ICN, Irvine, CA) as described. Stable chromosomal incorporation of the transgene was detected with a 1.0 kilobase BamHI/Hind II fragment of the tTA gene. Genotyping for the P210 BCR-ABL transgene was performed as described previously. Genotyping for the cre recombinase transgene was performed by PCR with primers 5’-ATGTTCAATTTACTGACCG-3’ and 5’CGCCGCATAACCAGTGAAAC-3’. Amplification conditions consisted of 35 cycles of 30 seconds denaturation at 94° C, 30 seconds annealing at 51° C, and 30 seconds extension at 72° C yielding an amplification product of 355 bp. RNA isolation and Northern blot analysis. Total RNA for Northern Blot analysis was isolated from murine tissue according to the CsCl method and expression analysis was performed as described, and a 1.02 kb fragment of the tTA gene isolated from pUHD 15-1 and the joining region of p210 BCR-ABL were used as probes. Expression of the gene for cre recombinase was detected using a 1.0 kb probe generated by digestion of the Tet-O-cre transgenic construct with Eco RV/Pvu II. Northern blots were stripped and reprobed with a GAPDH-specific probe. Western blotting Western blotting was performed essentially as described before. Briefly, cells in single suspension were washed with ice-cold PBS and lysed in RIPA buffer or 10% trichloracetate. After pH adjustment, sample buffer was added immediately, and the samples were boiled for 10 minutes. Proteins were separated on a only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Koschmieder et al, Page 8 7.5% SDS-PAGE gel, transferred onto a nitrocellulose membrane (BioRad, Hercules, CA), and probed with mouse anti-c-abl antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA). Analysis and monitoring of disease Peripheral blood was collected from the retro-orbital plexus with a heparinized capillary tube, and total white blood cell and differential counts were performed once a week. Peripheral blood films were stained with Wright-Giemsa for differential analysis and the number of white blood cells was determined with a hemocytometer after lysis of enucleated red blood cells with ACK lysis buffer (0.15 mol/L NH4Cl, 1 mmol/L KHCO3, 0.1 mmol/L Na2-EDTA ph 7.3). Histology Mice were sacrificed by CO2 inhalation, and organs were removed and fixed in neutrally buffered 10% formalin at room temperature for 16 hours prior to embedding in paraffin and sectioning. All tissues were stained with hematoxylin/eosin for light microscopy. Flow cytometry analysis Bone marrow cells were flushed from tibias and femurs with PBS. Enucleated red blood cells were lysed with ACK lysis buffer, and the remaining white blood cells were washed once with PBS. Lymph node and spleen tissue was squeezed through a 100 um cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ) with the plunger of a syringe. Cells were incubated with the appropriate antibodies. All analyses were performed on a dual-laser FACS (Becton Dickinson, Franklin Lakes, NJ). The following antibodies were used: anti-murine B220 (Caltag, San Francisco, CA), all other antibodies (murine CD34, CD41, Ter 119, Gr1, Mac-1, c-kit, Sca-1, BP-1, CD43, IgM) were purchased from Pharmingen (San Diego, CA). only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Koschmieder et al, Page 9 Multicolor FACS sorting and analysis of HSC and progenitor cells For sorting of myeloid progenitors, red cell-lysed bone marrow cells were stained with tricolor-conjugated rat antibodies specific for the following lineage markers: CD3 (RM3406), CD4 (MCD 0406), CD8a (MCD0806), CD19 (RM7706), Ly-6G (Gr-1) (RM3006), and CD45R (B220) (RM2606, all from Caltag, Burlingame, CA). Lin cells were removed with sheep anti-rat IgG-conjugated magnetic beads (Dynabeads M-450; Dynal A.S., Oslo, Norway), and the remaining cells were stained with rat antibodies anti-CD34-FITC (RAM34), anti-FcRγII/III-PE (2.4G2), anti-CD117 (c-kit)-APC (2B8), and anti-Sca-1 (Ly-6A/E)-Biotin (E13161.7, all from Pharmingen) and subsequently with anti-Streptavidin-APC/Cy7 (SA1014 from Caltag). Myeloid progenitors were sorted as LinSca-1c-KitCD34FcγRII/III (CMP), LinSca-1cKitCD34FcγRII/III (GMP), or LinSca-1c-KitCD34FcγRII/III (MEP) as described previously. For hematopoietic stem cells (HSC) and common lymphocyte progenitors (CLP), bone marrow cells were stained with lineage markers as well as anti-Sca-1 (Ly-6A/E)-FITC), anti-CD117 (c-kit)-APC (2B8), and anti-IL7Rα chain-PE (A7R34, e-Bioscience, San Diego, CA). HSC and CLP were sorted as LinSca-1ic-Kit and IL-7RαLinSca-1c-Kit populations, respectively. Cells were sorted using a highly modified double laser (488nm/350nm Enterprise II + 647nm Spectrum) high-speed FACS (Moflo-MLS, Cytomation, Fort Collins, CO, USA). Cells were sorted into 500 ul of buffer RLT with beta-mercaptoethanol, and RNA was extracted using the RNAEasy Micro kit including the carrier RNA step and DNAse on-column treatment according to the manufacturer ́s instructions (Qiagen). RT-PCR analysis of isolated HSC and progenitors Reverse transcription was performed using SuperScript reverse transcriptase (GIBCO BRL) with random primer hexamers according to the manufacturer’s instructions. 1 ul of each cDNA reaction was used for the detection of tTA gene expression. tTA was amplified with sense primer 5 ́-AAA AGC TGG ATC GAT CCT only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Koschmieder et al, Page 10 GAG AAC and antisense primer 5 ́-GGC GGC ATA CTA TCA GTA GTA GGT G. Murine GAPDH cDNA which served as a positive control was amplified using sense primer 5 ́-GGT GCT GAG TAT GTC GTG GAG TCT A and antisense primer 5 ́-CCT GGT TCA CCA CCT TCT TCT TGA TGT C. PCR conditions consisted of 35 cycles, with 30 seconds denaturation at 94°C, 30 seconds annealing at 55°C (tTA) or 58°C (GAPDH), and 90 seconds extension at 72°C. Colony forming unit (CFU) assay and megakaryocyte assay (CFU-MK) Bone marrow was flushed with IMDM containing 5% of FBS from femurs and tibias of double-transgenic and single-transgenic control animals. Spleen cells were isolated by squeezing tissue through a cell strainer. Enucleated red blood cells were removed with ACK cell lysis buffer, and colony assays were initiated according to the manufacturer instructions in Methocult GF M3434 supplemented with frowth factors as provided by the manufacturer (Stem Cell Technologies, Vancouver, Canada). Numbers of CFU-GM, BFU-E, and CFU-GEMM were determined 8 days after plating. Transplantation into NOD/SCID mice and real-time genomic PCR for engraftment assessment Bone marrow cells were harvested from the femurs and tibias of double-transgenic and control mice. Cells were resuspended in PBS and injected (2x10 cells/0.3 ml) into the tail veins of sublethally irradiated (300 cGy) NOD/SCID mice. Mice were housed in microisolator cages under sterile conditions. Real-time genomic PCR was performed on an ABIPrism 7700 Sequence Detector (Applied Biosystems) using the following parameters: 40 cycles of 94°C (15 s); and 60°C (60 s). Primers and probe (FAM-labeled) were as follows: murine CCAAT enhancer binding protein alpha (CEBPA) forward 5 ́-AAA GCC AAG AAG TCG GTG GAC, CEBPA reverse 5 ́-CTT TAT CTC GGC TCT TGC GC, CEBPA probe 5 ́-CGA GTA CCG GGT only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Koschmieder et al, Page 11 ACG GCG, BCR-ABL forward 5 ́-CTG GCC CAA CGA TGG CGA, BCR-ABL reverse 5 ́-CAC TCA GAC CCT GAG GCT CAA, BCR-ABL probe 5 ́-CCC TTC AGC GGC CAG TAG CAT CTG A. Statistical Analysis Data are expressed as mean ± standard deviation (SD) unless indicated otherwise and were compared using an unpaired Student ́s T test as provided in the result section. p<0.05 differences were considered significant. only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Koschmieder et al, Page 12