Oncogene-dependent engraftment of human myeloid ... - Nature

Leukemia (2001) 15, 814–818  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu

Oncogene-dependent engraftment of human myeloid leukemia cells in immunosuppressed mice M Kiser1, JA McCubrey2, LS Steelman2, JG Shelton2, J Ramage1, RL Alexander1, GL Kucera1, M Pettenati1, MC Willingham1, MS Miller1 and AE Frankel1 1

Department of Cancer Biology, Pediatrics and Pathology, Wake Forest University School of Medicine, Winston-Salem, NC; 2Department of Microbiology, Brody School of Medicine at East Carolina University, Greenville, NC, USA

We have developed an in vivo model of differentiated human acute myeloid leukemia (AML) by retroviral infection of the cytokine-dependent AML cell line TF-1 with the v-Src oncogene. When injected either intravenously or intraperitoneally into 300 cGy irradiated SCID mice, animals formed multiple granulocytic sarcomas involving the adrenals, kidneys, lymph nodes and other organs. The mean survival time was 34 ⴞ 10 days (n ⴝ 40) after intravenous injection and 24 ⴞ 3 days (n ⴝ 5) after intraperitoneal injection of 20 million cells. The cells recovered from leukemic animals continued to express interleukin-3 receptors and remained sensitive to the diphtheria fusion protein DT388IL3. Further, these granulocytic sarcomaderived cells grew again in irradiated SCID mice (n ⴝ 10). The cytogenetic abnormalities observed prior to inoculation in mice were stably present after in vivo passage. Similar to the results with v-Src transfected TF-1 cells, in vivo leukemic growth was observed with TF-1 cells transfected with the human granulocyte–macrophage colony-stimulating factor gene (n ⴝ 5) and with TF-1 cells recovered from subcutaneous tumors in nude mice (n ⴝ 5). In contrast, TF-1 cells expressing v-Ha-Ras (n ⴝ 5), BCR-ABL (n ⴝ 5), or activated Raf-1 (n ⴝ 44) did not grow in irradiated SCID mice. This is a unique, reproducible model for in vivo growth of a differentiated human acute myeloid leukemia and may be useful in the assessment of anti-leukemic therapeutics which have human-specific molecular targets such as the interleukin-3 receptor. Leukemia (2001) 15, 814–818. Keywords: TF1; acute myeloid leukemia; SCID mice; oncogenes; interleukin-3; cytogenetics

number of investigators have examined the in vivo growth potential of AML cell lines. Poorly differentiated cytokineindependent cell lines propagated well in cyclophosphamidetreated or irradiated SCID mice.2,5,9 Cytokine-dependent, differentiated AML cell lines only grew in cyclophosphamide treated and irradiated nude mice when the cells were retrovirally infected to express growth factors interleukin-3 (IL3) or granulocyte–macrophage colony-stimulating factor (GMCSF).10 Using a similar approach transgenic nude mice have been developed which overexpress human IL3 or GM-CSF. These animals yield subcutaneous tumors after implantation with the cytokine-dependent TF-1 AML cells.11,12 While these approaches have been successful, they result in receptor saturation and engagement. Therapeutics which target the IL3 or GM-CSF receptor may be blocked by the autocrine or paracrine cytokines. We sought to establish an in vivo model with differentiated human AML blasts expressing unoccupied growth factor receptors. Since cytokine receptor-mediated signaling pathways may be critical for blast survival and proliferation, we chose to transfect genes which would provide constitutive signaling through the cytokine receptor pathway bypassing the cytokine–cell surface receptor activation step. We now report the in vivo growth characteristics and cytokine receptor properties of a series of these cell lines.

Introduction Preclinical development of novel therapies for leukemia requires in vivo models to assess drug pharmacology, immune response and preliminary therapeutic index. Particularly with the emergence of agents targeted to human differentiation antigens, receptors and signal transduction pathway elements, there is a need for a reproducible, established human differentiated myeloid leukemia model in rodents. Methods currently available for engraftment of patient AML blasts include implantation of patient blasts into irradiated SCID or NOD/SCID mice.1–5 In some cases, improved growth has been observed in mice receiving regular intraperitoneal injections of growth factors (PIXY321 and human mast-cell growth factor).6 In other cases, AML growth only occurred in human fetal bone marrow subcutaneous implants in SCID mice.7 In most cases, engraftment was transient and subsequent in vivo passage was not shown. Attempts to grow human AML cells in NOD/SCID mice have also shown in 60% of cases that the phenotype of the AML graft showed major changes after outgrowth in NOD/SCID.8 Because of the need for a reproducible in vivo assay, a

Correspondence: AE Frankel, Hanes 4046, Wake Forest University School of Medicine, Med Center Blvd, Winston-Salem, NC 27157, USA; Fax: 336 716 0255 Received 2 November 2000; accepted 28 December 2000

Materials and methods

Cell lines The human cytokine-dependent TF-1 cells were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD, USA) + 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and supplemented with 1 ng/ml human GM-CSF (Immunex, Seattle, WA, USA).13 TF-1 cells were retrovirally infected as previously described to yield transfectants expressing v-Src, vHa-Ras, BCR-ABL, and activated Raf-1 (⌬Raf-1) as previously described.14,15 8A#2 cells were TF-1 cells transfected with the genomic GM-CSF gene linked to the Moloney murine leukemia virus long terminal repeat.16 The Tumor-1 cell line was derived from TF-1 cells collected from cells propagated as a subcutaneous tumor in nude mice.16 Table 1 details all the cell lines used. Cell lines, other than wild-type TF-1, were grown in RPMI 1640 + 10% FBS.

IL3 receptor densities Cell binding of 125I-labeled IL3 receptor agonist SC-65461 (Pharmacia, St Louis, MO, USA) in the presence and absence of unlabeled human IL-3 (SC-50341, Pharmacia) was measured as previously described.17 Nonlinear regression analysis

TF/v-Src engrafted SCID mice M Kiser et al

Table 1

Properties of assayed AML cell lines

Cell linea

TF-1 TF/v-Src TF/vHa-Ras TF/BCR/ABL TF/⌬Raf-1 8A#2 Tumor-1

Description

Erythroleukemia v-Src transformed TF-1 v-Ha-Ras transformed TF-1 BCR/ABL transformed TF-1 ⌬Raf-1 transformed TF-1 GM-CSF transfected TF-1 Nude mouse passaged TF-1

Growth Reference factor dependence + − −

12 14 14

−

13,14

−

14

−

15

−

15

a TF-1 grown in RPMI 1640 + 10% FBS + 1 ng/ml GM-CSF. Tumor1 grown in RPMI 1640 + 10% FBS. Other cells grown in RPMI 1640 + 10% FBS + 2 mg/ml G418.

using GraphPad Prism (GraphPad Software) was used to calculate high and low receptor affinities and densities.

DT388IL3 sensitivities Cells were incubated with varying concentrations of DT388IL3 and 3H-thymidine incorporation was measured after 48 h as previously described.18 The calculated IC50s were the concentrations of fusion protein that inhibited thymidine incorporation by 50% compared to control wells.

Cytogenetics For routine cytogenetic studies, cultured cells were exposed to 10 ␮g/ml of colcemid for 20 min, then spun down at 1000 g for 8 min. The cell pellet was resuspended in a hypotonic solution (0.075 m KCl) for 10 min. The spun down cells were fixed in 3:1 methanol:glacial acetic acid three times prior to slide preparation. Metaphase slides were banded using routine GTG-banding.19 Standard ISCN nomenclature was used to interpret the M-FISH/G-banded metaphases. For multi-color FISH, slides were prepared and then pretreated with RNase (10 mg/ml in 2 × SSC at 37°C) for 30 min followed by 10% pepsin in 0.01 N HCl for 5 min.20 After washing in PBS, slides were dehydrated in an ethanol series and allowed to air dry. Slides were denatured in 70% formamide/2 × SSC at 72°C for 1 to 3 min and then dehydrated in a cold ethanol series (70%, 80% and 100%) for 1 min. Vysis Spectrya Vysion M-FISH probe (10 ␮l) was denatured in a 72°C water bath for 5 min and then applied to a heated slide (42°C). Hybridization was allowed to proceed for 48 h in a humid chamber. Slides were post-washed according to the manufacturer’s instructions: 0.4 × SSC/0.3% NP-40 at 72°C for 2 min followed by 5 to 30 s rinses in 2 × SSC/0.1% NP-40 at room temperature. Slides were air dried and counterstained with DAPI II (Vysis, 125 ng/ml in anti-fade mounting solution). Analysis of the M-FISH chromosome metaphases was completed using the SpectraVysion Imaging System. This system’s software captures each of six different fluorescent planes of a single metaphase spread allowing for the identity of each of the 24 differently fluorescent tagged

probes that have hybridized to each chromosome. A karyotype is then generated based on the fluorescent labeling of the individual chromosomes. In addition, the software also generates DAPI bands permitting the chromosomes to be arranged in a standard karyotype.

815

Animals Balb-c-SCID/SCID mice (males and females; age 4–6 weeks) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and maintained in micro-isolator cages housed in laminar flow cubicles in a positive pressure room. The temperature of the room ranged from 22 to 25°C. The water was autoclaved and provided ad libitum. The food was irradiated and provided ad libitum. The mice were allowed to adjust to their new environment for 1 week. The mice were then irradiated with 300 cGy total body irradiation to diminish natural killer cell activity.21 After 24 h of observation, the mice were injected i.v. via tail vein with 2 × 107 TF-1 or modified TF-1 cells diluted in 0.2 ml of phosphate buffered saline (BioWhittaker, Walkersville, MD, USA) with 1% bovine serum albumin (Sigma, St Louis, MO, USA). After completion of all injections, cells from residual syringes were ⬎90% viable by Trypan Blue exclusion. A total of 119 mice were used. The mice were examined daily for overall activity and for the presence of masses. Moribund mice were sacrificed.

Autopsies Moribund SCID mice were sacrificed painlessly by standard CO2 asphyxiation, consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Samples from major organs and abdominal masses were removed, fixed in 4% buffered formaldehyde, dehydrated and embedded in paraffin. Sections were stained with hematoloxylin and eosin and examined under the microscope. In some animals, abdominal masses were resected, minced, separated by use of a cell strainer (Falcon 2340) and tested for the number of IL3 receptors, DT388IL3 sensitivity, and cytogenetics as described above.

Statistical analysis Survival was analyzed by the Kaplan–Meier method and mean survival time and standard deviation of the survival time was determined using GraphPad Prism software. For comparison of the number of IL3 receptors, dissociation constants, and IC50s, a Mann–Whitney test was utilized. The Mantel–Cox (logrank) test was used for comparison of survival between the groups. Results All of the TF-1 cell lines expressed high and low affinity IL3 receptors and were sensitive to DT388IL3 (Table 2). The TF-1 cell lines showed a complex but reproducible set of cytogenetic abnormalities (Figure 1). A final karyotype was generated based on the GTG banded metaphase chromosome spreads in conjunction with the M-FISH analysis. The cytogenetic nomenclature for the cell line was based on the G-banded Leukemia


816

Table 2 IL3 receptor densities and affinities and DT388IL3 sensitivities of AML cell lines

Table 3

Leukemia engraftment in irradiated SCID mice

Cell line Cell line

TF-1 TF/vSrc TF/v-Src#13 TF/v-Src#-16 Tumor-1 8A#2 TF/⌬Raf-1 TF/BCR/ABL TF/v-Ha-Ras

High High Low Low affinity DT388IL3 affinity affinity affinity receptor IC50 (pM) density KD (pM) receptor KD (pM) density (/cell) (/cell) 40 60 80 100 60 10 40 40 60

1325 847 1858 2444 305 179 987 1546 460

800 1200 800 1000 15000 1000 1100 800 600

5882 2123 4790 4553 21410 3661 1428 6280 3010

17 11 10 13 2 19 12 16 2

Non-linear regression analysis yielded R2 ⬎ 0.98 for each radiolabeled binding assay. Thymidine incorporation inhibition assays performed in duplicate with mean shown. IC50 values varied by ⬎30%.

karyotype (n = 20) and M-FISH chromosome preparations (n = 10). Exact breakpoints/chromosomal regions of some of the derived chromosomes could not be determined with certainty using G-banding and M-FISH and are so indicated within the nomenclature. The karyotype is as follows: 54,XY, der(1)(1qter-⬎p22::5?-⬎5?::1?-⬎1?::5?-⬎5?::8q13-⬎8qter),del (1)(q21),der(2)(2pter-⬎2q33::15q15-⬎q24::Xq26-⬎Xqter),-3,der (3)(3q21-⬎3p25::Xq26-⬎Xqter),der(6)t(6;14)(p21.2;?),der(7)t (3;7)(?;q36),dup(8q?),+del(8)(q13q22),+der(8)t(8;19)(q13;?),+der (11)(11qter-⬎11p11.2::19?-⬎19?::Yq11.2-⬎Yqter),der(12)t (10;12)(q21.2;q15),+der(12) (12qter-⬎12p11.2::13?-⬎13?::10 ?-⬎10?::3?-⬎3?),der(13)t(3;13)(?;p11.2),der(14)t(2;14)(q21;p11.2), der(15)t(11;15)(?;p11.2),der(17)t(3;17)(?;q25),der(19)t(16;19) (?;q13.3),+der(19)t(5;19)(?;q13.1)+der(20)t(3;20)(?;q11.2),dup (21q?),der(22)t(2;22)(p11.2;q13.3). Growth in irradiated SCID mice varied markedly between

TF/v-Src TF/v-Src#13 TF/v-Src#16 8A#2 Tumor-1 TF/⌬Raf-1 TF/BCR/ABL TF/v-Ha-Ras

No. granulocytic sarcomas/Total 25/25 5/5 5/5 4/5 3/5 2/44 0/5 0/5

Irradiated animals inoculated with 20 million cells in 0.2 ml PBS by tail vein injection and observed twice daily for 60 days. Five animals received 20 million TF/v-Src cells by intraperitoneal injections. Animals dying from other causes in ⬍60 days censored and included 15 animals in the TF/v-Src group. The TF/v-Src#13 and TF/v-Src#16 were cells collected from tumors after intravenous inoculation in irradiated SCID mice.

the different TF-1 lines (Table 3). Cells overexpressing v-Src or human GM-CSF propagated in mice. In addition, cells passaged as subcutaneous tumors in nude mice grew after intravenous injection in SCID mice. In contrast, cells transfected with v-Ha-Ras, BCR-ABL, or activated Raf-1 grew poorly or were completely unable to grow after intravenous injection into SCID mice. SCID mice inoculated with TF/v-Src cells intravenously died of progressive disease with a mean survival of 34 ± 10 days (Figure 3). Mice given intraperitoneal inoculations of TF/v-Src died of disseminated leukemia with a mean survival of 24 ± 3 days. Histopathology showed abdominal masses along the path of the vena cava and the thoracic aorta, and infiltration of the liver, kidney, adrenals, skeletal muscle, stomach and pancreas (Figure 2). Resected abdominal masses were assayed for IL3 receptors, DT388IL3 sensitivity and cytogenetics. Results closely matched those with cells prior to inoculation into SCID mice (Table 2 and Figure 1).

Figure 1 Karyotype of TF/v-Src cells. (a) G-banded karyotype prepared as described in the text. (b) Color M-FISH as described in the text. The karyotype is detailed in the Results. Leukemia


817

Figure 2 Histopathology of SCID mice moribund with progressive leukemia after intravenous inoculation of 20 million TF/v-Src cells. Hematoxylin and eosin stained 6 micron paraffin sections of indicated tissues. Leukemia infiltrates are shown by arrows.

Figure 3 Survival of SCID mice after i.v. injection of 2 × 107 TF/vSrc cells on day 1. GraphPad Prism software was used to generate the survival curve. Animals censored at time of death for nonleukemic causes confirmed by histopathology.

Discussion To establish an in vivo model of a differentiated AML, we modified the TF-1 cytokine-dependent cell line. Cell lines expressing activated oncogenes or the GM-CSF growth factor were prepared. These AML cells became cytokine-inde-

pendent but retained evidence of differentiation with IL3 receptors and DT388IL3 sensitivity. We also selected a cell line which propagated in nude mice. These cells had also lost cytokine-dependence but retained IL3 receptors. Karyotypic analysis suggested no gross changes in genetic material between these cell lines. We next sought to determine the in vivo growth characteristics of these cells in immunocompromised mice. The TF-1 cells transformed with v-Src grew best in mice. v-Src is a nonreceptor tyrosine kinase which may stimulate cytokine-independent proliferation and cell survival by activating Ras, and, subsequently, the Raf-MAPK signaling pathway.21 Interestingly, v-Src-transformed cells grew better in vivo than v-HaRas, activated Raf or BCR/ABL transformed cells. The observed difference in tumorigenicity between v-Src, v-HaRas and activated Raf is not likely due to a difference in growth rate as the cell lines displayed similar growth characteristics in vitro.15 Moreover, the differences in tumor formation between v-Src, v-Ha-Ras and activated Raf was not likely due to higher levels of autocrine cytokine production as the v-Src transformed cells produced lower levels of autocrine GM-CSF than the activated Raf-transformed cells. The enhanced in vivo growth may be due to the position of Src in signal transduction pathways as it can activate more downstream targets than Raf. Some v-Src transformed cells display constitutive activation of Jak and STAT proteins.22–25 It is conceivable that v-Src induces the activation of specific signalling molecules better than v-Ha-Ras or activated Raf. Moreover, v-Src may activate the phosphotidyl inositol-3 kinase (PI3K)/AKT survival pathway. Similar tumor growth in vivo Leukemia


818

was achieved by autocrine expression of GM- CSF in these cells. A number of investigators have postulated autocrine loops involving GM-CSF and GM-CSF receptors in the genesis of AML.21,26 GM-CSF likely stimulates non-receptor tyrosine kinases to also activate the Jak kinase and STAT transcription factors, as well as the Ras and the Raf-MAPK signaling pathway.21 Moreover, GM-CSF stimulates the PI3K/AKT survival pathway. The mechanism for the cytokine independence of the nude mouse tumor derived Tumor-1 cell line is currently unknown. Importantly, all these cell lines retained differentiated phenotypes with continued expression of IL3 receptors and DT388IL3 sensitivity. Further, the stability of the cytogenetic abnormalities of the TF/v-SRC cell line contrasts with the alterations in phenotypes observed with previous attempts to grow human AML cells in NOD/SCID mice.8 With the invasive, aggressive nature of the disease in the TF/v-Src leukemic mice and their retention of a differentiated AML phenotype, this model should serve as an excellent assay system to evaluate the in vivo anti-leukemic efficacy of a number of human IL3 receptor targeted therapeutics including DT388IL3 fusion protein27 and IL3 receptor agonists and antagonists.28

11

12

13

14

15

16

Acknowledgements This work was supported in part by the Leukemia and Lymphoma Society Grant No. 6114–99 (AEF), NIH CA76178 (AEF), NIH CA51025 (JAM) and the American Cancer Society Grant No. IRG-93–035–6 (GLK). The authors acknowledge the gift of SC-65461 and SC-50431 IL3 receptor agonist proteins from Dr Barbara Klein and Pharmacia, Inc. We thank Dr Douglas Case for input on statistical analyses.

17

References

20

1 Ailes LE, Gerhard B, Kawagoe H, Hogge DE. Growth characteristics of acute myelogenous leukemia progenitors that initiate malignant hematopoiesis in nonobese diabetic/severe combined immunodeficient mice. Blood 1999; 94: 1761–1772. 2 De Lord C, Clutterbuck R, Titley J, Ormerod M, Gordon-Smith T, Millar J, Powles R. Growth of primary human acute leukemia in severe combined immunodeficient mice. Exp Hematol 1991; 19: 991–993. 3 Chelstrom LM, Gunther R, Simon J, Raimondi SC, Krance R, Crist WM, Uckun FM. Childhood acute myeloid leukemia in mice with severe combined immunodeficiency. Blood 1994; 84: 20–26. 4 Ailles LE, Humphries RK, Thomas TE, Hogge DE. Retroviral marking of acute myelogenous leukemia progenitors that initiate longterm culture and growth in immunodeficient mice. Exp Hematol 1999; 27: 1609–1620. 5 Sawyers CL, Gishizky ML, Quan S, Golde DW, Witte ON. Propagation of human blastic myeloid leukemias in the SCID mouse. Blood 1992; 79: 2089–2098. 6 Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, CaceresCortes J, Minden M, Paterson B, Caligiuri MA, Dick JE. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367: 645–648. 7 Namikawa R, Ueda R, Kyoizumi S. Growth of human myeloid leukemias in the human marrow environment of SCID-hu mice. Blood 1993; 82: 2526–2536. 8 Rombouts WJC, Martens ACM, Ploemacher RE. Identification of variables determining the engraftment potential of human acute myeloid leukemia in the immunodeficient NOD/SCID human chimera model. Leukemia 2000; 14: 889–897. 9 Cesano A, Hoxie JA, Lange B, Nowell PC, Bishop J, Santoli D. The severe combined immunodeficient (SCID) mouse as a model for human myeloid leukemias. Oncogene 1992; 7: 827–836. 10 Thacker JD, Hogge DE. Cytokine-dependent engraftment of

Leukemia

18 19

21

22

23 24 25 26 27

28

human myeloid leukemic cell lines in immunosuppressed nude mice. Leukemia 1994; 8: 871–877. Miyakawa Y, Fukuchi Y, Ito M, Kobayashi K, Kuramochi T, Ikeda Y, Takebe Y, Tanaka T, Miyasaka M, Nakahata T, Tamaoki N, Nomura T, Ueyama Y, Shimamura K. Establishment of human granulocyte–macrophage stimulating factor producing transgenic SCID mice. Br J Haematol 1996; 95: 437–442. Fukuchi Y, Miyakawa Y, Kobayashi K, Kuramochi T, Shimamura K, Tamaoki N, Nomura T, Ueyama Y, Ito M. Cytokine dependent growth of human TF-1 leukemic cell line in human GM-CSF and IL-3 producing transgenic SCID mice. Leukemia Res 1998; 22: 837–843. Kitamura T, Tange T, Terasawa T, Chiba S, Kuwaiki T, Miyagawa K, Piao YF, Miyazono K, Urabe A, Takaku F. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3 or erythropoietin. J Cell Physiol 1989; 140: 323–334. McCubrey JA, Steelman LS, Hoyle PE, Blalock WL, WeinsteinOppenheimer C, Franklin RA, Cherwinski H, Bosch E, McMahon M. Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells. Leukemia 1998; 12: 1903–1929. McCubrey JA, Steelman LS, Wang X-Y, Davidian EW, Hoyle PE, White CR, Prevost KD, Algate PA, Robbins P, Mylott D, White MK. Autocrine growth factor secretion after transformation of human cytokine-dependent cells by viral and cellular oncogenes. Int J Oncol 1995; 7: 573–586. Hoyle PE, Steelman LS, McCubrey JA. Autocrine transformation of human hematopoietic cells after transfection with an activated granolocyte/macrophage colony stimulating factor gene. Cytokin Cell Molec Ther 1997; 3: 159–168. Alexander RL, Kucera GL, Klein B, Frankel AE. In vitro interleukin3 binding to leukemia cells predicts cytotoxicity of a diphtheria toxin/IL3 fusion protein. Bioconj Chem 2000; 11: 564–568. Frankel AE, Ramage J, Gannaway S, Kiser M, Alexander R, Kucera G, Miller MS. Characterization of diphtheria fusion toxins targeted to the interleukin-3 receptor. Protein Eng 2000; 13: 575–581. Abruzzese E, Radford JE, Miller JS, Vredenburgh JJ, Rao PN, Pettenati MJ, Cruz JM, Perry JJ, Amadori S, Hurd DD. Detection of abnormal pretransplant clones in progenitor cells of patients who developed myelodysplasia after autologous transplantation. Blood 1999; 94: 1814–1819. Rao PN, Flejter WL, Rahi S, Li H, Pettenati MJ. Identification of the inverted chromosome 16 using chromosome painting. Br J Haematol 1999; 104: 618–620. Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE, Wang X-Y, Algate PA, Franklin RA, Oberhaus SM, Steelman LS, McCubrey JA. Signal transduction, cell cycle regulatory, and antiapoptotic pathways regulated by IL-3 in hematopoietic cells: possible sites for intervention with anti-neoplastic durgs. Leukemia 1999; 13: 1109–1166. Yu CL, Meyer DJ, Campbell GS, Larner AC, Carter-Su C, Schwartz J, Jove R. Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science 1995; 269: 81–83. Campbell GS, Yu CL, Jove R, Carter-Su C. Constitutive activation of JAK1 in Src-transformed cells. J Biol Chem 1997; 272: 2591– 2594. Danial NN, Pernis A, Rothman PB. Jak-Stat signaling induced by the v-abl oncogene. Science 1995; 269: 1875–1877. Ilaria RL, VanEtten RA. P210 and p190 (BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific Stat family members. J Biol Chem 1996; 271: 31704–31710. Rogers SY, Bradbury D, Kozlowski R, Russell NH. Evidence for internal autocrine regulation of growth in acute myeloblastic leukemia cells. Exp Hematol 1994; 22: 593–598. Frankel AE, McCubrey JA, Miller MA, Delatte S, Ramage J, Kiser M, Kucera GL, Alexander RL, Beran M, Tagge EP, Kreitman RJ, Hogge DE. Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias. Leukemia 2000; 14: 576–585. Klein BK, Olins PO, Bauer SC, Caparon MH, Easton AM, Braford SR, Abrams MA, Klover JA, Paik K, Thomas JW, Hood WF, Shieh J-J, Polazzi JO, Donnelly AM, Zeng DL, Welply JK, McKearn JP. Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties. Exp Hematol 1999; 27: 1746–1756.