by Hiroyuki Kawagoe, and Gerard C. Grosveld Conditional MN1-TEL knock-in mice develop acute myeloid leukemia in conjunction with overexpression of HOXA9 by Hiroyuki Kawagoe, and Gerard C. Grosveld Blood Volume 106(13):4269-4277 December 15, 2005 ©2005 by American Society of Hematology

MN1-TEL enhances cytokine-dependent growth of myeloid progenitors. MN1-TEL enhances cytokine-dependent growth of myeloid progenitors. (A) In vitro hematopoietic differentiation of ES cells. MN1-TELKI/WT ES cells treated with Cre or not or WT ES cells were cultured in methylcellulose-based media (MC-1) with stem cell factor (SCF) to generate embryoid bodies (EBs). After 15 or 18 days, cells from dissociated EBs were plated into MC-2 in the presence of SCF, interleukin 3 (IL-3), and IL-6. Numbers of myeloid colonies were scored after 10 days of culture. Mean ± standard error (SE) is shown (n = 3). Experiments repeated with 3 different ES cell clones yielded identical results. (B) Colony-forming efficiency of MC-2 ES-derived hematopoietic progenitors in MC-3. Colony-forming cells (CFCs) were harvested from the MC-2 and replated into MC-3 with the same cytokines (replating assay). Numbers of myeloid colonies were scored after 10 days. Mean ± SE is shown (n = 3). Experiments repeated in triplicate using different ES clones yielded identical results. (C) Colonies in MC-3. Representative pictures captured on day 10 of culture are shown. Images were obtained as previously described.12 (D) GFP expression in ES-derived CFCs. GFP expression was analyzed by flow cytometry (FCM) after 10 days of culture in MC-2. Solid line indicates KI-ES–derived cells; broken line, control WT-ES–derived cells. (E) Cytokine-dependent growth of MN1-TEL+ myeloid progenitors. MN1-TEL/Mx1-Cre BM cells treated with polyinosinic-polycytidylic acid (pI-pC) or not were plated in MC. After the MC-3, cells were harvested and grown in liquid cultures. Numbers of viable cells are plotted. • indicates IL-3 + SCF; ○, IL-3; ▴, SCF; and ▵, no cytokine. Mean ± SE is shown (n = 3). Experiments repeated in triplicate using different cells yielded identical results. (F) May-Giemsa (M-G) staining of MN1-TEL+ cells in culture. Image obtained as previously described.12 (G) Fluorescence-activated cell sorter (FACS) analysis of Sca-1/c-Kit expression of an MN1-TEL+ cell line. Hiroyuki Kawagoe, and Gerard C. Grosveld Blood 2005;106:4269-4277 ©2005 by American Society of Hematology

MN1-TEL–expressing mice develop altered myelopoiesis de novo. MN1-TEL–expressing mice develop altered myelopoiesis de novo. (A) Increased GFP+ cells in BM of MN1-TEL–expressing mice, which died of severe anemia without T-lymphoid tumor (anemic cases). Numbers indicated are mean ± SE percentage of GFP+ cells (n = 3). MT/Cre indicates control MN1-TEL/Mx1-Cre BM cells 1 month (1 mo.) or 8 months (8 mo.) after treatment with pI-pC. (B) Increased Mac1+Gr1- cells in MN1-TEL–expressing mice. The entire population of BM cells was analyzed by FCM. (Left) Age-matched control MN1-TEL/Mx1-Cre BM cells not induced with pI-pC. (Right) BM cells of MN1-TEL–expressing mice with increased number of GFP+ cells. Numbers indicated are mean ± SE percentage (n = 3). (C) Mac1+/Gr1- cells in GFP+ or GFP- BM cells of MN1-TEL–expressing mice with increased number of GFP+ cells. Gates were set to select the 10% GFP-brightest or -dimmest population in MN1-TEL–expressing mice to clearly discriminate MN1-TEL+ and MN1-TEL- cells. Numbers indicated are mean ± SE percentage (n = 3). (Top) Mac1/Gr1 expression in GFP+ or GFP- BM cells. (Bottom) FSC and SSC of Mac1+/Gr1- cells in GFP+ or GFP- BM cells. Hiroyuki Kawagoe, and Gerard C. Grosveld Blood 2005;106:4269-4277 ©2005 by American Society of Hematology

MN1-TEL causes aggressive myeloproliferative disease when coexpressed with IL-3 in vivo. MN1-TEL causes aggressive myeloproliferative disease when coexpressed with IL-3 in vivo. (A) Retroviral transduction of MN1-TEL+ BM cells. BM cells were harvested from 6- to 8-week-old MN1-TEL/Mx1-Cre mice 2 weeks after the last injection with pI-pC (MN1-TEL+) or phosphate-buffered saline (PBS; MN1-TEL-). Sorted Lin- cells were transduced with pSRα–IL-3–IRES–YFP (IL-3) or pSRα-IRES-YFP (vector) retroviral vectors. Transduction efficiency was examined by FCM. A representative result of MN1-TEL+ cells transduced with IL-3 virus is shown. Uninfected WT BM cells were used as a negative control. Quadrants were determined using uninfected WT cells and GFP or YFP single-positive cells. (B) Survival analysis. Retrovirus transduced cells were transplanted into lethally irradiated syngeneic recipient mice (n = 10 in each group). Survival curves were generated according to the Kaplan-Meier method using StatView version 5.0 software (SAS Institute Inc, Cary, NC). Hiroyuki Kawagoe, and Gerard C. Grosveld Blood 2005;106:4269-4277 ©2005 by American Society of Hematology

Characteristics of the myeloproliferative disease induced by coexpression of MN1-TEL and IL-3. Characteristics of the myeloproliferative disease induced by coexpression of MN1-TEL and IL-3. (A) Myeloproliferation in mice that received transplants of MN1-TEL+/IL-3 cells. (Left) Hematoxylin-Eosin (H-E) staining of BM; (middle) myeloperoxidase staining of BM; (right) M-G staining of BM cells. An arrow indicates a cell with differentiated morphology. (B) Aggressive organ infiltration by myeloid cells in mice that received transplants of MN1-TEL+/IL-3 bone marrow. H-E staining of liver (left), spleen (middle), and lung (right). Arrows indicate infiltrations of the myeloid cells. Images were obtained as previously described.12 (C) GFP and YFP expression in BM cells from mice that received transplants of MN1-TEL+/IL-3 cells. FCM analysis was performed as described in Figure 3A. (D) Clonality of MN1-TEL+/IL-3+ myeloproliferative disease (MPD). Genomic DNA of MPD BM cells was digested with BglII and hybridized with an Il3 probe. Control indicates untransduced BM; and G, germ line band. Hiroyuki Kawagoe, and Gerard C. Grosveld Blood 2005;106:4269-4277 ©2005 by American Society of Hematology

HOXA9 and MN1-TEL cooperatively cause acute myeloid leukemia (AML) in mice. HOXA9 and MN1-TEL cooperatively cause acute myeloid leukemia (AML) in mice. (A) Expression of HOXA9 in AML patient samples with MN1-TEL. A quantitative RT-PCR result is shown. NBM indicates control normal BM (n = 3); and Pt, patient samples (n = 2). (B) Retroviral transduction of MN1-TEL+ BM cells. Sorted MN1-TEL+ or MN1-TEL- Lin- BM cells were transduced with pSRα-HOXA9-IRES-YFP (HOXA9) or pSRα-IRES-YFP (vector) retrovirus. The FCM analysis of GFP/YFP expression in MN1-TEL+ BM cells transduced with HOXA9 virus is shown on the right. Control indicates uninfected WT BM cells. (C) Survival analysis. Retrovirally transduced BM cells (MN1-TEL+/HOXA9+, MN1-TEL+/Vector, MN1-TEL-/HOXA9+, MN1-TEL-/Vector) were transplanted into lethally irradiated syngeneic recipients (n = 10 in each group). Survival curves were generated by the Kaplan-Meier method. ○ indicates AML; and •, T-lymphoid leukemia. Hiroyuki Kawagoe, and Gerard C. Grosveld Blood 2005;106:4269-4277 ©2005 by American Society of Hematology

Characteristics of AML caused by MN1-TEL+/HOXA9+ BM cells. Characteristics of AML caused by MN1-TEL+/HOXA9+ BM cells. (A) GFP and YFP expression in AML cells. BM cells of mice that received leukemic MN1-TEL+/HOXA9 transplants were analyzed by FCM. (B) Expression of MN1-TEL and HOXA9 in AML cells. Western blot of BM cells was incubated with TEL C-terminal or FLAG (for HOXA9) antibody. Gapdh detection was used as a loading control. Control indicates untransduced normal BM. (C) M-G staining of AML cells. Images were obtained as previously described.12 (D) Surface marker analysis of AML cells by FCM. (E) Aggressive organ infiltration of AML cells. H-E staining of liver and spleen sections is shown. (F) Clonality of AML cells. Genomic DNA of AML cells was digested with BglII and hybridized with a HOXA9 probe. Control indicates untransduced BM; and G, germ line band. Hiroyuki Kawagoe, and Gerard C. Grosveld Blood 2005;106:4269-4277 ©2005 by American Society of Hematology

Elevated expression of N-Myc in human and mouse MN1-TEL+/HOXA9+ AML Elevated expression of N-Myc in human and mouse MN1-TEL+/HOXA9+ AML. A quantitative RT-PCR result is shown. Elevated expression of N-Myc in human and mouse MN1-TEL+/HOXA9+ AML. A quantitative RT-PCR result is shown. (Left) Expression of N-Myc mRNA in mouse MN1-TEL+/HOXA9+ AML cells (BM); (right) expression of N-MYC mRNA in 2 human MN1-TEL AML patient samples. NBM indicates control normal BM (n = 3); and Pt, patient samples (n = 2). Hiroyuki Kawagoe, and Gerard C. Grosveld Blood 2005;106:4269-4277 ©2005 by American Society of Hematology