Requirement of c-Myb for p210BCR/ABL-dependent transformation of hematopoietic progenitors and leukemogenesis by Maria Rosa Lidonnici, Francesca Corradini, Todd Waldron, Timothy P. Bender, and Bruno Calabretta Blood Volume 111(9):4771-4779 May 1, 2008 ©2008 by American Society of Hematology

Loss of a c-Myb allele does not affect the frequency of hematopoietic progenitors. Loss of a c-Myb allele does not affect the frequency of hematopoietic progenitors. (A) Western blot analysis of c-Myb expression in enriched Lin− and Lin−Sca-1+Kit+ cells from c-Mybf/f and c-Mybf/d mice. GRB2 expression served as a control. (B) Frequency of Lin−Kit+, Lin−Sca-1+, and Lin−Sca-1+Kit+ cells (Q1, Q3, and Q2) in enriched Lin− cells of c-Mybf/f and c-Mybf/d mice, assessed by use of PE-conjugated anti–Sca-1 and APC-conjugated anti–c-Kit antibodies on enriched Lin− marrow cells. (C) Flow cytometric analysis of c-Kit expression in Lin−Sca-1+Kit+ cells from c-Mybf/f and c-Mybf/d mice. Representative of 3 experiments. Maria Rosa Lidonnici et al. Blood 2008;111:4771-4779 ©2008 by American Society of Hematology

Hematopoietic reconstitution in mice injected with c-Mybf/d or c-Mybf/f bone marrow cells. Hematopoietic reconstitution in mice injected with c-Mybf/d or c-Mybf/f bone marrow cells. Bone marrow cells (106) from a c-Mybf/f or a c-Mybf/d mouse were injected in 3 lethally irradiated syngenic mice (11 Gy, 2 split doses of 5.5 Gy rad/each). After 3 months, the proportion of bone marrow cells expressing the Sca-1, c-Kit, Gr-1, and Mac-1 antigens was tested by immunostaining. Histograms show frequency of Sca-1+c-Kit−, Sca-1−c-Kit+, Sca-1+c-Kit+, Gr-1+, and Mac-1+ cells in the bone marrow of mice injected with c-Mybf/f (A) or c-Mybf/d (B) bone marrow cells. None of the differences in the values of histograms in panels A,B is statistically significant. As control, lethally irradiated mice not injected with bone marrow cells died after 2 weeks; bone marrow cells purified from mice injected with c-Mybf/d cells were genotyped and showed the distinctive polymerase chain reaction (PCR) pattern of c-Mybf/d donor cells. Maria Rosa Lidonnici et al. Blood 2008;111:4771-4779 ©2008 by American Society of Hematology

Loss of a c-Myb allele has modest effects on colony formation of bone marrow progenitors. Loss of a c-Myb allele has modest effects on colony formation of bone marrow progenitors. Colony formation assays were performed using (A) enriched Lin− marrow cells (5 × 103 cells/plate) plated in methylcellulose in the presence of GM-CSF (P = .015); (B) Lin−Sca-1+ marrow cells (5 × 103cells/plate) plated in methylcellulose in the presence of IL-3, IL-6, and KL (P = .008); and (C) Lin−Sca-1+Kit+ marrow cells (2.5 × 105/plate) plated in methylcellulose in the presence of optimal (IL-3, 6 ng/mL; IL-6, 10 ng/mL; KL, 50 ng/mL) or suboptimal (1/10th) cytokine concentrations; P = .011 and P ≤ .001, respectively. Representative of 3 different experiments performed in duplicate. Maria Rosa Lidonnici et al. Blood 2008;111:4771-4779 ©2008 by American Society of Hematology

Low levels of c-Myb reduce colony formation of p210BCR/ABL-transduced marrow progenitors. Low levels of c-Myb reduce colony formation of p210BCR/ABL-transduced marrow progenitors. (A) Methylcellulose colony formation of p210BCR/ABL-transduced purified Lin− cells (2.5 × 103/plate) in the presence of optimal cytokine concentrations from c-Mybf/f or c-Mybf/d mice. This assay was performed once in duplicate plates. Inset shows c-Myb levels detected by Western blotting. (B) Methylcellulose colony formation of p210BCR/ABL-transduced Lin−Sca-1+ cells (5 × 103cells/plate) isolated from c-Mybf/f or c-Mybf/d mice, in the presence of optimal cytokine concentrations. Values (P ≤ .001) are representative of 3 different experiments performed in duplicate. Maria Rosa Lidonnici et al. Blood 2008;111:4771-4779 ©2008 by American Society of Hematology

Colony formation of p210BCR/ABL-transduced c-Mybf/d HSCs Colony formation of p210BCR/ABL-transduced c-Mybf/d HSCs. Lin−Sca-1+Kit+ cells from c-Mybf/f or c-Mybf/d mice were transduced with the MigRI-p210BCR/ABL retrovirus, GFP sorted, assessed for p210BCR/ABL levels, and plated in methylcellulose (2.5 × 103 cells/... Colony formation of p210BCR/ABL-transduced c-Mybf/d HSCs. Lin−Sca-1+Kit+ cells from c-Mybf/f or c-Mybf/d mice were transduced with the MigRI-p210BCR/ABL retrovirus, GFP sorted, assessed for p210BCR/ABL levels, and plated in methylcellulose (2.5 × 103 cells/plate) in the presence of an optimal cytokine concentration (A) or 1/10th of the full cytokine doses (B). In panel A, inset shows p210BCR/ABL levels detected by anti–c-Abl Western blotting. Colonies were scored after 7 days. Values (P ≤ .001) are expressed as the percentage of the number of colonies derived from c-Mybf/f HSCs. Secondary colony formation assays were performed using cells isolated from methylcellulose. Cells were replated onto new plates in the presence of an optimal cytokine concentration (C) or 1/10th of the cytokines (D) and scored again after 7 days. Values (P ≤ .001) are expressed as the percentage of the number of colonies derived from c-Mybf/f HSCs. Representative of 3 different experiments. Maria Rosa Lidonnici et al. Blood 2008;111:4771-4779 ©2008 by American Society of Hematology

Restoration of c-Myb expression rescues colony formation of c-Mybf/d p210BCR/ABL-transduced marrow progenitors. c-Mybf/d and c-Mybf/f Lin−Sca-1+Kit+ cells were transduced with the MigRI/p210BCR/ABL retrovirus and 24 hours after retroviral transduction sorte... Restoration of c-Myb expression rescues colony formation of c-Mybf/d p210BCR/ABL-transduced marrow progenitors. c-Mybf/d and c-Mybf/f Lin−Sca-1+Kit+ cells were transduced with the MigRI/p210BCR/ABL retrovirus and 24 hours after retroviral transduction sorted for GFP positivity. p210BCR/ABL-expressing c-Mybf/d Lin−Sca-1+Kit+ cells were subsequently transduced with a c-Myb/MSCV/puro retrovirus or the empty MSCV/puro vector as a negative control. p210BCR/ABL-expressing c-Mybf/f Lin−Sca-1+Kit+ cells were transduced with empty vector MSCV/puro as a positive control. After 24 hours, cells (8 × 104/plate) were plated in methylcellulose in the presence of puromycin (1 μg/mL). Colony formation was assessed 9 days later. Representative of 3 different experiments. P ≤ .001. Maria Rosa Lidonnici et al. Blood 2008;111:4771-4779 ©2008 by American Society of Hematology

Ectopic Bcl-2 expression does not enhance colony formation of c-Mybf/d p210BCR/ABL-transduced HSCs. (A) Expression of c-Myb–regulated genes in p210BCR/ABL-transduced c-Mybf/f and c-Mybf/d progenitors. Ectopic Bcl-2 expression does not enhance colony formation of c-Mybf/d p210BCR/ABL-transduced HSCs. (A) Expression of c-Myb–regulated genes in p210BCR/ABL-transduced c-Mybf/f and c-Mybf/d progenitors. Western blot analysis of c-Myb, c-Myc, cyclin B1, and Bcl-2 expression in nontransduced and p210BCR/ABL-transduced enriched Lin− cells from c-Mybf/f and c-Mybf/d mice. Actin expression served as a control. (B) Colony formation of Bcl-2–transduced p210BCR/ABL-expressing c-Mybf/d progenitors. c-Mybf/d Lin−Sca-1+Kit+ cells were transduced with the MigRI/p210BCR/ABL retrovirus and 24 hours after retroviral transduction sorted for GFP expression. p210BCR/ABL-expressing c-Mybf/d Lin−Sca-1+Kit+ cells were subsequently transduced with a Bcl-2/MSCV/puro retrovirus or the empty MSCV/puro vector as a negative control. After 24 hours, cells (4 × 104/plate) were plated in methylcellulose in the presence of puromycin (1 μg/mL). Colony formation was assessed 9 days later. Representative of 3 different experiments. Maria Rosa Lidonnici et al. Blood 2008;111:4771-4779 ©2008 by American Society of Hematology

c-Myb is required for p210BCR/ABL-dependent leukemogenesis. c-Myb is required for p210BCR/ABL-dependent leukemogenesis. (A) Colony formation assays of p210BCR/ABL p53−/−c-Mybww or p53−/−c-Mybw/d marrow progenitors. Lin−Sca-1+Kit+ HSCs isolated from 5-FU–treated p53−/−c-Mybww or p53−/−c-Mybw/d mice were retrovirally transduced with MigR1p210BCR/ABL and, after GFP sorting, assessed for colony formation after plating in methylcellulose in the presence of a full dose or 1/10th of cytokines (IL-3, IL-6, and KL). Colonies were counted after 7 days. Representative of 3 different experiments performed in duplicate. P ≤ .001. (B) Survival of recipient mice injected with p210BCR/ABL-transduced marrow cells Kaplan-Meier plot shows survival of C57BL/6J recipient mice after transplantation with p210BCR/ABL p53−/−c-Mybw/w bone marrow cells (n = 14) or p210BCR/ABL p53−/−c-Mybw/d bone marrow cells (n = 12). Bone marrow cells from 5-FU–treated (150 mg/kg, 4 days), p53−/−c-Mybw/w or p53−/−c-Mybf/d mice were retrovirally transduced with the MigR1p210Bcr/Abl retrovirus and injected in lethally irradiated (1100 rad in 2 split doses of 550 each) syngenic C57BL/6J recipient mice. (C) Secondary leukemia in mice injected with GFP-positive cells isolated from mice with p210BCR/ABL-induced primary leukemia. Kaplan-Meier plot shows survival of C57BL/6J recipient mice (n = 3) injected with GFP-positive cells from a mouse with leukemia induced by p210BCR/ABL p53−/−c-Mybw/w cells or of recipient mice (n = 3) injected with GFP-positive cells from a mouse with leukemia induced by p210BCR/ABL p53−/−c-Mybw/d cells. Sublethally irradiated (500 rad) recipient mice each received with 1.0 × 105 GFP+ bone marrow cells. Maria Rosa Lidonnici et al. Blood 2008;111:4771-4779 ©2008 by American Society of Hematology