An activated receptor tyrosine kinase, TEL/PDGFbetaR, cooperates with AML1/ETO to induce acute myeloid leukemia in mice

Abstract

The t(8;21)(q22;q22) translocation, occurring in 40% of patients with acute myeloid leukemia (AML) of the FAB-M2 subtype (AML with maturation), results in expression of the RUNX1-CBF2T1 [AML1-ETO (AE)] fusion oncogene. AML/ETO may contribute to leukemogenesis by interacting with nuclear corepressor complexes that include histone deacetylases, which mediate the repression of target genes. However, expression of AE is not sufficient to transform primary hematopoietic cells or cause disease in animals, suggesting that additional mutations are required. Activating mutations in receptor tyrosine kinases (RTK) are present in at least 30% of patients with AML. To test the hypothesis that activating RTK mutations cooperate with AE to cause leukemia, we transplanted retrovirally transduced murine bone marrow coexpressing TEL-PDGFRB and AE into lethally irradiated syngeneic mice. These mice (19/19, 100%) developed AML resembling M2-AML that was transplantable in secondary recipients. In contrast, control mice coexpressing with TEL-PDGFRB and a DNA-binding-mutant of AE developed a nontransplantable myeloproliferative disease identical to that induced by TEL-PDGFRB alone. We used this unique model of AML to test the efficacy of pharmacological inhibition of histone deacetylase activity by using trichostatin A and suberoylanilide hydroxamic acid alone or in combination with the tyrosine kinase inhibitor, imatinib mesylate. We found that although imatinib prolonged the survival of treated mice, histone deacetylase inhibitors provided no additional survival benefit. These data demonstrate that an activated RTK can cooperate with AE to cause AML in mice, and that this system can be used to evaluate novel therapeutic strategies.

Fig. 1.

Rapid mortality in mice coexpressing TP and AE. (A) Structure of retroviral constructs derived from the MSCV vector. LTR, long terminal repeats; Ψ, packaging signal. (B) Expression of retroviral constructs. Whole cell protein extracts from bone marrow (M) of the indicated mice or transfected 293T cells were analyzed by immunoblotting with antibodies to PDGFβR, ETO, or β-actin. TP-tag (32D), 32D cell line expressing His-tagged TEL-PDFGβR; Kasumi, AML1/ETO expressing cell line from a M2-AML patient. (C) Kaplan–Meier plot showing survival of mice after transplantation with bone marrow cells transduced with the retroviral constructs. MSCV-eGFP in wild-type B6129 mice (•, n = 4); MSCV-AE-eGFP in B6129 wild-type +/+ (▴, n = 5) or Runx1^+/- mice (▵, n = 8); MSCV-TP-AE in +/+ (▪, n = 10) or Runx1^+/- (□, n = 9); and MSCV-TP-148, +/+ (♦, n = 10), Runx1^+/- (⋄, n = 5).

Fig. 2.

MSCV-TP-AE mice develop transplantable AML. (A) Bone marrow cells stained with May–Grunwald/Giemsa. (B) Bone marrow differential counts. Averages of the different populations based on 200 total cells are shown ±SD. Wild type, n = 3; MSCV-TP-AE, n = 5; MSCV-TP-148, n = 5, MSCV-TP-GFP, n = 3. Blast+Pro, myeloblasts and promyelocytes; Myelo+Meta, myelocytes and metamyelocytes; Band+PMN, band and mature neutrophils; Eryth., erythroid precursors; Lymph., lymphocytes. (C) Survival of MSCV-TP-AE or MSCV-TP-148 secondary recipients. MSCV-TP-AE secondary recipients (▾, n = 5) develop a rapidly fatal myeloid leukemia similar to the primary recipients demonstrating transplantability of the leukemic phenotype. However, recipients of MSCV-TP-148 splenocytes remain disease-free (▴, n = 10).

Fig. 3.

Neoplastic cells from MSCV-TP-AE mice have an immature myeloid immunophenotype. Bone marrow cells isolated from MSCV-TP-AE, MSCV-TP-148, MSCV-TP-eGFP, and wild-type mice were analyzed by flow cytometry with antibodies to detect expression of the progenitor cell markers, CD 117 (c-Kit) and Sca-1.

Fig. 4.

Survival of MSCV-TP-AE secondary recipients treated with HDAC inhibitors and/or imatinib mesylate. (A) B6129 F₁ secondary recipient mice were transplanted with splenocytes from a primary MSCV-TP-AE mouse and then treated daily with vehicle (DMSO, ▴), 2mg/kg TSA (•), 5mg/kg TSA (○), or 50 mg/kg SAHA (⋄) per day. (B) B6129 F₁ secondary recipient mice were transplanted with splenocytes from a primary MSCV-TP-AE mouse and then treated daily with vehicle (10% DMSO/PBS) (•), 1mg/kg TSA per day (▵), 50 mg/kg imatinib per day (▪), or TSA plus imatinib (▴)(n = 5 for each arm). (C) Acetylation of histone H4 in the bone marrow of mice 2 h after treatment with vehicle, 5 mg/kg TSA per day, or 50 mg/kg SAHA per day.