The Myc/Max/Mxd Network Is a Target of Mutated Flt3 Signaling in Hematopoietic Stem Cells in Flt3-ITD-Induced Myeloproliferative Disease

Abstract

Acute myeloid leukemia (AML) has poor prognosis due to various mutations, e.g., in the FLT3 gene. Therefore, it is important to identify pathways regulated by the activated Flt3 receptor for the discovery of new therapeutic targets. The Myc network of oncogenes and tumor suppressor genes is involved in mechanisms regulating proliferation and survival of cells, including that of the hematopoietic system. In this study, we evaluated the expression of the Myc oncogenes and Mxd antagonists in hematopoietic stem cell and myeloid progenitor populations in the Flt3-ITD-knockin myeloproliferative mouse model. Our data shows that the expression of Myc network genes is changed in Flt3-ITD mice compared with the wild type. Mycn is increased in multipotent progenitors and in the pre-GM compartment of myeloid progenitors in the ITD mice while the expression of several genes in the tumor suppressor Mxd family, including Mxd1, Mxd2, and Mxd4, is concomitantly downregulated, as well as the expression of the Mxd-related gene Mnt and the transcriptional activator Miz-1. LSKCD150⁺CD48^- hematopoietic long-term stem cells are decreased in the Flt3-ITD cells while multipotent progenitors are increased. Of note, PKC412-mediated inhibition of Flt3-ITD signaling results in downregulation of cMyc and upregulation of the Myc antagonists Mxd1, Mxd2, and Mxd4. Our data provides new mechanistic insights into downstream alterations upon aberrant Flt3 signaling and rationale for combination therapies for tyrosine kinase inhibitors with Myc antagonists in treating AML.

Figure 1

Myeloid progenitor and hematopoietic stem cell populations are changed in Flt3-ITD mice. Analysis of hematopoietic stem cell (HSC) and multipotent progenitor (MPP) subpopulations within the Lin⁻Sca-1⁺Kit⁺ (LSK) population, as well as myeloid progenitors including pre-GM and granulocytic myeloid progenitors (GMPs), in the bone marrow of Flt3-ITD and wild-type (WT) mice, was performed using a staining procedure including endoglin and the SLAM receptor CD150.

Figure 2

Myeloid and multipotent progenitors have altered Myc network genes in Flt3-ITD mice. The expression of Myc network genes was carried out with reverse transcription quantitative PCR (RT-Q-PCR) in sorted subpopulations of murine hematopoietic stem and progenitor cells. Fold induction was calculated as a ratio of Flt3-ITD : WT samples. Statistical analysis was performed with Student's t-test on log-converted values (^∗p < 0.05). Significance was shown in many of the described altered expression levels; however, some changes showed low p values but did not quite reach statistically significance due to variances in the expression levels.

Figure 3

Flt3-ITD mice have altered frequencies of hematopoietic stem and progenitor cells. Next, we subdivided the hematopoietic stem cell compartment into long-term HSCs (LT-HSCs) and MPPs, utilizing the SLAM receptor staining with CD150 and CD48, and cells were sorted by flow cytometry for subsequent gene expression analysis.

Figure 4

Hematopoietic stem cells in Flt3-ITD mice have altered Myc network expression. The expression of Myc network genes was carried out with reverse transcription quantitative PCR (RT-Q-PCR) in hematopoietic stem cells. Fold induction was calculated as a ratio of Flt3-ITD : WT samples. Statistical analysis was performed with Student's t-test on log-converted values (^∗p < 0.05, ^∗∗p < 0.01).

Figure 5

Small molecule inhibitor, PKC412, modulates the expression of Myc network genes in human MV4-11 cells. The human Flt3-ITD mutated leukemia cell line, MV4-11, was treated with the Flt3-inhibitor PKC412 at two different concentrations (0.1 mM and 1 mM) for 15 minutes. Expression analysis of the Myc network genes was carried out with RT-Q-PCR. ND: not detected.