Research ArticleCancer

Integration of Hedgehog and mutant FLT3 signaling in myeloid leukemia

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Science Translational Medicine  10 Jun 2015:
Vol. 7, Issue 291, pp. 291ra96
DOI: 10.1126/scitranslmed.aaa5731
  • Fig. 1. GLI2 expression is increased in human FLT3-ITD AML.

    (A) GLI2 expression in human AML data sets (probe GLI2 − 228537_at) or TCGA. (B) Relative GLI2 and BCL2 expression in primary AML samples compared to normal CD34+ HSPCs [n = 5 for HSPCs, wild-type (WT) FLT3, and FLT3-ITD specimens]. Data represent mean ± SD. n.d., not detected.

  • Fig. 2. Flt3/ITD-SmoM2 mice develop rapidly fatal AML.

    (A) Kaplan-Meier survival curve of transgenic animals after poly(I:C) induction. Statistical significance determined by log-rank (Mantel-Cox) test comparing Flt3/ITD and Flt3/ITD-SmoM2 animals. (B) White blood cell (WBC) counts at 3 months after the completion of poly(I:C) treatment. Data represent mean ± SD. (C) Wright-Giemsa staining of peripheral blood smears. (D) Fluorescence-activated cell sorting (FACS) analysis of peripheral blood cells. Bar graph represents percentage of Mac1+Gr1+ cells (n = 5 to 7 per genotype). Data represent mean ± SD. (E) FACS analysis of bone marrow cells. Bar graph depicts bone marrow cellularity and percentage of Gr1+cKit+ cells (n = 3 to 7 per genotype). Data represent mean ± SD. (F) Spleen weights and representative spleen sizes. Data represent mean ± SD. (G) Hematoxylin and eosin staining of bone marrow and spleen sections. RP, red pulp; WP, white pulp.

  • Fig. 3. Myeloid progenitors are expanded and more proliferative in Flt3/ITD-SmoM2 mice.

    (A) Representative FACS plots depicting relative percentages of stem and progenitor KSL (LinSca1+cKit+), myeloid progenitor (LinSca1cKit+), GMP (LinSca1cKit+CD34+CD16/32+), CMP (LinSca1cKit+CD34+CD16/32), and MEP (LinSca1cKit+CD34CD16/32) cells in bone marrow of transgenic animals. (B) Total number of cells of each specific type per femur (n = 3 to 7 animals per genotype). Data represent mean ± SD. n.s., not significant. (C) Representative FACS plots of cells with BrdU incorporation in KSL, CMP, GMP, and MEP compartments. (D) Frequency of BrdU-positive cells from each population (n = 5). Data represent mean ± SD.

  • Fig. 4. STAT5 signaling is enhanced in Flt3/ITD-SmoM2 mice.

    (A) GSEA analysis comparing Flt3/ITD and Flt3/ITD-SmoM2 mice. NES, normalized enrichment score; FDR, false discovery rate. (B) Quantitative reverse transcription PCR (qRT-PCR) analysis of STAT5 target genes in sorted GMP cells (n = 3). Data represent mean ± SD.

  • Fig. 5. IPI-926 and sorafenib inhibit leukemic cell growth in vitro.

    (A) Viable cell counts of MV4-11 cells treated with IPI-926 and sorafenib for 3 days (n = 4). Data represent mean ± SD. (B) Viable cell counts of MV4-11 cell lines treated with recombinant SHH ligand and sorafenib for 3 days (n = 3). Data represent mean ± SD. (C) Viable cell counts of clinical FLT3-ITD AML specimens treated with IPI-926 and sorafenib for 3 days (n = 4 individual patients). Data represent mean ± SD. (D) Cell cycle distribution of MV4-11 cells treated with IPI-926 and sorafenib after 3 days (n = 3). Data represent mean ± SD. (E) Relative colony formation of MV4-11 cells in methylcellulose after treatment with IPI-926 and sorafenib for 3 days compared to vehicle control (n = 4). Data represent mean ± SD. (F) Western blot of pSTAT5, total STAT5, and tubulin from MV4-11 cells treated for 3 days. (G) Relative ratio of pSTAT5 to total STAT5 (n = 4). Data represent mean ± SD. (H) Relative levels of total STAT5 normalized to tubulin (n = 4). Data represent mean ± SD.

  • Fig. 6. Combined IPI-926 and sorafenib treatment improves survival.

    (A) Representative spleen sizes of animals after 16 days of drug treatment. (B) Frequency of abnormal Gr1+Mac1+ and cKit+Gr1+ cells in the peripheral blood and bone marrow, respectively, of animals treated with drugs for 16 days (n = 3 per group). Data represented as mean ± SD. (C) Kaplan-Meier survival curve of animals treated with IPI-926 and sorafenib (n = 3 to 5 per group). Statistical significance determined by log-rank (Mantel-Cox) test. (D) pSTAT5 and total STAT5 in bone marrow cells harvested after 24 hours of drug treatment. (E) Ratio of pSTAT5 to total STAT5, normalized to vehicle-treated cells (n = 3). Data represent mean ± SD.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/291/291ra96/DC1

    Fig. S1. GLI2 is expressed in FLT3-ITD AML clinical specimens.

    Fig. S2. Flt3/ITD-SmoM2 transgenic mice express Hh pathway genes and develop clinically relevant AML.

    Fig. S3. Specific bone marrow hematopoietic stem cell and myeloid progenitor compartments are maintained in Flt3/ITD-SmoM2 mice.

    Fig. S4. Leukemia formation in Flt3/ITD-SmoM2 mice is cell-intrinsic.

    Fig. S5. GSEA reveals increased STAT5 signaling in Flt3/ITD and antiapoptotic features in Flt3/ITD-SmoM2 mice.

    Fig. S6. The combination of sorafenib and IPI-926 limits the growth of FLT3-ITD AML.

    Fig. S7. Hh signaling affects FLT3-ITD at the level of STAT5.

    Fig. S8. IPI-926 inhibits the expression of Hh target genes in Flt3/ITD-SmoM2 bone marrow cells.

    Table S1. Leukemic organ infiltration.

    Table S2. PCR primers.

    Table S3. Original data (provided as a separate Excel file).

  • Supplementary Material for:

    Integration of Hedgehog and mutant FLT3 signaling in myeloid leukemia

    Yiting Lim, Lukasz Gondek, Li Li, Qiuju Wang, Hayley Ma, Emily Chang, David L. Huso, Sarah Foerster, Luigi Marchionni, Karen McGovern, David Neil Watkins, Craig D. Peacock, Mark Levis, Bruce Douglas Smith, Akil A. Merchant, Donald Small, William Matsui*

    *Corresponding author. E-mail: matsuwi{at}jhmi.edu

    Published 10 June 2015, Sci. Transl. Med. 7, 291ra96 (2015)
    DOI: 10.1126/scitranslmed.aaa5731

    This PDF file includes:

    • Fig. S1. GLI2 is expressed in FLT3-ITD AML clinical specimens.
    • Fig. S2. Flt3/ITD-SmoM2 transgenic mice express Hh pathway genes and develop clinically relevant AML.
    • Fig. S3. Specific bone marrow hematopoietic stem cell and myeloid progenitor compartments are maintained in Flt3/ITD-SmoM2 mice.
    • Fig. S4. Leukemia formation in Flt3/ITD-SmoM2 mice is cell-intrinsic.
    • Fig. S5. GSEA reveals increased STAT5 signaling in Flt3/ITD and antiapoptotic features in Flt3/ITD-SmoM2 mice.
    • Fig. S6. The combination of sorafenib and IPI-926 limits the growth of FLT3-ITD AML.
    • Fig. S7. Hh signaling affects FLT3-ITD at the level of STAT5.
    • Fig. S8. IPI-926 inhibits the expression of Hh target genes in Flt3/ITD-SmoM2 bone marrow cells.
    • Table S1. Leukemic organ infiltration.
    • Table S2. PCR primers.
    • Other Supplementary Material for

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3. Original data (provided as a separate Excel file).

    [Download Table S3]

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