Vehicle-treated mice succumbed to disease at 25 days posttransplant, ATRA- and sorafenib-treated mice survived an average of 30 and 32 days, respectively, and mice treated with sorafenib plus ATRA had significantly extended survival to a median of 42 days posttransplant (*** .001, Figure 5D). Open in a separate window Figure 5 ATRA and sorafenib improve disease progression of AML and survival in mouse xenograft models. of leukemic mice. Furthermore, engraftment of primary FLT3/ITD+ patient samples is reduced in mice following treatment with FLT3 TKI and ATRA in combination, with evidence of cellular differentiation occurring in vivo. Mechanistically, we provide evidence that the synergism of ATRA and FLT3 TKIs is at least in part due to the observation that FLT3 TKI treatment upregulates the antiapoptotic protein Bcl6, limiting the drugs apoptotic effect. However, cotreatment with ATRA reduces Bcl6 expression to baseline levels through suppression of interleukin-6 receptor signaling. These studies provide evidence of the potential of this drug combination to eliminate FLT3/ITD+ LSCs and reduce the rate of relapse in AML patients with FLT3 mutations. Introduction Acute myeloid leukemia (AML) is an AX-024 aggressive bone marrow (BM) malignancy. FMS-like tyrosine kinase 3 (in preleukemic HSC clones.12 However, there remains compelling evidence that FLT3 mutations are present in the LSC fraction as defined by the ability of the cells to propagate the disease in immunodeficient mice. Of note, ITD mutations of FLT3 have been shown to be present in the lineage-negative CD34+/CD38? LSC fraction of primary AML samples at the same allelic ratio as unsorted cells, and in most cases this sorted LSC population is able to engraft AX-024 NOD-SCID mice.13 Other investigators have demonstrated that FLT3/ITD is present in the CD34+/CD33? population in nearly 80% of cases harboring the mutation at diagnosis, and the presence of the mutation within this primitive population portends a poor prognosis.14 In order for any AML therapy to be curative, it needs to be effective against not only the bulk leukemia cells but against the LSCs as well. Data have also shown the importance of several stem cell pathways in maintaining LSCs AX-024 in AML, including the retinoic acid, WNT/-catenin, Notch, and Hedgehog (Hh) pathways.15-18 Some of these pathways have already been shown to be active specifically in FLT3-mutant AML whereas others have been shown to be active broadly in AML.15-20 Each of these pathways are candidates for combination therapy with FLT3 inhibitors. Retinoic acid (RA) has been shown to play an important role in the differentiation of normal HSCs, and treatment with all-RA (ATRA) has greatly improved the AX-024 cure rate for acute promyelocytic leukemia (APL).21 Stimulation of the RA pathway with ATRA has also been tried as monotherapy for the AX-024 treatment of nonpromyelocytic AML patients and has shown some clinical activity, although it has not been replicated in all studies.15,22-25 Our hypothesis is that LSCs may be susceptible to treatment with ATRA and, when combined with FLT3 inhibition, these drugs will be able to overcome the block in differentiation as well as negate the strong proliferative and survival signaling characteristic of FLT3 mutations. Interestingly, APL patients expressing FLT3/ITD mutations are successfully treated with ATRA combined with chemotherapy and/or arsenic trioxide.26,27 Moreover, previous studies have reported on the combinatorial effect of ATRA and FLT3 inhibitors on apoptosis in FLT3/ITD+ cell lines.28,29 We therefore explored the combination of molecularly targeting the RA pathway together with FLT3 TKIs to determine the effect on FLT3-mutant LSCs. Methods Growth inhibition Cells were seeded at a density of 1 1 105 to 2.5 105 cells per mL in the presence or absence of compounds for the indicated times. Cell proliferation was measured in quadruplicate using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay according to the manufacturers instructions (Roche Applied Science). Viable cell counts were performed by Trypan blue exclusion at 24-hour intervals. For colony-forming unit (CFU) assays, cells were plated at a density of 5 102 to 2 104 cells per mL in methylcellulose (Methocult H4230, H4435, or M3434; Stem Cell Technologies) and incubated at 37C. Total Rabbit polyclonal to IL25 colony counts and/or burst-forming unit erythroid, CFU-granulocyte/macrophage, CFU-granulocyte, and CFU-macrophage counts were obtained after 7 to 14 days. Transplantation experiments Transplantation of Molm14, FLT3/ITD+ primary patient sample, and leukemic NHD13;FLT3/ITD BM cells was performed as described previously.30-32 For details on transplantation, drug.