Combination of midostaurin and ATRA exerts dose-dependent dual effects on acute myeloid leukemia cells with wild type FLT3.

Midostaurin combined with chemotherapy is currently used to treat newly diagnosed acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 (FLT3)-mutations. However, midostaurin acts as an antagonist to some chemotherapeutic agents in leukemia cell lines without FLT3 mutations. All-trans retinoic acid (ATRA) induces apoptosis when used in combination with midostaurin in FLT3-mutated AML cells. This combination has been shown to be safe in AML patients. However, the effect of this combination has not been investigated in AML without FLT3 mutations. Cell proliferation was assessed by a cell counting assay. Cell death was evaluated by cell viability and Annexin-V assays. Cell differentiation was assessed by CD11b expression profiling and morphological analysis. To explore the underlying mechanisms, we studied the role of caspase3/7, Lyn, Fgr, Hck, RAF, MEK, ERK, AKT, PU.1, CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε by Western blot analysis and immunoprecipitation assays. Antitumor activity was also confirmed in mouse xenograft models established with AML cells. In this study, 0.1 - 0.25 μM midostaurin (mido(L)) combined with ATRA induced differentiation while 0.25 - 0.5 μM midostaurin (mido(H)) combined with ATRA triggered apoptosis in some AML cell lines without FLT3-mutations. Midostaurin combined with ATRA (mido-ATRA) also exhibited antitumor activity in mouse xenograft models established with AML cells. Mechanistically, mido(H)-ATRA-induced apoptosis was dependent on caspase-3/7. Mido(L)-ATRA inhibited Akt activation which was associated with decreased activity of Lyn/Fgr/Hck, resulted in dephosphorylation of RAF S259, activated RAF/MEK/ERK, along with upregulating the protein levels of C/EBPβ, C/EBPε and PU.1. A MEK specific inhibitor was observed to suppress mido(L)-ATRA-induced increases in the protein levels of C/EBPs and PU.1 and mido(L)-ATRA-induced differentiation. Furthermore, inhibition of Akt activity promoted mido(L)-ATRA-induced downregulation of RAF S259 phosphorylation and mido(L)-ATRA-induced differentiation. Therefore, Lyn/Fgr/Hck-associated Akt inhibition activated RAF/MEK/ERK and controlled mido(L)-ATRA-induced differentiation by upregulation of C/EBPs and PU.1. Mido(L)-ATRA also promoted assembly of the signalosome, which may facilitate RAF activation. Midostaurin combined with ATRA exerts antitumor activity against AML with wild-type FLT3 mutations in vitro and in vivo. These findings may provide novel therapeutic strategies for some AML patients without FLT3 mutations and imply a new target of midostaurin.

Lu H ，Weng XQ ，Sheng Y ，Wu J ，Xi HM ，Cai X ... -  《BMC CANCER》

Inhibition of Bcl-2 Synergistically Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Preclinical Models of FLT3-Mutated Acute Myeloid Leukemia.

To investigate the efficacy of the combination of the FLT3 inhibitors midostaurin or gilteritinib with the Bcl-2 inhibitor venetoclax in FLT3-internal tandem duplication (ITD) acute myeloid leukemia (AML) and the underlying molecular mechanism. Using both FLT3-ITD cell lines and primary patient samples, Annexin V-FITC/propidium iodide staining and flow cytometry analysis were used to quantify cell death induced by midostaurin or gilteritinib, alone or in combination with venetoclax. Western blot analysis was performed to assess changes in protein expression levels of members of the JAK/STAT, MAPK/ERK, and PI3K/AKT pathways, and members of the Bcl-2 family of proteins. The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the combination of gilteritinib and venetoclax. Lentiviral overexpression of Mcl-1 was used to confirm its role in cell death induced by midostaurin or gilteritinib with venetoclax. Changes of Mcl-1 transcript levels were assessed by RT-PCR. The combination of midostaurin or gilteritinib with venetoclax potently and synergistically induces apoptosis in FLT3-ITD AML cell lines and primary patient samples. The FLT3 inhibitors induced downregulation of Mcl-1, enhancing venetoclax activity. Phosphorylated-ERK expression is induced by venetoclax but abolished by the combination of venetoclax with midostaurin or gilteritinib. Simultaneous downregulation of Mcl-1 by midostaurin or gilteritinib and inhibition of Bcl-2 by venetoclax results in "free" Bim, leading to synergistic induction of apoptosis. In vivo results show that gilteritinib in combination with venetoclax has therapeutic potential. Inhibition of Bcl-2 via venetoclax synergistically enhances the efficacy of midostaurin and gilteritinib in FLT3-mutated AML.See related commentary by Perl, p. 6567.

Ma J ，Zhao S ，Qiao X ，Knight T ，Edwards H ，Polin L ，Kushner J ，Dzinic SH ，White K ，Wang G ，Zhao L ，Lin H ，Wang Y ，Taub JW ，Ge Y ... -  《-》

Combination of Ethacrynic Acid and ATRA Triggers Differentiation and/or Apoptosis of Acute Myeloid Leukemia Cells through ROS.

Li L ，Xi HM ，Lu H ，Cai X ... -  《-》

Dasatinib synergizes with ATRA to trigger granulocytic differentiation in ATRA resistant acute promyelocytic leukemia cell lines via Lyn inhibition-mediated activation of RAF-1/MEK/ERK.

All-trans retinoic acid (ATRA) resistance has been a critical problem in acute promyelocytic leukemia (APL) relapsed patients. In this study, dasatinib synergized with ATRA to trigger differentiation in ATRA-resistant APL cell lines. The combined treatment activated RAF-1, MEK and ERK as well as enhanced ATRA-promoted up-regulation of the protein level of PU.1, C/EBPβ and C/EBPε. U0126 (MEK specific inhibitor) and sorafenib tosylate (RAF-1 specific inhibitor) suppressed the combined treatment-induced differentiation, ERK phosphorylation and the up-regulation of C/EBPs and PU.1. Sorafenib tosylate also attenuated the MEK activity. However, the combined treatment did not enhance Ras activity and Ras inhibitor neither blocked MEK activation nor inhibited differentiation. Therefore, the combined treatment induced differentiation via Ras independent RAF-1/MEK/ERK. Earlier than RAF-1 activation, dasatinib suppressed Lyn activity, the predominant activated Src family kinase (SFK) and dephosphorylated RAF-1 at S259. Furthermore, SFK inhibitor, PP2 did suppress Lyn activity and mimicked the effect of dasatinib on ATRA-induced differentiation as well as decreased phosphorylation of RAF-1 at S259. Thus, it was suggested that Lyn inhibition might activate RAF-1 by the dephosphorylation of RAF at S259 and lead to differentiation. In conclusion, the combination of dasatinib and ATRA could overcome ATRA resistance through Lyn inhibition-mediated activation of RAF-1/MEK/ERK.

Ding M ，Weng XQ ，Sheng Y ，Wu J ，Liang C ，Cai X ... -  《-》

Effects of the multi-kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia.

Recently, several targeted agents have been developed for specific subsets of patients with acute myeloid leukaemia (AML), including midostaurin, the first FDA-approved FLT3 inhibitor for newly diagnosed patients with FLT3 mutations. However, in the initial Phase I/II clinical trials, some patients without FLT3 mutations had transient responses to midostaurin, suggesting that this multi-targeted kinase inhibitor might benefit AML patients more broadly. Here, we demonstrate submicromolar efficacy of midostaurin in vitro and efficacy in vivo against wild-type (wt) FLT3-expressing AML cell lines and primary cells, and we compare its effectiveness with that of other FLT3 inhibitors currently in clinical trials. Midostaurin was found to synergize with standard chemotherapeutic drugs and some targeted agents against AML cells without mutations in FLT3. The mechanism may involve, in part, the unique kinase profile of midostaurin that includes proteins implicated in AML transformation, such as SYK or KIT, or inhibition of ERK pathway or proviability signalling. Our findings support further investigation of midostaurin as a chemosensitizing agent in AML patients without FLT3 mutations.

Weisberg E ，Meng C ，Case AE ，Tiv HL ，Gokhale PC ，Buhrlage SJ ，Yang J ，Liu X ，Wang J ，Gray N ，Adamia S ，Sattler M ，Stone R ，Griffin JD ... -  《-》