Metformin synergistically sensitizes FLT3-ITD-positive acute myeloid leukemia to sorafenib by promoting mTOR-mediated apoptosis and autophagy.

Mutations of Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), accounting for approximately 30% of patients with acute myeloid leukemia (AML), results in poor therapeutic efficacy and short survival. Sorafenib, an oral multikinase inhibitor, can inhibit FLT3 and improve clinical outcome of FLT3 mutated leukemia. Our current studies have shown that, the antidiabetic drug metformin also exerts anti-leukemic effect by activating p-AMPK and synergistically sensitizes FLT3 mutated AML to sorafenib. Both agents suppress cell proliferation in a dose-dependent manner and induce apoptosis via cell cycle arrest, but does not obviously modulate autophagy marker, light chain 3 (LC3). Mechanistically, in the presence of metformin, the anticancer potential of sorafenib, accompanying with increased LC3 levels, is found to be synergistically enhanced with the remarkably reduced protein expression of the mTOR/p70S6K/4EBP1 pathway, while not appreciably altering cell cycle. Overall, these results show metformin in aid of sorafenib may represent a promising and attractive strategy for the treatment of FLT3-ITD mutated AML.

Wang F ，Liu Z ，Zeng J ，Zhu H ，Li J ，Cheng X ，Jiang T ，Zhang L ，Zhang C ，Chen T ，Liu T ，Jia Y ... -  《-》

Reversal of acquired drug resistance in FLT3-mutated acute myeloid leukemia cells via distinct drug combination strategies.

FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (FLT3-ITD) mutations are common in patients with acute myeloid leukemia (AML). These patients regularly develop resistance to FLT3 inhibitors suggesting that targeted combination drug strategies are needed to enhance AML therapy efficacy. Acquired point mutations of FLT3-ITD gene were screened using cDNA-based sequencing approach in vitro sorafenib-resistant cells, which were developed by long-term exposure of Ba/F3-ITD to increasing doses of sorafenib, and in FLT3-ITD mutated AML patients, who developed relapse following sorafenib therapy. Drug effects (e.g., proliferation inhibition, apoptosis induction, and changes in signal transduction protein expression) were assessed in AML cells harboring the point mutations in vitro and in FLT3-ITD-mutated AML patient samples. We identified several acquired point mutations in the tyrosine kinase domains (TKD) of the FLT3 gene in sorafenib-resistant murine leukemia cell line carrying human FLT3-ITD mutations, which were also detected in two of four sorafenib-resistant patient samples. Engineering these point mutations into Ba/F3-ITD cells generated sublines that demonstrated varying degrees of sorafenib [a type II tyrosine kinase inhibitor (TKI)] resistance. A similar pattern of resistance could be observed by exposing these sublines to the other type II TKIs AC220 and MLN518. However, these sublines retained sensitivity to the type I TKIs PKC412 or crenolanib. The combination of crenolanib with sorafenib demonstrated marked cytotoxic effects in all of the sorafenib-resistant sublines. These combination strategies could be clinically important in reversing acquired resistance to FLT3 inhibition in AML.

Zhang W ，Gao C ，Konopleva M ，Chen Y ，Jacamo RO ，Borthakur G ，Cortes JE ，Ravandi F ，Ramachandran A ，Andreeff M ... -  《-》

Sorafenib induces paradoxical phosphorylation of the extracellular signal-regulated kinase pathway in acute myeloid leukemia cells lacking FLT3-ITD mutation.

Fouladi F ，Jehn LB ，Metzelder SK ，Hub F ，Henkenius K ，Burchert A ，Brendel C ，Stiewe T ，Neubauer A ... -  《-》

Phosphoproteome Analysis Reveals Differential Mode of Action of Sorafenib in Wildtype and Mutated FLT3 Acute Myeloid Leukemia (AML) Cells.

Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD+ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy. Here, we studied the sorafenib effect on proliferation in a panel of 13 FLT3-ITD- and FLT3-ITD+ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 μm, whereas FLT3-ITD+ cells (MOLM-13, MV4-11) were found to be more sensitive to sorafenib than FLT3-ITD- cells. However, we identified two FLT3-ITD- cell lines (MONO-MAC-1 and OCI-AML-2) which were also sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in sorafenib sensitive FLT3-ITD- and FLT3-ITD+ cells. In MV4-11 cells sorafenib suppressed mTOR signaling by direct inhibition of FLT3. In MONO-MAC-1 cells sorafenib inhibited the MEK/ERK pathway. These data suggest that the FLT3 status in AML patients might not be the only factor predicting response to treatment with sorafenib.

Roolf C ，Dybowski N ，Sekora A ，Mueller S ，Knuebel G ，Tebbe A ，Murua Escobar H ，Godl K ，Junghanss C ，Schaab C ... -  《-》

Metformin synergizes with gilteritinib in treating FLT3-mutated leukemia via targeting PLK1 signaling.

Fms-like tyrosine kinase 3 (FLT3) mutations, present in over 30% of acute myeloid leukemia (AML) cases and dominated by FLT3-internal tandem duplication (FLT3-ITD), are associated with poor outcomes in patients with AML. While tyrosine kinase inhibitors (TKIs; e.g., gilteritinib) are effective, they face challenges such as drug resistance, relapse, and high costs. Here, we report that metformin, a cheap, safe, and widely used anti-diabetic agent, exhibits a striking synergistic effect with gilteritinib in treating FLT3-ITD AML. Metformin significantly sensitizes FLT3-ITD AML cells (including TKI-resistant ones) to gilteritinib. Metformin plus gilteritinib (low dose) dramatically suppresses leukemia progression and prolongs survival in FLT3-ITD AML mouse models. Mechanistically, the combinational treatment cooperatively suppresses polo-like kinase 1 (PLK1) expression and phosphorylation of FLT3/STAT5/ERK/mTOR. Clinical analysis also shows improved survival rates in patients with FLT3-ITD AML taking metformin. Thus, the metformin/gilteritinib combination represents a promising and cost-effective treatment for patients with FLT3-mutated AML, particularly for those with low income/affordability.

Chen M ，Shen C ，Chen Y ，Chen Z ，Zhou K ，Chen Y ，Li W ，Zeng C ，Qing Y ，Wu D ，Xu C ，Tang T ，Che Y ，Qin X ，Xu Z ，Wang K ，Leung K ，Sau L ，Deng X ，Hu J ，Wu Y ，Chen J ... -  《-》