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Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia.

Pillinger G, Abdul-Aziz A, Zaitseva L, Lawes M, MacEwan DJ, Bowles KM, Rushworth SA - Sci Rep (2015)

Bottom Line: The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised.In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells.These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Haematology, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, United Kingdom.

ABSTRACT
Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

No MeSH data available.


Related in: MedlinePlus

BTK targeted siRNA inhibits survival of MV4–11 but not OCI-AML3.(A) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) AML cell lines were transfected with control siRNA and 4 BTK siRNA and then cultured for 24 h before RNA extraction analysis for target gene expression using QRT-PCR. (B) Western blot analysis for total BTK in response to transfected control, BTK3 and BTK4 siRNA and (C) for pAKT and total AKT in response to transfected control and BTK4 siRNA. The presented blots were derived from multiple gels. The membranes were cut based on molecular weights and probed with the antibody of interest. (D) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were transfected with control siRNA and BTK siRNA3 and 4 and then cultured for 48 h and then assessed by CellTiterGlo. (E) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were transfected with control siRNA and BTK siRNA3 and then cultured for 24 h and then daunorubicin was added in increasing doses and then cultured for a further 48 hours and then assessed by CellTiterGlo. *Indicates P < 0.05 Mann Whitney test comparing the BTK siRNA transfected samples to control siRNA samples.
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f4: BTK targeted siRNA inhibits survival of MV4–11 but not OCI-AML3.(A) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) AML cell lines were transfected with control siRNA and 4 BTK siRNA and then cultured for 24 h before RNA extraction analysis for target gene expression using QRT-PCR. (B) Western blot analysis for total BTK in response to transfected control, BTK3 and BTK4 siRNA and (C) for pAKT and total AKT in response to transfected control and BTK4 siRNA. The presented blots were derived from multiple gels. The membranes were cut based on molecular weights and probed with the antibody of interest. (D) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were transfected with control siRNA and BTK siRNA3 and 4 and then cultured for 48 h and then assessed by CellTiterGlo. (E) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were transfected with control siRNA and BTK siRNA3 and then cultured for 24 h and then daunorubicin was added in increasing doses and then cultured for a further 48 hours and then assessed by CellTiterGlo. *Indicates P < 0.05 Mann Whitney test comparing the BTK siRNA transfected samples to control siRNA samples.

Mentions: It has been reported that ibrutinib has other kinase targets27. Therefore to establish an anti-BTK role for ibrutinib in FLT3-ITD AML we evaluated the survival of AML cell lines using BTK KD siRNA experiments. Figure 4A and B show the BTK mRNA and protein expression of AML cell lines, after transfection with siRNA targeted to different parts of the BTK gene. It shows that BTK siRNA3 and 4 are the most effective at BTK gene knockdown in both MV4–11 (FLT3-ITD) cells and OCI-AML3 (non-FLT3-ITD) mutated cells. Figure 4C shows that BTK siRNA4 can inhibit downstream pAKT in MV4–11 but not OCI-AML3. BTK-KD with BTK siRNA3 and 4 compared to control siRNA results in decreased cell survival in MV4–11 (FLT3-ITD) but not in OCI-AML3 (non-FLT3-ITD) mutated cells (Fig. 4D). Finally, in results similar to those observed with ibrutinib, BTK-KD with siRNA3 enhanced daunorubicin induced cell death (Fig. 4E). These data confirm that BTK plays a key role in the survival of MV4–11 FLT3-ITD mutated cells.


Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia.

Pillinger G, Abdul-Aziz A, Zaitseva L, Lawes M, MacEwan DJ, Bowles KM, Rushworth SA - Sci Rep (2015)

BTK targeted siRNA inhibits survival of MV4–11 but not OCI-AML3.(A) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) AML cell lines were transfected with control siRNA and 4 BTK siRNA and then cultured for 24 h before RNA extraction analysis for target gene expression using QRT-PCR. (B) Western blot analysis for total BTK in response to transfected control, BTK3 and BTK4 siRNA and (C) for pAKT and total AKT in response to transfected control and BTK4 siRNA. The presented blots were derived from multiple gels. The membranes were cut based on molecular weights and probed with the antibody of interest. (D) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were transfected with control siRNA and BTK siRNA3 and 4 and then cultured for 48 h and then assessed by CellTiterGlo. (E) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were transfected with control siRNA and BTK siRNA3 and then cultured for 24 h and then daunorubicin was added in increasing doses and then cultured for a further 48 hours and then assessed by CellTiterGlo. *Indicates P < 0.05 Mann Whitney test comparing the BTK siRNA transfected samples to control siRNA samples.
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f4: BTK targeted siRNA inhibits survival of MV4–11 but not OCI-AML3.(A) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) AML cell lines were transfected with control siRNA and 4 BTK siRNA and then cultured for 24 h before RNA extraction analysis for target gene expression using QRT-PCR. (B) Western blot analysis for total BTK in response to transfected control, BTK3 and BTK4 siRNA and (C) for pAKT and total AKT in response to transfected control and BTK4 siRNA. The presented blots were derived from multiple gels. The membranes were cut based on molecular weights and probed with the antibody of interest. (D) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were transfected with control siRNA and BTK siRNA3 and 4 and then cultured for 48 h and then assessed by CellTiterGlo. (E) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were transfected with control siRNA and BTK siRNA3 and then cultured for 24 h and then daunorubicin was added in increasing doses and then cultured for a further 48 hours and then assessed by CellTiterGlo. *Indicates P < 0.05 Mann Whitney test comparing the BTK siRNA transfected samples to control siRNA samples.
Mentions: It has been reported that ibrutinib has other kinase targets27. Therefore to establish an anti-BTK role for ibrutinib in FLT3-ITD AML we evaluated the survival of AML cell lines using BTK KD siRNA experiments. Figure 4A and B show the BTK mRNA and protein expression of AML cell lines, after transfection with siRNA targeted to different parts of the BTK gene. It shows that BTK siRNA3 and 4 are the most effective at BTK gene knockdown in both MV4–11 (FLT3-ITD) cells and OCI-AML3 (non-FLT3-ITD) mutated cells. Figure 4C shows that BTK siRNA4 can inhibit downstream pAKT in MV4–11 but not OCI-AML3. BTK-KD with BTK siRNA3 and 4 compared to control siRNA results in decreased cell survival in MV4–11 (FLT3-ITD) but not in OCI-AML3 (non-FLT3-ITD) mutated cells (Fig. 4D). Finally, in results similar to those observed with ibrutinib, BTK-KD with siRNA3 enhanced daunorubicin induced cell death (Fig. 4E). These data confirm that BTK plays a key role in the survival of MV4–11 FLT3-ITD mutated cells.

Bottom Line: The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised.In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells.These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Haematology, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, United Kingdom.

ABSTRACT
Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

No MeSH data available.


Related in: MedlinePlus