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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

Ibrutinib inhibits downstream FLT3 signalling.(A) Primary AML blasts which were FLT3-ITD and non FLT3-ITD were treated with increasing doses of ibrutinib for 3 hour and then whole cell extracts were prepared and Western blot analysis was conducted for pBTK, BTK. and (B) pAKT, AKT, pMAPK and MAPK protein levels. (C) AML cell lines which were FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) were treated with 500 nM of ibrutinib for 3 hour and then whole cell extracts were prepared and Western blot analysis was conducted for pFLT3, FLT3, pAKT, AKT, pMAPK, MAPK, pSTAT and STAT protein levels. The presented blots were derived from multiple gels. The membranes were cut based on molecular weights and probed with the antibody of interest.
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f2: Ibrutinib inhibits downstream FLT3 signalling.(A) Primary AML blasts which were FLT3-ITD and non FLT3-ITD were treated with increasing doses of ibrutinib for 3 hour and then whole cell extracts were prepared and Western blot analysis was conducted for pBTK, BTK. and (B) pAKT, AKT, pMAPK and MAPK protein levels. (C) AML cell lines which were FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) were treated with 500 nM of ibrutinib for 3 hour and then whole cell extracts were prepared and Western blot analysis was conducted for pFLT3, FLT3, pAKT, AKT, pMAPK, MAPK, pSTAT and STAT protein levels. The presented blots were derived from multiple gels. The membranes were cut based on molecular weights and probed with the antibody of interest.

Mentions: To determine if ibrutinib inhibits downstream signalling in FLT3-ITD mutated primary AML blasts, we compared AML with FLT3-ITD to AML without FLT3-ITD in response to increasing concentrations of ibrutinib for 3 h. Figure 2A shows that in primary AML associated with FLT3-ITD mutations showed decreased expression of pBTK in response to ibrutinib when compared to total BTK expression. Figure 2B shows that ibrutinib inhibits downstream phosphorylated proteins, including AKT and MAPK in primary AML cells with FLT-ITD. Comparing AML cell lines, MV4–11 (FLT3-ITD) but not OCI-AML3 (non-FLT3-ITD) show reduced activity of downstream phosphorylated AKT, MAPK and STAT5 in response to ibrutinib treatment (Fig. 2C). Moreover, to determine if ibrutinib can target FLT3 directly we examined the effect of ibrutinib on FLT3 phosphorylation and Fig. 2C shows that ibrutinib does not target FLT3 directly. Supplementary Figure 3 shows that AC220 can also inhibit pSTAT5 in MV4–11 cells. Together, these results confirm that ibrutinib inhibits FLT3 mutation associated downstream signalling in FLT3-ITD AML.


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)

Ibrutinib inhibits downstream FLT3 signalling.(A) Primary AML blasts which were FLT3-ITD and non FLT3-ITD were treated with increasing doses of ibrutinib for 3 hour and then whole cell extracts were prepared and Western blot analysis was conducted for pBTK, BTK. and (B) pAKT, AKT, pMAPK and MAPK protein levels. (C) AML cell lines which were FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) were treated with 500 nM of ibrutinib for 3 hour and then whole cell extracts were prepared and Western blot analysis was conducted for pFLT3, FLT3, pAKT, AKT, pMAPK, MAPK, pSTAT and STAT protein levels. The presented blots were derived from multiple gels. The membranes were cut based on molecular weights and probed with the antibody of interest.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4544001&req=5

f2: Ibrutinib inhibits downstream FLT3 signalling.(A) Primary AML blasts which were FLT3-ITD and non FLT3-ITD were treated with increasing doses of ibrutinib for 3 hour and then whole cell extracts were prepared and Western blot analysis was conducted for pBTK, BTK. and (B) pAKT, AKT, pMAPK and MAPK protein levels. (C) AML cell lines which were FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) were treated with 500 nM of ibrutinib for 3 hour and then whole cell extracts were prepared and Western blot analysis was conducted for pFLT3, FLT3, pAKT, AKT, pMAPK, MAPK, pSTAT and STAT protein levels. The presented blots were derived from multiple gels. The membranes were cut based on molecular weights and probed with the antibody of interest.
Mentions: To determine if ibrutinib inhibits downstream signalling in FLT3-ITD mutated primary AML blasts, we compared AML with FLT3-ITD to AML without FLT3-ITD in response to increasing concentrations of ibrutinib for 3 h. Figure 2A shows that in primary AML associated with FLT3-ITD mutations showed decreased expression of pBTK in response to ibrutinib when compared to total BTK expression. Figure 2B shows that ibrutinib inhibits downstream phosphorylated proteins, including AKT and MAPK in primary AML cells with FLT-ITD. Comparing AML cell lines, MV4–11 (FLT3-ITD) but not OCI-AML3 (non-FLT3-ITD) show reduced activity of downstream phosphorylated AKT, MAPK and STAT5 in response to ibrutinib treatment (Fig. 2C). Moreover, to determine if ibrutinib can target FLT3 directly we examined the effect of ibrutinib on FLT3 phosphorylation and Fig. 2C shows that ibrutinib does not target FLT3 directly. Supplementary Figure 3 shows that AC220 can also inhibit pSTAT5 in MV4–11 cells. Together, these results confirm that ibrutinib inhibits FLT3 mutation associated downstream signalling in FLT3-ITD AML.

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