Limits...
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 enhances daunorubicin induced apoptosis.(A) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were treated with 500 nM of ibrutinib for 1 hour and washed and daunorubicin was added in increasing doses and then cultured for 48 hours and then assessed by CellTiterGlo. (B) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were treated with 500 nM of ibrutinib for 1 hour and washed and daunorubicin added in increasing doses and then cultured for 24 hours and then assessed by annexin V and PI staining. (C) Primary AML (FLT3-ITD) were pre-treated with ibrutinib for 1 h and then treated 50 nM daunorubicin and then cultured for 48 hours on BMSC and then assessed by CD45 and annexin V staining. Cells expressed as percent Annexin V positive. *indicates P < 0.05 Mann Whitney test comparing the ibrutinib plus daunorubicin to daunorubicin alone treated samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4544001&req=5

f3: Ibrutinib enhances daunorubicin induced apoptosis.(A) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were treated with 500 nM of ibrutinib for 1 hour and washed and daunorubicin was added in increasing doses and then cultured for 48 hours and then assessed by CellTiterGlo. (B) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were treated with 500 nM of ibrutinib for 1 hour and washed and daunorubicin added in increasing doses and then cultured for 24 hours and then assessed by annexin V and PI staining. (C) Primary AML (FLT3-ITD) were pre-treated with ibrutinib for 1 h and then treated 50 nM daunorubicin and then cultured for 48 hours on BMSC and then assessed by CD45 and annexin V staining. Cells expressed as percent Annexin V positive. *indicates P < 0.05 Mann Whitney test comparing the ibrutinib plus daunorubicin to daunorubicin alone treated samples.

Mentions: We next examined the effect of ibrutinib in combination with front line AML chemotherapy daunorubicin by using increasing concentrations of daunorubicin, alone and then in combination with Ibrutinib at 500 nM. Figure 3A and B shows that ibrutinib enhances daunorubicin induced apoptosis in FLT3-ITD AML cell line but not non-FLT3-ITD mutated cell lines. From this observation we hypothesise that in FLT3-ITD AML ibrutinib may permit a dose reduction in daunorubicin treatment while maintaining anti-leukaemic cytotoxicity.


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 enhances daunorubicin induced apoptosis.(A) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were treated with 500 nM of ibrutinib for 1 hour and washed and daunorubicin was added in increasing doses and then cultured for 48 hours and then assessed by CellTiterGlo. (B) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were treated with 500 nM of ibrutinib for 1 hour and washed and daunorubicin added in increasing doses and then cultured for 24 hours and then assessed by annexin V and PI staining. (C) Primary AML (FLT3-ITD) were pre-treated with ibrutinib for 1 h and then treated 50 nM daunorubicin and then cultured for 48 hours on BMSC and then assessed by CD45 and annexin V staining. Cells expressed as percent Annexin V positive. *indicates P < 0.05 Mann Whitney test comparing the ibrutinib plus daunorubicin to daunorubicin alone treated samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Ibrutinib enhances daunorubicin induced apoptosis.(A) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were treated with 500 nM of ibrutinib for 1 hour and washed and daunorubicin was added in increasing doses and then cultured for 48 hours and then assessed by CellTiterGlo. (B) FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3) cells were treated with 500 nM of ibrutinib for 1 hour and washed and daunorubicin added in increasing doses and then cultured for 24 hours and then assessed by annexin V and PI staining. (C) Primary AML (FLT3-ITD) were pre-treated with ibrutinib for 1 h and then treated 50 nM daunorubicin and then cultured for 48 hours on BMSC and then assessed by CD45 and annexin V staining. Cells expressed as percent Annexin V positive. *indicates P < 0.05 Mann Whitney test comparing the ibrutinib plus daunorubicin to daunorubicin alone treated samples.
Mentions: We next examined the effect of ibrutinib in combination with front line AML chemotherapy daunorubicin by using increasing concentrations of daunorubicin, alone and then in combination with Ibrutinib at 500 nM. Figure 3A and B shows that ibrutinib enhances daunorubicin induced apoptosis in FLT3-ITD AML cell line but not non-FLT3-ITD mutated cell lines. From this observation we hypothesise that in FLT3-ITD AML ibrutinib may permit a dose reduction in daunorubicin treatment while maintaining anti-leukaemic cytotoxicity.

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