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. 2015 Aug 21:5:12949.
doi: 10.1038/srep12949.

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

Genevra Pillinger  1 , Amina Abdul-Aziz  1 , Lyubov Zaitseva  1 , Matthew Lawes  2 , David J MacEwan  3 , Kristian M Bowles  1   2 , Stuart A Rushworth  1
Affiliations

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

Genevra Pillinger et al. Sci Rep. .

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.

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Figures

Figure 1
Figure 1. Ibrutinib inhibits survival of FLT3-ITD positive AML.
Primary AML blasts from 5 patients with FLT3-ITD and 6 patients with non FLT3-ITD were treated with increasing doses of ibrutinib for 1 hour and then washed and cultured for 72 hours and then assessed by CellTiterGlo. Data were normalised to DMSO treated cells. Line indicates median and *indicates statistical significance of P < 0.05 using the Mann-Whitney test comparing ibrutinib treated samples compared to control. (B) AML cell lines which were FLT3-ITD (MV4–11) and non FLT3-ITD (OCI-AML3 and THP-1) were treated with increasing doses of ibrutinib for 1 hour and then washed and cultured for 72 hours and then assessed by CellTiterGlo. *indicates statistical significance of P < 0.05 using the Mann-Whitney test comparing ibrutinib treated samples compared to control. (C) AML blasts and CD34+ control cells were treated with 0, 500 and 1000 nM of ibrutinib and colony forming assays were performed to show the number of colonies In all panels line indicates the median and *indicates statistical significance of P < 0.05 between ibrutinib treated groups and control using the Mann-Whitney test. (D) Untreated primary AML blasts which were FLT3-ITD and non FLT3-ITD and (E) untreated AML cell lines were examined for the expression of pBTK, total BTK and β-actin. The presented blots were derived from multiple gels. The membranes were cut based on molecular weights and probed with the antibody of interest.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. 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.
Figure 4
Figure 4. 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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