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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 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.
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f1: 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.

Mentions: We have previously shown that ibrutinib inhibits factor induced AML blast proliferation and downstream AKT and MAPK signalling pathways9. Here we looked to characterise whether FLT3-ITD mutated primary AML and AML cell lines, which accounts for approximately 20% of all AML14, responds to BTK inhibition by ibrutinib treatment. First we identified 5 AML patients with FLT3-ITD mutation and 6 AML patients with no FLT3 mutation. Next we treated the primary AML blast with increasing concentrations of ibrutinib for 48 hours followed by analysis of cell survival. AML with FLT3-ITD mutations show increased sensitivity to increasing doses of ibrutinib compared to non-FLT3 mutated AML (Fig. 1A), Similar experiments in AML cell lines shows that MV4–11 (FLT3-ITD), but not OCI-AML3 or THP-1 (non- FLT3-ITD), have reduced survival in response to increasing concentrations of ibrutinib up to a maximum of 5000 nM (Fig. 1B). Finally we used colony forming cell assay to determine the effect to ibrutinib on FLT3-ITD primary AML colony formation compared to normal CD34+ HPC and non-FLT3 mutated AML blasts. Fig. 1C shows that AML with FLT3-ITD showed increased sensitivity to ibrutinib compared to normal CD34+ HPC and non-FLT3 mutated AML. Supplementary figure 1 shows that our positive control AC220 can also inhibit proliferation in MV4–11 and FLT-ITD primary AML. Moreover, AC220 in combination with ibrutinib showed no additive inhibitory effect on MV4–11 suggesting that these inhibitors are working through the same pathway (supplementary figure 2). To confirm the expression of pBTK in human FLT3-ITD AML compared to AML blasts without mutation we examined pBTK and total BTK in primary AML and AML cell lines. Figure 1D shows that pBTK is highly expressed in FLT3-ITD AML compared to no FLT3-ITD mutation. Furthermore, analysis of pBTK in AML cell lines confirms that FLT3-ITD mutated cells have high pBTK expression (Fig. 1E). However we did observe expression of pBTK in non-FLT3 mutated AML cell lines. Together these results suggest that ibrutinib is particularly effective at inhibiting FLT3-ITD mutated AML cell survival.


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 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f1: 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.
Mentions: We have previously shown that ibrutinib inhibits factor induced AML blast proliferation and downstream AKT and MAPK signalling pathways9. Here we looked to characterise whether FLT3-ITD mutated primary AML and AML cell lines, which accounts for approximately 20% of all AML14, responds to BTK inhibition by ibrutinib treatment. First we identified 5 AML patients with FLT3-ITD mutation and 6 AML patients with no FLT3 mutation. Next we treated the primary AML blast with increasing concentrations of ibrutinib for 48 hours followed by analysis of cell survival. AML with FLT3-ITD mutations show increased sensitivity to increasing doses of ibrutinib compared to non-FLT3 mutated AML (Fig. 1A), Similar experiments in AML cell lines shows that MV4–11 (FLT3-ITD), but not OCI-AML3 or THP-1 (non- FLT3-ITD), have reduced survival in response to increasing concentrations of ibrutinib up to a maximum of 5000 nM (Fig. 1B). Finally we used colony forming cell assay to determine the effect to ibrutinib on FLT3-ITD primary AML colony formation compared to normal CD34+ HPC and non-FLT3 mutated AML blasts. Fig. 1C shows that AML with FLT3-ITD showed increased sensitivity to ibrutinib compared to normal CD34+ HPC and non-FLT3 mutated AML. Supplementary figure 1 shows that our positive control AC220 can also inhibit proliferation in MV4–11 and FLT-ITD primary AML. Moreover, AC220 in combination with ibrutinib showed no additive inhibitory effect on MV4–11 suggesting that these inhibitors are working through the same pathway (supplementary figure 2). To confirm the expression of pBTK in human FLT3-ITD AML compared to AML blasts without mutation we examined pBTK and total BTK in primary AML and AML cell lines. Figure 1D shows that pBTK is highly expressed in FLT3-ITD AML compared to no FLT3-ITD mutation. Furthermore, analysis of pBTK in AML cell lines confirms that FLT3-ITD mutated cells have high pBTK expression (Fig. 1E). However we did observe expression of pBTK in non-FLT3 mutated AML cell lines. Together these results suggest that ibrutinib is particularly effective at inhibiting FLT3-ITD mutated AML cell survival.

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