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. 2014 Apr;99(4):688-96.
doi: 10.3324/haematol.2013.093187. Epub 2013 Oct 31.

CHK1 and WEE1 inhibition combine synergistically to enhance therapeutic efficacy in acute myeloid leukemia ex vivo

Leena Chaudhuri  1 , Nicole D Vincelette, Brian D Koh, Ryan M Naylor, Karen S Flatten, Kevin L Peterson, Amanda McNally, Ivana Gojo, Judith E Karp, Ruben A Mesa, Lisa O Sproat, James M Bogenberger, Scott H Kaufmann, Raoul Tibes
Affiliations

CHK1 and WEE1 inhibition combine synergistically to enhance therapeutic efficacy in acute myeloid leukemia ex vivo

Leena Chaudhuri et al. Haematologica. 2014 Apr.

Abstract

Novel combinations _targeting new molecular vulnerabilities are needed to improve the outcome of patients with acute myeloid leukemia. We recently identified WEE1 kinase as a novel _target in leukemias. To identify genes that are synthetically lethal with WEE1 inhibition, we performed a short interfering RNA screen directed against cell cycle and DNA repair genes during concurrent treatment with the WEE1 inhibitor MK1775. CHK1 and ATR, genes encoding two replication checkpoint kinases, were among the genes whose silencing enhanced the effects of WEE1 inhibition most, whereas CDK2 short interfering RNA antagonized MK1775 effects. Building on this observation, we examined the impact of combining MK1775 with selective small molecule inhibitors of CHK1, ATR and cyclin-dependent kinases. The CHK1 inhibitor MK8776 sensitized acute myeloid leukemia cell lines and primary leukemia specimens to MK1775 ex vivo, whereas smaller effects were observed with the MK1775/MK8776 combination in normal myeloid progenitors. The ATR inhibitor VE-821 likewise enhanced the antiproliferative effects of MK1775, whereas the cyclin-dependent kinase inhibitor roscovitine antagonized MK1775. Further studies showed that MK8776 enhanced MK1775-mediated activation of the ATR/CHK1 pathway in acute leukemia cell lines and ex vivo. These results indicate that combined cell cycle checkpoint interference with MK1775/MK8776 warrants further investigation as a potential treatment for acute myeloid leukemia.

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Figures

Figure 1.
Figure 1.
RNAi screen. (A) Graphical representation of the RNAi screen is shown for TF-1 and HEL cells treated with 250 nM MK1775. Data are plotted as the delta between the median of each (siRNA + 250 nM MK1775)/non-silencing siRNA (NS) and siRNA/NS. Each graph is representative of two screens. Graphical representation of the RNAi screen is shown for TF-1, THP-1, HEL and MDSL-2 for both the screens at (B) 250 nM MK1775 and (C) 750 nM MK1775. The RNAi screens were run in duplicate.
Figure 2.
Figure 2.
ATR inhibition sensitizes and CDK inhibition antagonizes WEE1 inhibiition by MK1775. (A) TF-1, HEL and THP-1 cells were treated with nine doses of MK1775 alone and in combination with 0.011–8 μM VE-821, a selective small molecule inhibitor of ATR kinase. (B) TF-1, HEL and THP-1 cells were treated with nine doses of MK1775 alone and in combination with 0.01–40 μM roscovitine, a selective small molecule inhibitor of CDK1, CDK2 and CDK5. Viability was measured 96 h after drug treatment by CellTiter Glo. A representative result is shown from two independent experiments, each conducted in quadruplicate.
Figure 3.
Figure 3.
CHK1 kinase inhibition sensitizes to WEE1 kinase inhibition in AML in vitro. (A) Drug dose-response curves show a shift in MK1775 EC50 upon co-treatment with 0.010–10 μM MK8776, a selective small molecule inhibitor for CHK1 in four AML cell lines, a myelodysplastic syndrome and a B-acute lymphoblastic leukemia cell line. A representative result is shown from two independent experiments, each conducted in quadruplicate.
Figure 4.
Figure 4.
MK1775 activates the ATR/CHK1 pathway but does not induce cell cycle arrest. U937 (A), HL-60 (B) or ML-1 cells (D) were treated for 24 h with diluent, 100 nM MK8776, 100 nM MK1775, or the combination of 100 nM MK8776 + MK1775. At completion of the incubation, cells were stained with propidium iodide and subjected to flow microfluorimetry. Insets: cell cycle distribution as determined by CellQuest software after the indicated treatments. Arrow in (D), subdiploid cells suggesting apoptosis. (C) After U937 cells had been treated with MK1775 at 0, 25, 50, 100 and 200 nM in the absence of MK8776 (lanes 1–5) or presence of 100 nM MK8776 (lanes 6–10), whole cell lysates (50 μg of total cellular protein) were subjected to sodium dodecyl polyacrylamide gel electrophoresis followed by immunoblotting with antibodies that recognize the indicated antigen. Loss of the Chk1 signal in lanes 9 and 10 likely reflects previously described activation-induced degradation.
Figure 5.
Figure 5.
MK8776 enhances MK1775-induced apoptosis. (A) U937 cells were treated for 48 h with diluent, 100 nM MK8776, 100 nM MK1775, or the combination of 100 nM MK8776 + MK1775; stained with prodium iodide; and subjected to flow microfluorimetry. U937 (B), HL-60 (C) or ML-1 cells (D) were treated for 48 h with the indicated concentrations of MK1775 in the absence or presence of 100 nM MK 8776, stained with propidium iodide, subjected to flow microfluorimetry, and analyzed as illustrated in panel (A). Error bars, ± SEM from three or four independent experiments in each cell line. Experiments showing annexin V binding are presented in Online Supplementary Figure S3.
Figure 6.
Figure 6.
MK8776 enhances MK1775-induced antiproliferative effects in colony-forming assays of AML cell lines and primary AML specimens. U937 (A) and HL-60 (B) cells were plated in 0.3% agar containing the indicated concentrations of MK1775 in the absence or presence of 100 nM MK8776. Colonies were counted at 14 days and compared to those of samples containing diluent (0.2% DMSO). Upper insets in (A) and (B), the same cells were plated in 0.3% agar containing the indicated concentrations of MK8776. Lower insets in (A) and (B), combination index values calculated from data in the main panel and upper inset under the assumption that effects of the two agents are mutually exclusive. Note that combination values <1.0 indicate synergy. Error bars in (A) and (B): ± SEM from quadruplicate samples. (C–E). Three different AML samples were plated in Methocult® methycellulose containing the indicated concentrations of MK1775 in the absence or presence of 100 nM MK8776. Leukemic colonies were counted at 14 days and compared to those in samples containing diluent (0.2% DMSO). Error bars in (C–E): range of values from duplicate plates. As indicated in the text, four additional AML samples and three additional normal controls were examined.
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