Dual Aurora A and JAK2 kinase blockade effectively suppresses malignant transformation

Abstract

Aurora A and JAK2 kinases are involved in cell division and tumor cell survival, respectively. Here we demonstrate that ectopic expression of Aurora A and JAK2 together is more effective than each alone at inducing non-transformed cells to grow in an anchorage-independent manner and to invade. Furthermore, siRNA silencing or pharmacological inhibition of Aurora A and JAK2 with Alisertib and Ruxolitinib, respectively, is more effective than blocking each kinase alone at suppressing anchorage-dependent and -independent growth and invasion as well as at inducing apoptosis. Importantly, we have developed dual Aurora and JAK inhibitors, AJI-214 and AJI-100, which potently inhibit Aurora A, Aurora B and JAK2 in vitro. In human cancer cells, these dual inhibitors block the auto-phosphorylation of Aurora A (Thr-288) and the phosphorylation of the Aurora B substrate histone H3 (Ser-10) and the JAK2 substrate STAT3 (Tyr-705). Furthermore, AJI-214 and AJI-100 inhibit anchorage dependent and independent cell growth and invasion and induce G2/M cell cycle accumulation and apoptosis. Finally, AJI-100 caused regression of human tumor xenografts in mice. Taken together, our genetic and pharmacological studies indicate that targeting Aurora A and JAK2 together is a more effective approach than each kinase alone at inhibiting malignant transformation and warrant further advanced pre clinical investigations of dual Aurora A/JAK2 inhibitors as potential anti tumor agents.

Figure 1. Co-expression of Aurora-A and JAK2 is more effective than single expression at inducing malignant transformation in HPNE cells

HPNE cells were transfected with vector, Aurora A, JAK2 or both Aurora A and JAK2 as described in Materials and Methods. Cells were harvested 48 h post-transfection, and the resulting lysates were immunoblotted with indicated antibodies (A), processed for soft agar growth assays (B) or invasion assays (C) as described in Materials and Methods. White, light gray, dark gray and black colors represent vector, Aurora A (Aur), JAK2 and Aurora A + JAK2 (A+J), respectively. For the soft agar (B), the data are the average +/− SE of three wells per condition. For the invasion (C), the data are the average +/− SE of three independent experiments. The data for (A) and (B) are representative of three independent experiments, respectively.

Figure 2. Depletion of Aurora A and JAK2 kinases is highly effective at inducing apoptosis and at inhibiting anchorageindependent growth and invasion

MDA-MB-468 and HT-29 cells were transfected with NT siRNA, Aurora A siRNA, JAK2 siRNA or siRNAs to both aurora A and JAK2, and processed for Western blotting (A), soft agar (B) or invasion (C) assays as described under Materials and Methods. For the soft agar (B), the data are the average +/− SE of three wells per condition. For the invasion (C), the data are the average +/− SE of three independent experiments. The data for (A) and (B) are representative of three independent experiments, respectively.

Figure 3. Combination treatment with the Aurora A inhibitor, Alisertib, and the JAK2 inhibitor, Ruxolitinib, is more effective than single treatment at inhibiting anchorage-independent growth and invasion and at inducing apoptosis

MDA-MB-468 and HT-29 cells were treated with vehicle control (white), Alisertib (light gray), Ruxolitinib (dark gray) or the combination (black), and the Cells were processed for Western blotting (A) and (B), soft agar (C) and invasion (D) assays as described under Materials and Methods. For the soft agar (C), the data are the average +/− SE of three wells per condition. For the invasion (D), the data are from one invasion chamber per condition. The data for (A), (B), (C) and (D) are representative of three, two, three and two independent experiments, respectively.

Figure 4. AJI-214 and AJI-100 are dual Aurora A/B and JAK2 inhibitors

AJI-214 and AJI-100 synthesized as described by us previously [30] and submitted to Reaction Biology Inc. for determination of IC50 values against Aurora A, Aurora B and JAK2 using the HotSpot method as described previously [30].

Figure 5. AJI-214 and AJI-100 inhibit the phosphorylation of Aurora A, Histone H3 and STAT3, induce apoptosis and inhibit anchorage-dependent proliferation in cancer cells

(A) MDA-MB-468 Cells were synchronized by treatment with nocodazole (100 ng/mL) for 20 h, treated with AJI-214 or AJI-100 for 2 h and processed for Western immunoblotting with p-Aurora-A (Thr288) antibody as described under Materials and Methods. VX-680 was used as a control for Aurora-A inhibition. (B) MDA-MB-468 Cells were treated with AJI-214 or AJI-100 for 2 h and processed for Western immunoblotting with P-HH3, P-STAT3, PARP, C-Caspase3 and GAPDH antibodies as described under Materials and Methods. (C) MDA-MB-468 Cells were plated in 96-well plates and treated with the indicated concentrations of AJI-214 and AJI-100 for 48 h and processed for MTT assays as described in “Materials and Methods”. The data for (A), (B) and (C) are representative of two, two and three independent experiments, respectively.

Figure 6. Aurora A and JAK2 dual inhibitors are highly effective at inhibiting anchorage-independent growth and invasion as well as in vivo tumor growth of MDA-MB-468 xenografts in nude mice

MDA-MB-468 cells were treated with vehicle control, AJI-214 or AJI-100 and the cells were processed for soft agar (A) and invasion (B) assays as described under Materials and Methods. For the soft agar (A), the data are the average +/− SE of three wells per condition. For the invasion (B), the data are from one invasion chamber per condition. The data for (A) and (B) are representative of three and two independent experiments. (C and D) Effects of AJI-100 on tumor growth of MDA-MB-468 xenografts in nude mice. Mice bearing MDA-MB-468 tumor xenografts were treated (once a day for 14 days) with vehicle (50% PG+15%HPCD) or AJI-100 (50 mpk/day) as described under Materials and Methods. (C) Representative tumor growth curve from vehicle treated mouse (square) and AJI-100 treated mouse (triangle). (D) Average (+/− SE) percent change in tumor volumes for each treatment group (vehicle squares, AJI-100 triangles).