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G2-arrested B lymphoma is prone to Wee1 inhibition
enhanced the mitotic entry of doxorubicin-induced G2 phase-arrested lymphomas (Figure 6A,B), whereas it did not affect the non-G2 phase-arrested lymphomas (Figure 6B). In summary, our data showed that Ramos and Ly7 lymphomas were arrested in G1 or S phase upon Ara-C treatment (Online Supplementary Figure S7); however, dox- orubicin treatment promoted G2 phase arrest in these two lymphoma lines, which correlated with their newly acquired sensitivity to Wee1 inhibitor.
Combined Ara-C and Wee1 inhibitor profoundly suppressed tumor growth in vivo
To test the therapeutic effects of combined Ara-C and Wee1 inhibitor treatment, we established an in vivo trans- plant model in which G1XP lymphoma cells injected into syngeneic recipient mice develop into secondary B-cell lymphomas (Figure 7A,B). These secondary G1XP lym- phomas were initially sensitive to Ara-C treatment, but became unresponsive to continuous Ara-C treatment and relapsed ∼20 days after the initial Ara-C treatment (Figure 7A,B). Wee1 inhibitor alone had no obvious effects on tumor growth or recipient survival compared with the vehicle control (Figure 7A,B). In contrast, combined treat- ment of Wee1 inhibitor and Ara-C significantly prolonged recipients’ survival (Figure 7A) and effectively suppressed tumor growth (Figure 7B). Of note, some of the recipient mice in the Ara-C/MK1775 group died early from unknown causes other than lymphoma. At day 34, tumor size was significantly reduced in the combined treatment group compared with that in the group treated with Ara- C alone (Figure 7C); animals in the groups treated with the vehicle control or Wee1 inhibitor were terminated before this time point due to tumor size exceeding the limit of institutional guidelines. In addition, combined treatment with Ara-C and Wee1 inhibitor attenuated the side effects of Ara-C treatment alone since the weight of recipients was significantly greater in the combined treatment group (Figure 7D). We conclude that, when combined with other chemotherapeutic agents, Wee1 inhibitor may provide a novel therapeutic intervention for B-cell lymphomas that can be arrested in the G2 phase of the cell cycle.
teins included in the SOMAscan assay, only three were differentially expressed between untreated and Ara-C- treated primary B cells, including cyclinB1/A2. However, we failed to identify the functional consequence of cyclinB1/A2 upregulation since double knockdown did not perturb the cell cycle or cause more apoptosis. Perhaps upregulation of these cyclins is indeed a stereotyped uni- versal response to DNA damage, but is only functionally significant in certain types of DNA damage, such as irradi- ation.35 Another possibility is that other cyclins may play a compensatory role when cyclins A2 and B1 are knocked down. Our preliminary RNA-seq data did not show the upregulation of cyclinB1/A2 transcripts in the Ara-C-treat- ed group. Hence, we predict that Ara-C treatment may somehow regulate the protein level of cyclinB1/A2, for example, via post-translational modulation of cyclinB1/A2 that leads to high and stabilized protein levels being main- tained. Future studies are needed to elucidate the mecha- nisms that upregulate these cyclins upon Ara-C treatment, independently from blocking the cell cycle.
Previous studies showed that DNA damage often elicits innate immune responses, such as upregulation of NKG2D ligands or interferon responses, in macrophages36
A
B
Figure 6. Wee1 inhibitor (MK1775) enhances premature mitotic entry of G2 phase-arrested lymphomas upon treatment with doxorubicin. CH12, Ramos, Ly1, Ly7 and DHL-16 lymphoma cells were treated as described in Figure 5. Cells were collected and fixed by 70% ethanol. After propidium iodide (PI) and pH3 staining, cell cycles were determined by flow cytometry (FL1-H/FL2-A). (A) Representative FACS data of Ly7 lymphoma cells are shown. (B) Statistical analysis of premature mitotic entry in various lymphoma lines. Statistical sig- nificance was calculated with ANOVA. Tukey multiple comparison test, *P≤0.05. Data are representative results of three independent experiments.
Discussion
Althrough Ara-C and doxorubicin have been used in clinics for several decades, it remains incompletely under- stood how primary B cells or B-cell lymphomas respond to such DNA-damaging agents. In the current study, we employed Ara-C and doxorubicin to treat primary mouse B cells and various B-cell lymphoma lines and present four novel findings: (i) upregulation of cyclinA2 and cyclinB1 appears to be an intrinsically programmed DNA damage response; (ii) Ara-C or doxorubicin induces differential cell cycle arrest in different types of B cells; (iii) Wee1 inhibitor sensitizes G2 phase-arrested B lymphoma cells to Ara-C treatment by inducing premature mitotic entry and mitot- ic catastrophe. Furthermore, Ara-C-induced G1 phase- arrest can be converted to G2 phase-arrest by doxorubicin treatment in certain B-cell lymphomas (e.g., Ramos cells), which correlates with newly acquired sensitivity to Wee1 inhibitor, and (iv) combined treatment with Ara-C and Wee1 inhibitor profoundly suppressed the tumor growth of transplanted G1XP lymphomas in vivo.
We present unexpected findings that, among 1310 pro-
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