The clinical and molecular heterogeneity of AML remain major challenges in improving outcomes for this disease. In order to objectively and rapidly identify proteins which can be modulated to enhance the activity of current standard therapy, we queried the entire genome using complementary functional and descriptive assays. The integration of data from these experiments strongly implicates cell cycle checkpoints as critical determinants of AML cell fate after exposure to cytarabine. WEE1 was among the proteins identified in the genome-wide functional screen, which also appears to be over-expressed in AML cells as compared to normal cells. Genetic and pharmacologic inhibition of WEE1 together with cytarabine inhibited proliferation and induced apoptosis in AML cell lines and primary AML samples, validating the potential of WEE1 as a therapeutic target in AML. Cell cycle analyses and western blotting confirm that the effect is due to the abrogation of the intra-S phase checkpoint, a function of WEE1 that to our knowledge has not been previously considered from a therapeutics perspective.
These data highlight the power of integrating genome-scale experimentation, and functional genomic screening in particular. While gene expression profiling of tumors and analysis of mutations within cancer genomes has provided a wealth of data and may eventually be used prognostically or to guide therapy, the descriptive nature of the data is difficult to interpret and apply clinically. That is, whether the over-expression or mutation of any given gene in a particular tumor may have functional significance or is a viable therapeutic target remains unknown until further experimentation is pursued. In contrast, functional genetic screening identifies molecules based on functional relevance a priori. The approach that we have developed allows for cost-effective, high-throughput validation, rapidly narrowing focus for detailed analyses of true positives. In this particular screen, the false positive rate was higher among shRNAs expected to confer chemoresistance, as compared to chemosensitivity (). The reasons for this are not entirely clear, as others have performed positive selection screening with shRNA libraries with good results (44
). Differences in methodology may have contributed to our observations, as continuous or repeated drug exposure may be a more effective method for detecting drug resistance conferring shRNAs (44
While cell cycle checkpoints have been previously studied in AML, to our knowledge, this is the first report to document the functional importance of WEE1 in AML. Chk1 has similar effects in arresting the cell cycle in the context of chemotherapy, and its inhibition has been explored therapeutically (46
). WEE1 may prove to be a better therapeutic target, though, as its effects in cell cycle regulation are direct and specific, while Chk1 integrates from and propagates signals through multiple other molecules, including WEE1. MK1775 is in early phase clinical trials in combination with chemotherapy for solid tumors, and the side effect profile is manageable in preliminary reports (47
We have demonstrated the importance of WEE1 in inducing the intra-S phase checkpoint in the context of cytarabine, which we propose is critical for AML cell survival during treatment. These data support a model whereby cytarabine treatment results in stalled DNA replication; while some cells will commit to apoptosis, others will recover and successfully complete cell cycle progression. But if inhibition of WEE1 prevents proper recognition of stalled replication, we speculate that replication forks will collapse, leading to S phase arrest and consequent apoptosis. Our results are somewhat surprising, in that the primary function of WEE1 is considered to be the inhibition of cell cycle progression at the G2/M transition of the cell cycle via inhibitory phosphorylation of CDK1 at Y15 (35
), while we observed abrogation of CDK2 Y15 phosphorylation with WEE1 inhibition in addition to inhibitory phosphorylation of CDK1 (not shown). Our data are consistent with the conserved function of WEE1 orthologs from Arabidopsis
, and human cells in phosphorylation of CDK2 to regulate progression through S-phase (49
). The S-phase function of WEE1 in AML cells broadens the therapeutic relevance of this molecule, particularly for treatment of AML, which is heavily dependent upon the S phase effects of cytarabine.
Inhibition of WEE1 is primarily being explored in patients with abnormal p53 function, since p53 is primarily responsible for the G1 checkpoint and cells with impaired p53 function are highly dependent on the G2/M checkpoint to maintain genomic integrity (52
). Indeed, cells with impaired p53 function can be sensitized to DNA damage by impairing the G2/M checkpoint via inhibition of Chk1 or WEE1 (53
). While combining WEE1 inhibition with anthracycline has been shown to be synergistic in solid tumor models (54
), whether inhibition of WEE1 in the context of anthracycline is synergistic in AML cells has yet to be demonstrated. While TP53
is not frequently mutated in AML, its function is often impaired (55
); thus, it is reasonable to hypothesize that WEE1 in combination with anthracyclines would result in synergistic AML cell death. Nonetheless, our data implicating the importance of WEE1 in combination with cytarabine were generated in cell lines that are reported to have functional p53, suggesting that the inhibition of WEE1 can be considered for tumors with functional p53.
We have demonstrated that WEE1 inhibition sensitizes AML cells to cytarabine in vitro; however, we cannot conclude that these findings will be broadly applicable to all patients with AML. Indeed, not all of the primary patient samples we tested provided evidence of combinatorial effects, suggesting underlying molecular susceptibility to this combination. Thus, it will be important to develop appropriate, practical biomarkers with which to predict which patients may benefit the most from such a therapeutic strategy. Animal modeling of AML with assessment of pharmacokinetics will provide further data in evaluating the effectiveness of WEE1 inhibition with cytarabine in eliminating AML cells and prolonging survival.
In summary, using integrated genomic analyses, we have identified and validated WEE1 as a potential therapeutic target in AML. These data highlight the power of integrated genomic analyses which may vastly accelerate the pace of discovery, validation and translation of molecular biology phenomena.