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CUDC-907 monotherapy in AML
5C), the upregulation of Bim (Figure 3B) was likely due to transcriptional regulation mediated by the HDAC inhibitor moiety of CUDC-907. However, given the evi- dence that the ERK pathway regulates Bim degrada- tion,33,34 post-transcriptional mechanisms cannot be ruled out. Additionally, inactivation of AKT and ERK may also contribute to the antileukemic activity of CUDC-907 through other downstream targets.12,14
HDAC inhibitors have been shown to induce differenti- ation, cell cycle arrest, DNA damage, and apoptosis in AML cells.20,26,35-37 One mechanism through which HDAC inhibitors exert their anticancer activity is through down- regulation of DNA damage response proteins, such as CHK1 and Wee1, as we and others have reported.23-26 In agreement, we detected downregulation of CHK1 and Wee1 protein and transcript levels (Figures 3C and 6G, I, and J). HDAC inhibitor-induced downregulation of CHK1 and Wee1 has been shown to be mediated through down- regulation of E2F1.37,38 However, the decrease of E2F1 was not consistent in the AML cell lines and primary AML patient sample. CUDC-907 treatment caused decreases of E2F1, CHK1, and Wee1 in three AML cell lines and one pri- mary AML patient’s sample. However, in the other pri- mary AML patient sample, CUDC-907 treatment did not result in a decrease of E2F1 protein but did decrease both CHK1 and Wee1 protein levels. These results suggest that downregulation of CHK1 and Wee1 was probably mediat- ed through transcript regulation, though it may not have been entirely mediated through downregulation of E2F1.
CUDC-907 treatment also decreased RRM1 protein and transcript levels (Figures 3C and 6H, J), suggesting that downregulation of this gene was probably mediated by a transcriptional mechanism. Based on our results using hydroxyurea, RRM1 likely played an important role in CUDC-907-induced DNA damage. Inhibition of RR decreases dNTP pools, resulting in DNA replication fork stalling, impaired DNA repair, and DNA damage.39 In agreement with Sun et al.,16 we found that CUDC-907 treatment reduced expression of c-Myc protein prior to induction of apoptosis (Figures 4C, D and 7C). Given its role in cell growth, proliferation, and survival, the early downregulation of c-Myc may play a more prominent role in CUDC-907-induced apoptosis since changes in CHK1, Wee1, RRM1, Bim, and Mcl-1 levels occur after c-Myc downregulation.
FLT3-ITD AML has been shown to be associated with increased DNA damage and misrepair,40 potentially making such leukemias more sensitive to DNA replication fork stalling, impaired DNA repair, and DNA damage. Interestingly, we found that primary AML samples from patients with FLT3-ITD were significantly more sensitive to CUDC-907 ex vivo (Figure 2A). FLT3-ITD has also been shown to constitutively activate downstream PI3K and ERK pathways, conferring resistance to PI3K and ERK inhibitors. However, HDAC inhibitors have been shown to upregulate ubiquitin conjugase41 and inhibit HSP90 resulting in proteasomal degradation of FLT3.42,43 Consistent with those reports, CUDC-907 treatment did indeed decrease FLT3 protein levels in the FLT3-ITD AML cell line MOLM-13 (Online Supplementary Figure S10). Thus, the HDAC inhibitor moiety of CUDC-907 reduces FLT3 levels, relieving constitutive activation of the PI3K and ERK pathways, and allowing the PI3K inhibitor function of
CUDC-907 to induce AML cell death. This may explain the superior response of FLT3-ITD AML cells to CUDC- 907 (Figure 2A), although the effects of CUDC-907 on AML cell apoptosis, colony-formation capacity, and FLT3 protein levels need to be further elucidated in additional primary samples from patients with FLT3-ITD AML.
Results of the first phase I trial of CUDC-907 were recently published, outlining the safety, tolerability, and preliminary activity in patients with lymphoma or multi- ple myeloma.17 In that study, the recommended dosing for further clinical studies was identified to be 60 mg admin- istered orally, once daily for 5 days, followed by 2 days off treatment, as there were no dose-limiting toxicities at this dosing and schedule. In addition, side effects were consis- tent with the known profile of HDAC or PI3K inhibitors and deemed manageable. Our data show promising in vivo efficacy against an AML cell line-derived xenograft mouse model, supporting further clinical development of CUDC- 907 as an AML-focused therapy. While modest weight loss was seen after CUDC-907 treatment (nadir: -5.4% on day 22, 2 days after last treatment), it was completely reversible within 4 days. This fact coupled with the observed modest survival benefit produced from the inter- rupted treatment schedule indicate that either or both the dosing and schedule can be further optimized (i.e. in hind- sight, the drug was so well tolerated that the 4-day inter- ruption of treatment may not have been necessary). Furthermore, the tolerability of CUDC-907 suggests that it may be used in combination with other therapies. Conventional chemotherapy drugs, such as cytarabine or daunorubicin, may synergize with CUDC-907 as these drugs induce DNA damage and would likely add further insult to the stressed DNA repair system following CUDC-907 treatment.
In summary, our study demonstrates that CUDC-907 induces DNA damage and apoptosis in AML cell lines and primary patients’ samples, and targets AML progenitor cells while sparing normal hematopoietic cells in vitro. In addition, our initial in vivo study generated a promising increase in survival following CUDC-907 monotherapy. As a dual inhibitor, CUDC-907 lends itself to the possibil- ity of combination therapies to further eliminate AML and prevent disease relapse. Our findings provide new insights into the mechanism of action of CUDC-907 in AML cells (Figure 7G) and support its clinical development for the treatment of AML.
Acknowledgments
This study was supported by Jilin University, Changchun, China, the Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, and by grants from the National Natural Science Foundation of China, NSFC 31671438 and NSFC 31471295, Hyundai Hope on Wheels, LaFontaine Family/U Can-Cer Vive Foundation, Kids Without Cancer, Children’s Hospital of Michigan Foundation, Decerchio/Guisewite Family, Justin’s Gift, Elana Fund, Ginopolis/Karmanos Endowment and the Ring Screw Textron Endowed Chair for Pediatric Cancer Research. The Animal Models and Therapeutics Evaluation Core is supported, in part, by NIH Center grant P30 CA022453 to the Karmanos Cancer Institute at Wayne State University. The funders had no role in study design, data collection, analysis and interpretation of data, decision to publish, or preparation of the manuscript.
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