Inhibitory effects of Tomivosertib in acute myeloid leukemia

The MAPK-interacting kinases 1 and 2 (MNK1/2) have generated increasing interest as therapeutic targets for acute myeloid leukemia (AML). We evaluated the therapeutic potential of the highly-selective MNK1/2 inhibitor Tomivosertib on AML cells. Tomivosertib was highly effective at blocking eIF4E phosphorylation on serine 209 in AML cells. Such inhibitory effects correlated with dose-dependent suppression of cellular viability and leukemic progenitor colony formation. Moreover, combination of Tomivosertib and Venetoclax resulted in synergistic anti-leukemic responses in AML cell lines. Mass spectrometry studies identified novel putative MNK1/2 interactors, while in parallel studies we demonstrated that MNK2 - RAPTOR - mTOR complexes are not disrupted by Tomivosertib. Overall, these findings demonstrate that Tomivosertib exhibits potent anti-leukemic properties on AML cells and support the development of clinical translational efforts involving the use of this drug, alone or in combination with other therapies for the treatment of AML.


INTRODUCTION
Acute myeloid leukemia (AML) is the second most common form of leukemia in adults, and has a very poor overall survival rate [1,2]. For many years, chemotherapy based on the administration of anthracycline/cytarabine combinations has been the mainstay of AML treatment [3,4]. More recently, advancements in next-generation sequencing technologies and understanding of genomic alterations involved in leukemogenesis have allowed the development of several novel targeted therapy approaches that complement the action of the currently available chemotherapeutic treatments. Among them are targeted drugs for specific mutations found in AML, such as the FLT3 inhibitors, IDH1/IDH2 inhibitors, pro-apoptotic agents, Hedgehog pathway inhibitors and others [5][6][7]. Even though these therapies have increased at variable degrees the response rates and survival benefit of subgroups of AML patients [8][9][10][11]; the development of resistance towards these novel drugs and subsequent relapse remains one of the major challenges for the treatment of this disease [12]. Therefore, there continues to be a need for new therapeutic modalities, including approaches targeting negative-feedback signaling pathways that may be activated in response to antileukemic treatments, leading to resistance.
Tomivosertib, also known as eFT-508, is a potent, highly selective and orally bioavailable MNK1 and MNK2 inhibitor that is currently under investigation in Phase 1/2 clinical trials for the treatment of patients with advanced solid tumors and lymphomas [42]. This drug inhibits the activity of MNK1 and MNK2 with half-maximal inhibitory concentration (IC 50 ) values of 2.4 nM and 1 nM; and has demonstrated potent anti-neoplastic effects against several cell lines and tumor models including diffuse large B-cell lymphoma (DLBCL), breast cancer, and lung cancer [42]. However, its potential activity against AML has not been explored. In this study, we investigated the efficacy of Tomivosertib in pre-clinical models of AML. We demonstrate that Tomivosertib suppresses eIF4E phosphorylation in AML cells and decreases leukemic cell survival and proliferation. We also provide evidence for synergy of Tomivosertib with Venetoclax, in vitro.

Tomivosertib inhibits eIF4E phosphorylation and decreases leukemic cell survival and proliferation
Initially, we determined whether Tomivosertib blocks phosphorylation of the MNK effector eIF4E in AML cells. U937, MV411 and MM6 cells were treated with increasing doses of Tomivosertib for a period of 1 and 4 hours and eIF4E phosphorylation was assessed. Treatment with Tomivosertib abrogated the phosphorylation of eIF4E on serine 209 at a concentration of 0.1 µM, supporting potent inhibition of MNK1/2 activity in AML cells ( Figure 1A-1C). To determine the potential antileukemic effects of Tomivosertib, we employed cellular viability assays as well as clonogenic assays in methylcellulose, using a panel of leukemia cell lines. Tomivosertib treatment resulted in dose-dependent inhibition of cellular viability in the AML lines tested. The compound showed the greatest suppressive activity against MV411, MM6 and KG-1 cells ( Figure 1D-1F). Conversely, higher concentrations of the drug were required to suppress the viability of the myelomonocytic THP-1 and U937 cell lines ( Figure 1G and 1H). A similar pattern was observed when the effects of Tomivosertib on leukemic progenitor cells were assessed. Treatment with Tomivosertib significantly inhibited KG-1-, MV411-, and MM6-derived leukemic progenitor (CFU-L) colony formation, while the effects were less pronounced on THP1-and U937-derived colony formation (Figure 2A-2E). Inhibitory effects were also seen on primary leukemic precursors from AML patients ( Figure 2F). Taken together, these studies demonstrate that Tomivosertib inhibits eIF4E phosphorylation in AML cells, resulting in decreased leukemic cell survival and proliferation.

Tomivosertib synergistically enhances the antileukemic effects of Venetoclax against AML cells in vitro
In consequent studies, we investigated the antileukemic activity of a combined treatment of Tomivosertib with Venetoclax in AML cell lines. Venetoclax is a novel BCL-2 inhibitor that was recently approved by the FDA for the treatment of elderly patients with AML due to its significant clinical activity in combination with cytarabine or hypomethylating agents [43,44]. However, the development of resistance to Venetoclax can occur through upregulation of other pro-survival proteins such as Myeloid cell leukemia 1 (MCL-1) [45,46]. MNK1/2 was previously shown to be required for mRNA translation of MCL-1 [24,33], and therefore, we hypothesized that concomitant treatment of cells with Venetoclax and a MNK1/2 inhibitor may help to overcome the resistance to this drug. We first investigated the effects of combination treatment with Tomivosertib and Venetoclax on cellular viability of different AML cell lines. The combination of Tomivosertib with Venetoclax resulted in synergistic inhibitory effects on cellular viability ( Figure 3A and 3B). Similarly, the combination of Tomivosertib and Venetoclax resulted in potent inhibition of U937-and KG-1-derived CFU-L growth as compared to each agent alone ( Figure 3C and 3D). Altogether, these studies provide evidence that the Tomivosertib/Venetoclax combination induces potent anti-leukemic responses.

LC-MS/MS analysis identifies putative MNK1/2 targets and interactors
To identify novel binding partners of MNK1/2, liquid-chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on protein-MNK1/2 complexes immunoprecipitated from 293T cells. We found that 16 proteins interacted with MNK1, 10 proteins interacted with MNK2 and 24 proteins interacted with both MNK1 and MNK2 ( Figure 4A). Pathway and process enrichment analysis of the putative MNK1/2 interactors identified translation as one of the most significantly represented pathway activity, consistent with the role of MNK1/2 in this process ( Figure 4B).
Among the different proteins, RAPTOR and mTOR were identified as interacting partners of MNK2 in the LC-MS/MS studies ( Figure 4A). We first confirmed the interaction between MNK2, mTOR and RAPTOR observed in the proteomic analysis. For this purpose, we overexpressed FLAG-MNK2 and FLAG-MNK1 in 293T cells and assessed the ability of MNK1 or MNK2 to interact with RAPTOR and mTOR by co-immunoprecipitation. Only MNK2 interacted with both RAPTOR and mTOR ( Figure 4C). We then determined whether this interaction between MNK2 and RAPTOR was altered by treatment with Tomivosertib ( Figure 4D and 4E). We also then further confirmed this interaction by overexpressing HA-RAPTOR and assessed the ability of RAPTOR to bind to MNK2 by co-immunoprecipitation ( Figure 4E). Together, these findings indicate that MNK2 interacts with mTORC1 but that Tomivosertib does not affect the interaction.
In the current study, we assessed the anti-leukemic effects of Tomivosertib [42], a highly selective MNK1/2 inhibitor that has recently entered phase 1/2 clinical trials for the treatment of patients with several types of solid tumors and lymphomas. Our studies demonstrate that Tomivosertib induces potent suppressive effects on leukemic cell viability and clonogenicity through inhibititon of eIF4E phosphorylation on serine 209 in different AML cell lines. Similar to what was observed with other less potent or specific MNK inhibitors; Tomivosertib was most effective at suppressing the growth of M5 subtype AML cells that acquire FLT3 activating mutations, such as MV4-11 and MM6 [32][33][34][35][36]. This is consistent with the fact that the M5 subtype of AML is characterized by the expression of high One-way ANOVA analysis followed by Tukey's test was used to evaluate statistically significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. levels of phosphorylated eIF4E, which correlates with poor prognosis in this cancer [18,19]. Moreover, the MAPK signaling cascade can be activated downstream of the FLT3 receptor [62]. Therefore, the concomitant presence of mutant proteins upstream of MNK1/2 such as FLT3 and elevated eIF4E levels, may constitute the best model for exploring the potential clinical utility of Tomivosertib. In contrast, THP-1, U937 and KG-1 cells, which do not express FLT3 mutations, were less sensitive to the compound. These cell lines, however, all have p53 mutations which results in loss of function of p53. In our LC/MS/MS analysis of proteins bound to MNK1 and MNK2, we discovered transcription regulation of p53 as one of the pathways significantly represented. Therefore it may be relevant in future studies to focus on the role that MNKs play in the regulation of the p53 pathway and determine the effects of the p53 mutational status on responses to MNK inhibitors.
Our data also demonstrate that the combination of Tomivosertib with Venetoclax enhances the anti-leukemic responses in mutant p53-expressing AML cell lines. The combination of these two drugs resulted in synergistic suppression of cellular viability and CFU-L growth in U937 and KG-1 cells. It has recently been proposed that AML cells can develop resistance to BH3 mimetics, such as Venetoclax, through increased levels of the anti-apoptotic protein MCL-1 via regulation of MCL-1 expression and stability [45,46,63,64]. Moreover, studies involving the use of Venetoclax and MEK inhibitors to simultaneously block BCL-2 and MAPK pathways have resulted in synergistic induction of apoptosis and suppression of cell proliferation in AML models [65]. The precise mechanism for the synergistic effect of Tomivosertib and Venetoclax remains to be elucidated in the future.
It is well-established that MNK1/2 drives mRNA translation through phosphorylation of its thoroughlycharacterized substrate eIF4E [15][16][17]. Phosphorylation of eIF4E by MNK1/2 has been shown to promote capdependent translation and the nuclear export of mRNAs with oncogenic potential [26][27][28][29][30][31]. However, only a few other MNK1/2 substrates are known beyond eIF4E.  We identified several potential novel interactors to both MNK1 and MNK2 in our LC/MS/MS analysis which require future validation studies to determine if they are bona fide substrates or regulatory partners. Notably, our LC-MS/MS studies identified mTOR and RAPTOR as interacting partners of MNK2. We demonstrated an interaction between MNK2, mTOR and RAPTOR through co-immunoprecipitation. A previous study has shown that MNK1/2 binds to mTORC1 and helps regulate mTORC1 substrate binding [66]. We only observed mTOR and RAPTOR binding to MNK2 in our LC/MS/MS analysis which supports previous published data that MNK2 binds with much more affinity than MNK1 [66]. This also supports previously published data that MNK2, not MNK1, is responsible for the activation of the feedback loop induced by the mTORC1 inhibitor rapamycin and that MNK2 plays a role in rapamycin insensitive mTORC1 complexes [67,68]. We did not see an effect on binding of RAPTOR to MNK2 upon MNK kinase inhibition with Tomivosertib similarly to what was observed with another MNK inhibitor CGP 57380 [66]. Overall, our data further supports previously published data that MNK proteins may play a role in regulating mTORC1.
Viewed altogether, these studies indicate that MNK1/2 inhibition would most likely be a successful strategy in only a subset of AML patients. In future studies it will be crucial to ascertain what pathways are responsible for sensitivity to MNK inhibitors. These studies will help to identify potential regulatory programs through which MNK1/2 modulates cell signaling pathways critical for leukemic cell survival and may lead to the development of novel therapeutic interventions for AML.

Chemical reagents
Tomivosertib (eFT-508) and Venetoclax (ABT-199) were purchased from TargetMol. All compounds were dissolved in dimethyl sulfoxide (DMSO) and used at the indicated doses.

Clonogenic leukemic progenitor assays in methylcellulose
Clonogenic assays in methylcellulose were conducted as in previous studies [69][70][71][72][73]77]. For AML patient samples, informed consent was obtained prior to collection of peripheral blood or bone marrow cells as approved by the Institutional Review Board of Northwestern University.

Co-immunoprecipitation assays
Cells were treated with either DMSO (vehiclecontrol) or Tomivosertib at the indicated doses and time points. Samples were processed and immunoprecipitation was performed using anti-FLAG-M2 agarose conjugated beads (Sigma-Aldrich) or anti-HA Sepharose conjugated beads (Cell Signaling) as previously described [77].

Proteomics immunoprecipitation analysis using LC-MS/MS
Samples were prepared and processed by LC-MS/MS analysis as previously reported [77]. Immunoprecipitation was performed with anti-FLAG-M2 agarose conjugated beads. www.oncotarget.com

Gene annotation and protein function enrichment analysis
This was performed as previously described using the Metascape database [77,78].

Statistical analyses
All experiments were performed in triplicate and repeated at least twice; variations about mean were presented as standard error. One-way ANOVA was used to compare more than two groups followed by Tukey's multiple comparison test. Student's t-test was used to assess differences between two groups. Differences were considered statistically significant when p values were less than 0.05. All statistical analyses were performed using Prism GraphPad 6.0.

ACKNOWLEDGMENTS
We would like to thank Dr. Rikiro Fukunaga from Osaka University for kindly providing the cDNA for MNK1 and MNK2a. The authors also thank Northwestern University's Proteomics Center of Excellence Core Facility for assistance.