Anlotinib suppresses MLL-rearranged acute myeloid leukemia cell growth through inhibiting SETD1A/AKT-mediated DNA damage response CURRENT STATUS: POSTED

Background: Leukemias driven by chromosomal translocation of the mixed-lineage leukemia gene (MLL) are highly prevalent in hematological malignancy. The poor survival rate and lack of an effective targeted therapy for patients with MLL-rearranged (MLL-r) leukemias emphasize an urgent need for improved knowledge and novel therapeutic approaches for these malignancies. In this study, we investigated the potential effectiveness and mechanism of Anlotinib, a novel receptor tyrosine kinase inhibitor, in MLL-r acute myeloid leukemia(AML). Methods: MLL-r AML cell lines were treated with different concentration of Anlotinib, then cell viability, apoptosis and cell cycle were analyzed. Next, the anti-leukemia effect of Anlotinib was further evaluated in vivo in a xenograft model of AML-carrying MLL-rearrangement. Finally, we performed RNA-seq analysis, Gene Set Enrichment Analysis (GSEA) and western bolting to explore the underlying mechanism of the anti-leukemia effect of Anlotinib. Results: Our findings revealed that Anlotinib significantly suppresses the growth, promotes robust apoptosis and induces G2/M arrest in MLL-r AML cells. Moreover, Anlotinib effectively inhibited the growth of MLL-r AML cells in a xenograft murine model. In mechanism, we find that the inhibitive role of Anlotinib in MLL-r AML could be largely attributed to the dysfunction of DNA damage and repair. Furthermore, we confirmed that Anlotinib impaired DNA damage response via inhibiting SETD1A and AKT. damage MLL-fusion-driven DNA repair enzymes the AML, leukemias the a novel RTK inhibitor, Anlotinib, MLL-r AML cell growth vivo preclinical targeting DNA damage response.


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The normal MLL1 gene encodes an approximately 500-kDa nuclear protein with multiple functional domains and binding partners, which was historically recognized as transcriptional deregulators of distinct HOX genes in normal hematopoietic differentiation 5 . In addition to MLL1, five more MLL family members (MLL2, MLL3, MLL4, SETD1A, and SETD1B) are found in mammals 6 . All of the MLL proteins physically associate with other protein factors to form larger macromolecular complexes, which stimulates the H3K4 methyltransferase activity of MLL proteins 6 . Leukemia-associated translocations involving 11q23 have been shown to generate in-frame fusions of the MLL1 gene to more than 80 different partner genes 7 . According to the report, the five most frequent fusion partners of MLL are AF4 (∼36%), AF9 (∼19%), ENL (∼13%), AF10 (∼8%), PTD (∼5%), which together represents more than 80% of the MLL-Fusion Proteins(MLL-FPs) found in MLL-r leukemia patients 7 . MLL-FPs have different chromatin-modifying activities than normal MLL proteins. Thecatalytic SET domain of wild type MLL that harbors H3K4 methyltransferase activity is lost in MLL-FPs 2,6 . Moreover, N-terminally truncated MLL alone is not sufficient to transform cells 2 . These findings argue for a non-catalytic function of MLL and redundancies such that other MLL homologs might contribute to leukemogenesis.
For this reason, unexpected and major roles for wild-type MLL and its paralogs (MLL2 and SETD1A) have been observed in MLL-r leukemia 8,9,10 . Therefore, drugs targeting MLL homolog binding partners have been identified as potential therapeutic tools against MLL-r leukemia 11,12,13,14,15,16 . In addition, several inhibitors of signaling pathways such as PI3K/AKT/mTOR and MAPK show a promising antileukemia activity for treatment of MLL-r leukemia 17,18 .
Anlotinib, a new oral small-molecule receptor tyrosine kinases(RTK) inhibitor, is designed to primarily inhibit multi-targets including VEGFR1, VEGFR2/KDR, VEGFR3, c-Kit, PDGFR-α, and the fibroblast growth factor receptors (FGFR1, FGFR2, and FGFR3) 19 . Amazing results obtained from the preclinical study have demonstrated that Anlotinib has anti-tumor activity across a broad spectrum of advanced cancers with little side effects 19 . For this reason, Anlotinib has been approved in China for the treatment of patients with advanced or metastatic non-small cell lung cancer 20 , and also is undergoing phase II and/or III clinical development for various sarcomas and carcinomas in China, USA and Italy 21 . Besides angiogenesis associated targets, new mechanisms and targets have been reported for Anlotinib inhibitory action on tumor growth 22,23 . Since Anlotinib can inactivate both AKT and ERK pathway, it is reasonable to assume that Anlotinib might also have a potential antileukemia activity.
In this study, we investigated the antitumor effects of Anlotinib in MLL-r leukemia and explored its potential mechanisms and downstream targets.

Materials And Methods
Reagents. The assay was performed as reported previously 16 . In brief, cells were cultured and treated with Anlotinib for 24, 48, and 72 h in a same condition as described above. Cells were incubated with Anlotinib for 24, after which total RNA was isolated as described previously 24 . RNA sequencing (RNA-seq) was then carried out via a commercially available service (Sangon Biotech, Shanghai). GSEA was used to enrich the signal pathways involved in differentially expressed genes. The gene set is c2.cp.kegg.v6.0.symbols.gmt[curated].
Animal study.
Animal studies were performed according to the Xiamen University Animal Guideline and approved by the Animal Welfare Committee. In brief, a total of 5 × 10 6 Molm-13 cells were inoculated subcutaneously into nude mice. After 3 days, mice were randomly divided into vehicle control and Anlotinib groups (n = 5, each group), and then treated respectively with either vehicle (PBS) or Anlotinib (6 mg/kg/day) by oral gavage for 9 days. Tumor size and body weight were measured daily.
Mice were sacrificed at the end of drug treatment or tumor size reached 1000 mm 3 . All tumors was removed, measured, and weighted. Tumor volume was calculated using the formula: V = (L × W 2 )/2, where L is the longest and W the shortest diameter of the tumor.
Statistical analysis.
All statistical analyses were carried out using the SPSS 23.0 and GraphPad Prism 6.0 softwares. The differences between two groups were analyzed using the two-tailed Student's t test. P < 0.05 was considered as statistically significant.

Results
Anlotinib effectively suppresses the growth and induces robust apoptosis of MLL-rearranged AML cells.
To assess the therapeutic action of Anlotinib in MLL-r AML, we first investigated its growth inhibition effect against two MLL-r AML cell lines (Molm13 and MV4-11) using the CCK8 assay. As shown in To explore whether the cytotoxicity of Anlotinib is also associated with cell cycle arrest, we analyzed cell cycle distribution upon Anlotinib treatment.At the early phase (8 hours) after Anlotinib administration, a dose-dependent cell cycle arrest in G2/M checkpoint was observed in both Molm-13 and MV4-11 ( Fig. 2A and C). This effect was continued until the late phase (24 hours) of Anlotinib treatment ( Fig. 2B and D).
The regimen using Anlotinib is active in vivo in a xenograft model of AML-carrying MLLrearrangement.
Then, the anti-tumor activity of the regimen using Anlotinib was examined in a mouse xenograft model established by subcutaneous inoculation with Molm-13 cells. After 3 days, mice were randomly divided into vehicle control group and Anlotinib treatment group. Anlotinib was administered orally once a day at a dose of 6 mg/kg/day for 9 days. During the drug administration, tumor volumes and mouse body weights were measured every day to evaluate the antitumor efficacy and side effects of Anlotinib. Our results showed that Anlotinib significantly reduced tumor burden of mice bearing MLL-r AML cells, as reflected by a remarkable decrease of tumor mass and weight ( Fig. 3A-C). Otherwise, there was no notable distinction between the mouse body weights of the Anlotinib and control groups ( Fig. 3D), suggesting a good tolerance for the in vivo use of Anlotinib. Together, these findings indicate that the regimen of Anlotinib is safe and effective in vivo against MLL-r AML.
Inactivation of DNA damage response contributes to the anti-leukemia effect of Anlotinib.
To delineate the molecular basis of tumor suppression role for Anlotinib in MLL-r AML, we performed RNA-seq analysis and found a different pattern of gene expression between Anlotinib treated group and control group (Fig. 4A).These differentially expressed genes(DEGs)were further analyzed with GSEA to identify their biological functions. The results showed that Anlotinib can affect many biological processes including spliceosome, DNA repair, metabolism, cell cycle and so on (Fig. 4B).
Among them, genes involved in DNA damage and repair pathway were particularly enriched (Fig. 4C).
Through Venn diagrams, we identified five critical genes for the DNA damage response (Fig. 4D). They are mainly composed of POLD1 POLD2 POLD3 LIG1 and PCNA which were downregulated by Anlotinib (Fig. 4E),and these findings were confirmed by western blotting (Fig. 4F). Our results indicate that the inhibitive role of Anlotinib in MLL-r AML could be largely attributed to the dysfunction of DNA damage and repair.
Anlontinib impairs DNA damage responses by downregulating SETD1A and AKT.
Recent studies have shown that MLL-fusion-driven leukemia requires SETD1A and AKT for appropriate expression of DNA damage response genes to safeguard genomic integrity 10,25,26 . Moreover, MLLfusion proteins engage a large number of distinct protein-protein interactions that are highly relevant for the biology of MLL-fusion proteins 27 . Thus, we sought to revealthe molecular logic of how modular protein-protein interactions between SETD1A/AKT and DNA damage response genes using two online databases, Gene Expression Profiling Interactive Analysis(GEPIA) and STRING (https://string-db.org/).
Our bioinformatics analysis showed that both SETD1A and AKT were strongly correlated with POLD1, POLD2, and POLD3 (Fig. 5A-H). These results further confirmed that SETD1A and AKT are required to regulate the expression of DNA repair specific genes in MLL-r leukemia. For this reason, we wondered whether Anolotinib is able to target SETD1A and AKT. As showed in Fig. 5I, both SETD1A and AKT were significantly downregulated in Anlotinib treatment group. Collectively, these results demonstrate that Anlotinib inhibit MLL-r leukemia cell growth through targeting SETDA1 and AKT mediated DNA damage response.

Discussion
Leukemias driven by MLL-rearrangement are highly prevalent in acute leukemia with a dismal prognosis 2,3,4 . The poor survival rate and lack of an effective targeted therapy for patients with MLL-r leukemias emphasize an urgent need for improved knowledge and novel therapeutic approaches for these malignancies. Oncogenic MLL-fusion proteins often hijack essential molecular mechanisms during leukemogenesis 6 . Recently, a considerable amount of studies have revealed that MLL and its chaperones are required for genomic integrity in MLL-r leukemia 10,27,28,29,30,31 and lead to chemoresistance 32 . For example, SETD1A is required for survival of AML cells depending on its FLOS domain but not the enzymatic SET domain 10 . The FLOS domain of SETD1A acts as a cyclin-K-binding site that is responsible for chromosomal recruitment of cyclin K and for DNA-repair-associated gene expression 10 . In addition, SETD1A is found functionally found to interact with the DNA damage repair protein RAD18 to safeguard genome stability 30 . These data suggest a pivotal role of DNA damage response for MLL-fusion-driven leukemia. Therefore, targeting DNA repair enzymes represent promising therapeutic strategies for the treatment of AML, especially MLL-driven leukemias 33 . In the present study, we showed that a novel RTK inhibitor, Anlotinib, inhibited MLL-r AML cell growth and progression in both in vitro and in vivo preclinical models by targeting DNA damage response.
Anlotinib has a broad spectrum of inhibitory action on tumor angiogenesis and growth, and has been approved for the treatment of several advanced refractory solid tumors 19,20,21 . Although it was designed to primarily inhibit receptor tyrosine kinase related to tumor vasculogenesis 34 , amounts of evidences show that Anlotinib suppresses tumor cells growth via inhibition of multiple pathways and targets 19,20,21 . Some of its targets, such as c-Kit 35 , Aurora-B 36,37 , AKT 18 and MEK pathway 17 , are involved in MLL-r leukemia. In this context, our study extends the field of indications for Anlotinib to the treatment of MLL-r AML by inducing apoptosis and cell cycle arrest ( Fig. 1 and Fig. 2). Moreover, our functional analysis on RNA sequencing data unravel an important role of DNA damage response for Anotinib antitumor activity (Fig. 4),which in line with cell cycle checkpoint arrest in G2/M by Anotinib (Fig. 2). In our experimental setting, we confirmed that Anlotinib is able to induce dysfunction of DNA damage response as manifested by downregulating DNA damage response genes (POLDs) (Fig. 4). Our results provide a novel insight into the mechanism and clinical application of Anlotinib.
DNA polymerase delta (POLD), a member of family B polymerases in eukaryotes, is essential for the leading and lagging strand synthesis 38,39,40 , which includes four subunits in mammals: POLD1-4 41 .
POLD1 functions as the catalytic subunit, which plays critical roles in DNA-replicate and DNA-repair processes, and POLD2 serves as a scaffold by interacting with POLD1 and the other POLD subunits 42 .
Multiple studies suggest that the abnormality of POLD1 is related to the poor prognosis of many tumors 43,44 . However, its role in AML is little known. Here, we disclose POLD1 as a prognostic predictor in AML by GEPIA, where patients with high POLD1 expression have an inferior survival (data not shown). In our research, Anlotinib could downregulate the expression of POLD1 and its subunits, highlighting a potential application of Anlotinib to the subgroup of AML with high POLD1 expression.
Since SETD1A and AKT are both required for appropriate expression of DNA damage response 10,25,26 , we further unravel the relationship between SETD1A/AKT and POLD family proteins by using bioinformatics (Fig. 5). Importantly, our results point to a molecular logic for SETD1A and AKT to control POLD family genes expression. Although much work is still needed to define the exact mechanisms by which SETD1A/AKT interact POLDs, according to our current findings, Anlnotib hampers DNA damage response via inhibition of SETD1A and AKT in MLL-r AML cells.

Conclusion
Taken together, our work shows that Anlotinib effectively suppresses the growth and induces robust apoptosis in MLL-r AML cells, probably through inhibiting SETD1A and AKT mediated DNA damage response. The present study also highlights a novel mechanism and a promise of using Anlotinib in the treatment of MLL-r leukemia.