Abstract

   T‐cell lymphoma (TCL) is a group of non‐Hodgkin's lymphoma with high
   heterogeneity and unfavorable prognosis. Current standard treatments
   have demonstrated limited efficacy in improving the outcomes for TCL
   patients. Therefore, identification of novel drug targets is urgently
   needed to improve the prognosis of TCL patients. Through multi‐omics
   analysis, aberrant expression of threonine tyrosine kinase (TTK) in TCL
   is identified. High expression of TTK is closely associated with poor
   prognosis in TCL patients. Targeting TTK through gene knockdown exerts
   anti‐tumor effects in vitro and in vivo, including inhibiting the cell
   proliferation, inducing G2/M phase arrest, enhancing DNA damage and
   cell apoptosis. Mechanically, p38α is identified as the potential
   phosphorylation substrate of TTK through phosphoproteomic
   quantification and motif prediction. Furthermore, inhibition of TTK
   suppresses activation of p38α through dephosphorylating it at
   Thr180/Tyr182, thereby promoting the activation of AMPK/mTOR pathway.
   In addition, targeting TTK enhances the autophagy in TCL cells through
   dephosphorylating p38α. CFI‐402257, a specific inhibitor of TTK, is
   found to exhibit anti‐tumor effects and exerted synergistic efficacy
   with PI3K inhibitor, Duvelisib, in TCL. The study shows that TTK
   contributes to the development of TCL by regulating p38α‐mediated
   AMPK/mTOR pathway. CFI‐402257 is expected to be a promising strategy
   for TCL treatment.

   Keywords: p38α, phosphorylation, T‐cell lymphoma, threonine tyrosine
   kinase, ubiquitination
     __________________________________________________________________

   In the study, aberrant expression of threonine tyrosine kinase (TTK) in
   TCL is identified. High expression of TTK is closely associated with
   poor prognosis in TCL patients. Inhibition of TTK, by either knockdown
   or specific inhibitor CFI‐402257, exerts anti‐tumor effects in vitro
   and in vivo. Mechanically, targeting TTK induces autophagy through
   dephosphorylating p38α at Thr180/Tyr182, which further promotes the
   activation of AMPK/mTOR pathway.

   graphic file with name ADVS-12-2413990-g002.jpg

1. Introduction

   T‐cell lymphoma (TCL) is a group of malignancies that arises from T
   cells, which is characterized by high aggressiveness and poor
   prognosis.^[ [42]^1 ^] The complex molecular subtyping has resulted in
   the absence of specific and highly effective targeted therapy for TCL
   patients. Despite the development of new drugs including CD30
   antibody‐drug conjugate, histone deacetylase inhibitors (HDACi), and
   folic acid antagonists,^[ [43]^2 ^] the therapeutic outcome and
   prognosis of TCL patients remain unsatisfactory with 5‐year
   progression‐free survival (PFS) rate less than 40% and overall survival
   (OS) rate less than 50% in peripheral T‐cell lymphoma (PTCL), while the
   5‐year OS of T‐cell acute lymphoblastic leukemia/lymphoma (T‐ALL) was
   48%.^[ [44]^3 ^] Given the lack of effective therapeutic interventions,
   advancing our understanding of the molecular biology in TCL and
   identifying novel drug targets are urgently needed to optimize the
   clinical outcome of TCL patients.

   Genomic instability is a hallmark of tumor cells, contributing to
   aberrant cell cycle control and indeterminate proliferation through the
   dysregulation of essential cellular pathways.^[ [45]^4 ^] The spindle
   assembly checkpoint (SAC) has been reported as a critical biological
   function to ensure accurate chromosome segregation in mitosis and
   prevent genomic instability.^[ [46]^5 ^] Targeting SAC proteins could
   induce high levels of genomic instability and aneuploidy that tumor
   cells cannot tolerate, ultimately leading to senescence and
   apoptosis.^[ [47]^6 ^]

   Threonine tyrosine kinase (TTK) is the central molecule that plays a
   crucial role in recruiting other SAC proteins to unattached
   kinetochores in prometaphase.^[ [48]^6 ^] More importantly, TTK has
   been found to regulate the phosphorylation of various substrates
   including c‐Abl,^[ [49]^7 ^] MDM2,^[ [50]^8 ^] and CHK2^[ [51]^9 ^]
   through kinase activity, thereby mediating cellular oxidative stress
   and DNA damage repair. Upregulation of TTK has been observed in various
   tumors, such as breast cancer and hepatocellular carcinoma (HCC), and
   was associated with worse prognosis of tumor patients.^[ [52]^6 ,
   [53]^10 ^] Moreover, TTK specific inhibitors, including NMS‐P715,
   MPI‐0479605, MPS‐BAY2b, and MPS‐IN‐3, have been found to exert
   anti‐tumor effects in various tumors.^[ [54]^11 , [55]^12 , [56]^13 ^]
   CFI‐402257 is a novel developed TTK inhibitor with higher
   selectivity,^[ [57]^14 ^] which has obtained approval for a phase 2
   clinical trial involving patients with ER+/HER2‐ advanced breast cancer
   ([58]NCT05251714).^[ [59]^15 , [60]^16 ^] However, the roles and
   mechanisms of TTK and its inhibitors in hematologic malignancies,
   particularly TCL, have not been clarified and require further
   investigation.

   In this study, we identified TTK as a pivotal contributor to the
   development of TCL. Targeted inhibition of TTK exerted anti‐tumor
   effects both in vitro and in vivo. Mechanically, inhibiting TTK
   dephosphorylated p38α at Thr180/Tyr182, thereby promoting the
   activation of AMPK/mTOR pathway. TTK inhibitor, CFI‐402257, exerted
   anti‐tumor effects in TCL and synergized with the PI3K inhibitor
   Duvelisib. These results elucidated the oncogenic role of TTK in TCL
   and provided a novel potent target and combinatorial therapeutic
   strategy for TCL patients.

2. Results

2.1. TTK Expression Was Upregulated in TCL and Associated with Tumor
Progression

   To screen for differentially expressed genes (DEGs) in TCL, analysis
   using multi‐omics data from publicly available databases was conducted.
   Through comparing 142 TCL clinical samples to 26 normal T cell samples
   from healthy donors, DEGs analysis revealed significant upregulation of
   3967 genes and downregulation of 5969 genes in TCL (Fold Change > 1.2,
   P < 0.05, Figure [61]S1A,B, Supporting Information). Furthermore, Kyoto
   Encyclopedia of Genomes (KEGG) pathway enrichment analysis demonstrated
   significant enrichment of DEGs in tumor‐associated signaling pathways
   including PI3K/AKT pathway and mTOR pathway (Figure [62]S1C, Supporting
   Information). Kaplan–Meier survival analysis of 192 TCL patients
   identified 2040 prognostic‐associated genes in TCL (P < 0.05, Figure
   [63]S1D, Supporting Information). Moreover, 1203 survival‐critical
   genes in four TCL cell lines (Karpas‐299, Ki‐JK, SUP‐M2, SUP‐T1) were
   identified using CRISPR screen data (Figure [64]S1E, Supporting
   Information). Combined with the above results, we disclosed 13 genes
   that might play key roles in TCL progression, including TTK, CDCA8,
   BUB1B, HJURP, HIST1H2BM, TICAM2, KIF23, CDT1, RPP40, CRCP, SDC1, HAMP,
   RAD51 (Figure [65]S1F, Supporting Information).

   Interestingly, cell cycle has been enriched in the gene function
   enrichment of DEGs, prognostic‐associated genes and survival‐critical
   genes (Figure [66]S2A–C, Supporting Information), suggesting that cell
   cycle regulation may be closely associated with the development of TCL.
   To further investigate the effect of cell cycle regulation in TCL
   development, drug screening was performed based on FDA Anti‐tumor Drug
   Library and 110 drugs were identified to effectively inhibit TCL cell
   viability. Among them, drugs targeting cell cycle, cytoskeletal
   signaling, and NF‐κB showed the highest effective ratio (Figure
   [67]S2D, Supporting Information). Consequently, we specifically focused
   on 4 cell cycle‐related genes, including TTK, BUB1B, CDT1, RAD51, and
   further performed qPCR to verify their expression levels in TCL (Figure
   [68]S2E, Supporting Information; Figure [69]1A). Consequently, TTK mRNA
   was consistently upregulated in TCL compared to peripheral blood normal
   T cells from healthy donors and displayed the highest expression level
   among the four genes (Figure [70]1A).

Figure 1.

   Figure 1
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   TTK expression was upregulated in TCL and associated with tumor
   progression. A) qPCR analysis showed that TTK expression was higher in
   TCL cells compared with normal T cells from healthy donors (n = 3). The
   level of TTK mRNA is shown on the y‐axis, while the cells are displayed
   on the x‐axis. B) The levels of TTK mRNA were significantly increased
   in TCL tissues (n =  142) from [72]GSE6338 and [73]GSE45712 compared
   with normal T cells (n  = 26). C) Kaplan–Meier analysis showed that
   high TTK expression level was associated with poor OS in TCL patients
   (n = 192) from [74]GSE58445. D) WB showed that the expression level of
   TTK was higher in TCL cell lines compared with T cells from healthy
   donors. E) Representative IHC staining images of TTK in TCL tissues and
   reactive hyperplasia samples. Bar  =  50 µm. F) TTK expression was
   higher in TCL tissues (n  =  92) compared to reactive hyperplasia
   samples (n  =  40, Control) when detected by IHC. G‐H) Kaplan–Meier
   survival analysis showed the association between TTK expression and OS
   and PFS in TCL patients, respectively. Data are shown as the mean ± SD.
   *P <  0.05; **P <  0.01; ***P <  0.001.

   Given the aberrant expression of TTK, we further explored the potential
   value of TTK in TCL. Up‐regulated TTK mRNA level was found in TCL
   patients (Figure [75]1B). Survival analysis indicated that TTK mRNA
   expression was associated with poor prognosis (Figure [76]1C).
   Significantly higher protein level of TTK was observed in TCL cell
   lines, compared to normal T cells from healthy donors (Figure [77]1D).
   The elevated TTK protein expression was verified by immunohistochemical
   (IHC) staining in TCL primary tissues (n = 92) compared to reactive
   hyperplasia (n = 40) (Figure [78]1E,F).

   Further survival analysis revealed the correlation between high TTK
   expression and worse OS and PFS in Shandong Provincial Hospital cohort
   (Figure [79]1G,H). The relevance between TTK expression and
   clinicopathological parameters of TCL patients was evaluated and TTK
   positive expression was associated with elevated plasma salivary acid
   (Table [80]S1, Supporting Information). Collectively, above results
   suggested that the expression of TTK might serve as a promising
   prognostic factor in TCL patients.

2.2. Knockdown of TTK Suppressed the Growth of TCL In Vitro and In Vivo
Through Regulating Cell Cycle, DNA Damage and Apoptosis

   The above findings motivated us to further explore the biological
   function of TTK in TCL. TTK knockdown and overexpression models were
   established in both Jurkat and Myla 3676 cells (Figure [81]S3A–C,
   Supporting Information). Knockdown of TTK significantly inhibited the
   cell proliferation of TCL cells (Figure [82]2A). The expression of
   proliferation marker, c‐myc and Ki‐67, were also found to be inhibited
   after TTK knockdown (Figure [83]2B). To further confirm the regulatory
   role of TTK on TCL progression in vivo, we constructed TCL xenograft
   mouse models (n = 6 per group). Compared with the control group, mouse
   bearing Sh‐TTK cells displayed reduced growth rate of tumor
   (Figure [84]2C), tumor weight (Figure [85]2D), and tumor volume
   (Figure [86]2E). Besides, mouse receiving Sh‐TTK TCL cells displayed
   decreased level of tumor burden when estimated by in vivo imaging
   system (Figure [87]2F). IHC results showed that Sh‐TTK group displayed
   reduced Ki‐67 expression level (Figure [88]S3D, Supporting
   Information). Taken together, our findings indicated that TTK
   contributed to TCL growth both in vitro and in vivo.

Figure 2.

   Figure 2
   [89]Open in a new tab

   Knockdown of TTK suppressed the growth of TCL in vitro and in vivo
   through regulating cell cycle, DNA damage and apoptosis. A) TTK
   knockdown inhibited the cell proliferation of TCL cells when assessed
   using CCK8 assay (n = 3). B) WB assay was used to detect the expression
   level of c‐myc and Ki‐67. TTK knockdown inhibited c‐myc and Ki‐67
   expression in TCL cells. C) Time course of tumor growth in Sh‐Control
   and Sh‐TTK TCL xenograft models (n  =  6 per group). D) Tumor weights
   of xenografts taken from TCL xenograft models in Sh‐Control and Sh‐TTK
   groups (n  =  6 per group). E) Tumor volumes of xenografts taken from
   TCL mouse models in Sh‐Control and Sh‐TTK groups (n  =  6 per group).
   F) Bioluminescence of tumor in Sh‐Control and Sh‐TTK TCL xenograft
   models (n  =  6 per group). G‐H) Detection of cell cycle in TCL cells
   after TTK knockdown by flow cytometry (n = 3). I‐J) Immunofluorescence
   analysis showed that TTK knockdown increased the number of micronuclei
   in TCL cells. Bar  =  20 µm. K) Immunofluorescence analysis showed that
   TTK knockdown enhanced the intensity of p‐H2AX in TCL cells.
   Bar  =  20 µm. L) WB analysis of the expression level of p‐H2AX in TCL
   cells after TTK knockdown. M) Representative images of cell death after
   TTK knockdown in TCL cells. Bar  =  20 µm. N‐O) Detection of cell
   apoptosis in TCL cells after TTK knockdown by flow cytometry (n = 3).
   P) WB analysis of the expression level of apoptotic markers in TCL
   cells after TTK knockdown. Data are shown as the mean ± SD. **P < 0.05;
   **P < 0.01; ***P < 0.001.

   To further explore the potential effects of TTK in TCL, functional
   enrichment analysis of DEGs between TCL patients with high and low TTK
   expression was performed in [90]GSE6338 and [91]GSE45712. Gene ontology
   (GO) analysis revealed that TTK appeared to be closely related to cell
   proliferation, apoptosis, cell cycle, chromosome segregation and DNA
   damage repair (Figure [92]S3E, Supporting Information). To verify the
   biological role of TTK in TCL, flow cytometry was employed and the
   results revealed that TTK knockdown led to the accumulation of cells in
   G2/M phase in TCL cells (Figure [93]2G,H). Given the crucial role of
   TTK in normal chromosome segregation during mitosis, immunofluorescence
   (IF) staining of α‐tubulin was performed and showed enhanced chromosome
   missegregation in TCL cells after TTK knockdown (Figure [94]S3F,
   Supporting Information). As micronuclei, chromosome fragments encased
   by the nuclear membrane in the cytoplasm, is usually acknowledged to
   arise from mitotic defects, we also found that TTK knockdown promoted
   the formation of micronuclei in TCL (Figure [95]2I,J), which may be
   mediated by chromosome missegregation and resulted in enhanced DNA
   damage. Furthermore, TTK knockdown also increased the expression level
   of p‐H2AX, a marker of DNA damage, in TCL (Figure [96]2K,L).

   It has been reported that DNA damage could induce p53‐dependent and
   independent apoptosis,^[ [97]^17 ^] the effect of TTK knockdown on
   apoptosis in TCL cells was further investigated. Microscopic
   examination found that TTK knockdown caused cell death manifested by
   the formation of solidified chromatin and cleaved nuclei
   (Figure [98]2M). Flow cytometry also revealed a significant increase in
   the apoptotic ratio of TCL cells after TTK knockdown (Figure [99]2N,O).
   Additionally, TCL cells with TTK knockdown displayed increased
   expression of apoptosis‐related molecules, including cleaved‐PARP,
   cleaved‐Caspase‐3 and cleaved‐Caspase‐9, indicating the pro‐apoptosis
   role of TTK knockdown (Figure [100]2P). Above results suggested that
   targeting TTK might suppress the growth of TCL through inducing cell
   cycle arrest, DNA damage and apoptosis.

2.3. TTK Specific Inhibitor CFI‐402257 Displayed Anti‐Tumor Effects in TCL

   To further investigate the potential anti‐tumor effects of targeting
   TTK in TCL, we used CFI‐402257 to selectively inhibit the activity of
   TTK. Time‐ and dose‐dependent inhibitory effect of CFI‐402257 treatment
   on the proliferation was found in TCL cells (Figure [101]3A). The
   inhibitory effect of CFI‐402257 on cell viability was also observed in
   TCL cells (Figure [102]S4A, Supporting Information). Besides,
   CFI‐402257 was found to induce the G2/M phase arrest in TCL cells
   (Figure [103]3B,C). Moreover, CFI‐402257 treatment resulted in abnormal
   chromosome segregation in TCL cells (Figure [104]S4B, Supporting
   Information). Elevated micronuclei formation and p‐H2AX expression were
   also observed through IF staining and WB (Figure [105]S4C–E, Supporting
   Information). CFI‐402257 induced cell death manifested by solidified
   chromatin and cleaved nuclei (Figure [106]3D). In addition, flow
   cytometry results revealed increased rate of apoptotic TCL cells after
   CFI‐402257 treatment (Figure [107]3E). Furthermore, downregulation of
   c‐myc expression and upregulation of cleaved‐PARP, cleaved‐Caspase‐3
   expression supported the anti‐proliferation and apoptosis‐inducing
   effects of CFI‐402257 in TCL cells (Figure [108]3F).

Figure 3.

   Figure 3
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   TTK specific inhibitor CFI‐402257 displayed anti‐tumor effects in TCL.
   A) CCK8 assay showed that CFI‐402257 exerted time‐ and dose‐dependent
   inhibitory effect on the proliferation in TCL cells (n = 3). B‐C)
   Detection of cell cycle in TCL cells after CFI‐402257 treatment (10 µM
   or 20 µM, 48 h) by flow cytometry (n = 3). D) Representative images of
   cell death after CFI‐402257 treatment (10 µM, 48 h) in TCL cells.
   Bar  =  20 µm. E) Detection of cell apoptosis in TCL cells after
   CFI‐402257 treatment (10 µM or 20 µM, 48 h) by flow cytometry (n = 3).
   F) WB analysis showed that CFI‐402257 treatment (10 µM or 20 µM, 48 h)
   decreased the expression of c‐myc and increased the expression of
   apoptotic markers in TCL cells. Data are shown as the mean ± SD. *P <
    0.05; **P <  0.01; ***P <  0.001.

2.4. TTK Interacted with p38α Through C‐Terminal and Phosphorylated p38α in
TCL

   Previous studies have shown that TTK could regulate the progression of
   esophageal squamous cell carcinoma and melanoma through its kinase
   activity.^[ [110]^18 , [111]^19 ^] To delve deeper into the kinase
   regulatory mechanism of TTK in TCL, we conducted the quantification of
   the phosphoproteome in Jurkat cell and identified 1129 differentially
   phosphorylated proteins after TTK knockdown (Figure [112]S5A,B,
   Supporting Information). These proteins were enriched in GO functions
   related to cell cycle, protein ubiquitination, autophagy, and so on
   (Figure [113]4A). Additionally, to pinpoint the direct kinase
   substrates regulated by TTK, we integrated the results of motif
   prediction^[ [114]^20 ^] for TTK substrates and phosphoproteomics
   quantification, and identified 12 potential kinase substrates of TTK
   (Figure [115]4B).

Figure 4.

   Figure 4
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   TTK interacted with p38α through C‐terminal and phosphorylated p38α in
   TCL. A) GO enrichment analysis of differentially phosphorylated
   proteins. The circle area indicated the number of genes, while the
   circle color represented the range of the corrected P values. B) 12
   potential kinase substrates of TTK were identified by integrating the
   results of motif prediction percentile and phosphoproteomics
   quantification. Bar plot showed the fold change of differentially
   phosphorylated proteins. C) The predicted binding mode of TTK (blue)
   and p38α (green). D) Co‐IP assay showed the interaction between TTK and
   p38α in TCL. E) Immunofluorescence co‐staining of TTK and p38α in TCL.
   Bar  =  20 µm. F) Immunofluorescence distribution showed the
   co‐localization of TTK (red) and p38α (green). G) The schematic of TTK
   truncated fragments. H) Co‐IP assay confirmed the interaction between
   TTK 525–857aa and p38α in TCL. I) WB analysis showed the expression
   level of p38α and p‐p38α after TTK knockdown in TCL. J) WB analysis
   showed the expression level of p38α and p‐p38α after CFI‐402257
   treatment (10 µM, 48 h) in TCL. K) WB analysis showed the expression
   level of p38α and p‐p38α after transfected with TTK kinase inactivated
   mutation plasmid. L‐M) CCK8 assay showed that the proliferation of TCL
   cells was inhibited by TTK kinase inactivation and p38 phosphorylation
   inactivation plasmids, which was reversed by p38 phosphorylation
   activation plasmid (n = 3). N) Detection of cell apoptosis in TCL cells
   after TTK knockdown and p38α phosphorylation activation plasmid
   transfection by flow cytometry (n = 3). O) Detection of cell cycle in
   TCL cells after TTK knockdown and p38α phosphorylation activation
   plasmid transfection by flow cytometry (n = 3). Data are shown as the
   mean ± SD. *P <  0.05; **P <  0.01; ***P <  0.001.

   Subsequently, to explore the possible interactions between TTK and
   potential substrates, HDOCK molecular docking analysis was performed
   and the Confidence Score of TTK and p38α was the highest
   (Figure [117]4C, Confidence Score = 0.9955, Docking score = ‐419.88,
   Ligand rmsd = 0.33). Co‐Immunoprecipitation (Co‐IP) further validated
   the protein‐protein interaction between TTK and p38α (Figure [118]4D).
   The fluorescence distributions in IF co‐staining of TTK and p38α
   supported their co‐localization within TCL cells (Figure [119]4E,F).
   Moreover, based on the reported TTK domains and their functions, we
   constructed three TTK truncated plasmids, N‐terminal‐K192 (including
   N‐terminal extension and N‐terminal tetratricopeptide repeat domains,
   associated with the subcellular localization of TTK to the
   centrosomes), Q193‐I524 (including MR domain, associated with the
   binding to NDC80 complex), and Y525‐C‐terminal (including kinase
   domain).^[ [120]^21 , [121]^22 , [122]^23 ^] Subsequently, full‐length
   and truncated plasmids of TTK were separately transfected in TCL cells
   companied by p38α‐HA plasmid (Figure [123]4G). It was disclosed by
   Co‐IP results that TTK interacted with p38α based on the C‐terminal
   region (Y525‐C‐terminal) (Figure [124]4H). These results suggested the
   interaction between TTK and p38α and their structural basis in TCL.

   To further explore the regulatory mechanism between TTK and p38α, we
   assessed the relative expression level of p‐p38α after TTK knockdown,
   and found decreased expression of both p38α and p‐p38α in TCL cells,
   and the expression of p‐p38α was decreased with more prominent tendency
   (Figure [125]4I). Additionally, CFI‐402257 treatment was also found to
   decrease the expression of p38α and p‐p38α with the similar tendency
   (Figure [126]4J). Above results indicated that TTK had regulatory
   effect on both expression and phosphorylation of p38α in TCL. Given
   that TTK was initially identified as bispecific protein kinase
   phosphorylating tyrosine and threonine, to elucidate whether TTK
   regulated the expression of p38α through kinase activity, the
   phosphorylation inactivated (p38α‐HA, T180A/Y182A) and activated mutant
   (p38α‐HA, T180E/Y182E) plasmid of p38α and the kinase inactivated
   mutant plasmid of TTK (TTK‐Flag/His, K553A) were constructed. It was
   found that the transfection of TTK kinase inactivated mutant plasmid
   led to the downregulation of both p38α and p‐p38α expression in TCL,
   indicating that TTK regulated the expression and phosphorylation of
   p38α through kinase activity (Figure [127]4K). Collectively, the above
   results suggested that targeting TTK could inhibit p38α phosphorylation
   at T180/Y182.

   Further rescue experiments were performed to explore whether the
   regulatory role of TTK on TCL proliferation depended on the
   phosphorylation of p38α. The proliferation of TCL cells was inhibited
   by the transfection of TTK kinase inactivation and p38 phosphorylation
   inactivation plasmids, whereas the p38α phosphorylation activation
   plasmid reversed the inhibitory effect of TTK kinase inactivation
   plasmid on proliferation (Figure [128]4L,M), suggesting that TTK
   exerted pro‐proliferative effects in TCL through the phosphorylation of
   p38α. Moreover, we also found that p38α phosphorylation activation
   plasmid reversed the pro‐apoptotic effect of TTK knockdown in TCL when
   detected by flow cytometry (Figure [129]4N). However, p38α
   phosphorylation activation failed to reverse the G2/M phase arrest
   after TTK knockdown, indicating that TTK regulated the cell cycle
   mainly in a p38α‐independent manner (Figure [130]4O).

   Notably, as TTK inhibition and kinase inactivated mutation also
   decreased the level of p38α, to further explore the regulatory
   mechanism of TTK on p38α expression, qPCR was performed and TTK
   knockdown did not significantly change the mRNA expression level of
   MAPK14 (p38α) (Figure [131]S6A, Supporting Information), indicating
   that TTK might directly regulate the protein level of p38α. To explore
   whether TTK affected the protein degradation of p38α, we conducted
   Cycloheximide (CHX) assay and illustrated that the protein degradation
   of p38α was expedited after TTK knockdown, which could be reversed by
   MG132 (Figure [132]S6B, Supporting Information). Given that p38α
   phosphorylation has been reported to regulate the activity of ubiquitin
   protein ligase including PJA1 and Ube3c,^[ [133]^24 , [134]^25 ^] it
   was hypothesized that the phosphorylation of p38α could affect its own
   ubiquitination degradation. We subsequently conducted CHX assay after
   transfecting WT p38α (p38α‐HA), phosphorylation‐inactivated (p38α‐HA,
   T180A/Y182A), and activated p38α plasmids (p38α‐HA, T180E/Y182E) to
   investigate the association between p38α phosphorylation and protein
   degradation. The results revealed that phosphorylation inactivation of
   p38α promoted the proteasome degradation of p38α while phosphorylation
   activation of p38α inhibited it (Figure [135]S6C, Supporting
   Information). Additionally, Co‐IP analysis revealed that both TTK
   kinase inactivation and p38α phosphorylation inactivation promoted the
   ubiquitination of p38α in TCL (Figure [136]S6D,E, Supporting
   Information). Subsequently, we predicted potential ubiquitination sites
   for p38α using GPS‐Uber^[ [137]^26 ^] and found that K152, K15, and
   K295 were the most promising sites for ubiquitination modifications
   (Table [138]S2, Supporting Information). The above results implied that
   TTK regulated the ubiquitin‐proteasomal degradation of p38α via
   dephosphorylation‐coupled way.

2.5. Inhibiting TTK Activated the AMPK/mTOR Pathway Through p38α to Restrain
TCL Development

   To further pursue the downstream TTK‐regulated signaling pathway of
   TCL, KEGG enrichment analysis of differentially phosphorylated proteins
   after TTK knockdown revealed significant enrichment in the AMPK pathway
   (Figure [139]5A). Subsequently, we found increased expression level of
   p‐AMPK and downregulation of p‐mTOR after inhibiting TTK, indicating
   the activation of AMPK/mTOR pathway (Figure [140]5B,C). Meanwhile,
   elevated expression levels of p‐TSC2 and p‐Raptor were also observed
   after TTK inhibition (Figure [141]S7, Supporting Information).
   Furthermore, the expression level of p‐AMPK was also increased while
   p‐mTOR expression was decreased after transfection with p38α
   phosphorylation inactivation plasmid (Figure [142]5D). Importantly, the
   activation of AMPK/mTOR pathway after TTK knockdown could be rescued by
   the transfection with p38α phosphorylation activation plasmid
   (Figure [143]5E), indicating that TTK regulated the AMPK/mTOR pathway
   in TCL through phosphorylating p38α.

Figure 5.

   Figure 5
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   Inhibiting TTK activated the AMPK/mTOR pathway through p38α to restrain
   TCL development. A) KEGG enrichment analysis of differentially
   phosphorylated proteins. The circle area indicates the number of genes
   in the pathway, while the circle color represents the range of the
   corrected P values. B,C) WB analysis showed the expression level of
   p‐AMPK and p‐mTOR after TTK knockdown and CFI‐402257 treatment (10 µM,
   48 h). D) WB analysis showed the expression level of p‐AMPK and p‐mTOR
   after transfection with p38α phosphorylation inactivation plasmid in
   TCL. E) WB analysis showed the expression level of p‐AMPK and p‐mTOR
   after TTK knockdown and transfection with p38α phosphorylation
   activation plasmid. F) CCK8 assay showed that the combined treatment
   with Rapamycin (mTOR inhibitor, 10 µM, 48 h) enhanced anti‐tumor
   effects of CFI‐402257 (10 µM, 48 h) in TCL (n = 3). G) WB analysis
   showed the expression level of p‐AMPK and p‐mTOR after TTK knockdown
   and Dorsomorphin (AMPK inhibitor) treatment (10 µM, 48 h). H) CCK8
   assay showed that Dorsomorphin (AMPK inhibitor, 10 µM, 48 h) and
   MHY1485 (mTOR activator, 20 µM, 48 h) rescued the inhibitory effect of
   TTK knockdown on TCL cell proliferation (n = 3). I) Mechanism diagram
   summarized that TTK contributed to TCL development through regulating
   the p38α/AMPK/mTOR axis. Data are shown as the mean ± SD. *P <  0.05;
   **P <  0.01; ***P <  0.001.

   Additionally, considering the potential anti‐tumor effects of Rapamycin
   (mTOR inhibitor), we evaluated the combinational inhibitory effects of
   CFI‐402257 and Rapamycin on the proliferation of TCL cells, and found
   that the combined treatment with Rapamycin enhanced anti‐TCL effect of
   CFI‐402257 (Figure [145]5F). Furthermore, as TTK also regulated the
   expression of AKT, which could promote mTOR activation,^[ [146]^27 ,
   [147]^28 ^] we used Dorsomorphin (AMPK inhibitor) to elucidate the main
   mechanism by which TTK regulated mTOR. The results displayed that
   Dorsomorphin treatment rescued the downregulation of p‐mTOR expression
   induced by TTK knockdown, suggesting that TTK mainly regulated mTOR in
   TCL via AMPK (Figure [148]5G). Subsequently, both Dorsomorphin and mTOR
   activator MHY1485 were found to rescue the inhibitory effect of TTK
   knockdown on TCL cell proliferation, suggesting that TTK modulated TCL
   proliferation through AMPK/mTOR pathway (Figure [149]5H). Collectively,
   above results suggested that targeting TTK activated the AMPK/mTOR
   pathway through p38α dephosphorylation to inhibit TCL development.

2.6. TTK Regulated the Autophagy in TCL via Modulating p38α Phosphorylation

   Differentially phosphorylated proteins after TTK knockdown were
   significantly enriched in biological processes including autophagy as
   observed in the functional enrichment. Meanwhile, it has been reported
   that p38α was involved in the autophagy regulation.^[ [150]^29 ,
   [151]^30 ^] To elucidate whether TTK regulated autophagy in TCL cells
   through p38α, we detected the autophagic vesicles after TTK knockdown.
   IF staining revealed the augmentation of LC3B in the autophagic
   vesicles after CFI‐402257 treatment (Figure [152]6A). LC3B IF staining
   also revealed that TTK knockdown and p38α phosphorylation inactivation
   plasmid transfection promoted autophagy process in TCL cells
   (Figure [153]6B,C; Figure [154]S8, Supporting Information). CFI‐402257
   treatment and TTK knockdown reduced the expression level of p62 and
   elevated the expression level of Beclin and LC3B‐II (Figure [155]6D,E).
   Chloroquine was used to block autophagosome degradation and it was
   found that chloroquine treatment further enhanced the promotion of
   autophagy by TTK knockdown, suggesting that TTK knockdown enhanced the
   autophagic flux in TCL (Figure [156]6F). Moreover, assessment of p62,
   Beclin, LC3B‐II also revealed enhanced autophagy process in TCL cells
   after p38α phosphorylation inactivation (Figure [157]6G). Notably, p38α
   phosphorylation activation plasmid rescued the effect of TTK knockdown
   on autophagy, indicating that TTK regulated autophagy by
   phosphorylating p38α in TCL (Figure [158]6H). Taken together, our
   results clarified that TTK could regulate the autophagy in TCL via
   modulating p38α phosphorylation.

Figure 6.

   Figure 6
   [159]Open in a new tab

   TTK regulated the autophagy in TCL via modulating p38α phosphorylation.
   A) IF analysis of LC3B autophagic vesicles (red) after CFI‐402257
   treatment (10, 20µM, 48 h) in TCL cells. Bar  =  20 µm. B) IF analysis
   of LC3B autophagic vesicles (red) after TTK knockdown in TCL cells.
   Bar  =  20 µm. C) IF analysis of LC3B autophagic vesicles (red) after
   transfection with p38α phosphorylation inactivation plasmid in TCL.
   Bar  =  20 µm. D) WB analysis showed the expression level of p62,
   Beclin and LC3B‐II after CFI‐402257 treatment (10 µM, 48 h) in TCL
   cells. E) WB analysis showed the expression level of p62, Beclin, and
   LC3B‐II after TTK knockdown in TCL. F) WB was used to determine the
   expression level of p62, Beclin, and LC3B‐II after TTK knockdown and
   chloroquine treatment (10µM, 12 h) in TCL. G) WB analysis showed the
   expression level of p62, Beclin, and LC3B‐II after transfection with
   p38α phosphorylation inactivation plasmid in TCL. H) WB analysis showed
   the expression level of p62, Beclin and LC3B‐II after TTK knockdown and
   transfection with p38α phosphorylation activation plasmid in TCL. Data
   are shown as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

2.7. CFI‐402257 Exerted Synergetic Anti‐Tumor Effects with Duvelisib in TCL
Cells and Xenograft Models

   To further explore the potential clinical application of CFI‐402257 in
   TCL treatment, 211 drugs with anti‐tumor effects in TCL from FDA
   Anti‐tumor Drug Library were selected to uncover the potential
   synergetic drugs with CFI‐402257 according to single drug anti‐TCL
   effect and drug recommendation in clinical practice. The combinational
   inhibitory effect of 211 drugs with 10µM CFI‐402257 in TCL cell was
   evaluated by CellTiter‐Glo Luminescent (CTG) assay and the top20 drugs
   were shown in Figure [160]7A. Subsequently, 9 TCL clinical recommended
   drugs with potential combination effects were used to assess the
   Chou‐Talalay Combination Index (CI) with CFI‐402257 at three
   concentration gradients (Figure [161]7B). Three drugs (Duvelisib,
   Lenalidomide, Bortezomib) with the smallest mean CI at all three
   concentration gradients were selected to further evaluate the
   synergetic effect with different combinational concentration ratios of
   CFI‐402257. It was found that Duvelisib (the PI3K δ/γ inhibitor)
   exhibited the highest synergetic scores, indicating its intense and
   stable synergetic effect with CFI‐402257 in TCL (Figure [162]7C; Figure
   [163]S9A–C, Supporting Information). Moreover, TTK knockdown was found
   to enhance the sensitivity of TCL cells to Duvelisib, indicating that
   TTK was involved in the mechanism of synergistic effects
   (Figure [164]7D).

Figure 7.

   Figure 7
   [165]Open in a new tab

   CFI‐402257 exerted synergetic anti‐tumor effects with Duvelisib in TCL
   cells and xenograft models. A) Combinational inhibitory effect of drugs
   in FDA Anti‐tumor Drug Library with 10 µM CFI‐402257 in Jurkat cell
   after 48 h treatment. Plot showed the top 20 drugs with the lowest
   relative cell viability. B) Chou–Talalay Combination Index of 9 drugs
   with 10 µM CFI‐402257 in Jurkat cell after 48 h treatment. C) HSA
   synergetic scores of Duvelisib with 10 µM CFI‐402257 in Jurkat cell
   after 48 h treatment. D) CCK8 assay showed that TTK knockdown increased
   the sensitivity of TCL cells to Duvelisib (n = 3). E) WB analysis
   showed the expression of p‐PI3K and p‐AKT after TTK knockdown in TCL.
   F) IF analysis of p‐AKT on cell membrane after TTK knockdown in TCL. G)
   The predicted binding mode of TTK (blue) and AKT (green). H) Co‐IP
   analysis showed the interaction between TTK and AKT in TCL cells. I) IF
   co‐staining of TTK and AKT in TCL. Bar  =  5 µm. J) Schematic diagram
   of the TCL xenograft mouse models treated with CFI‐402257 (6mg kg^−1
   day^−1) and Duvelisib (10mg kg^−1 day^−1). K) Time course of tumor
   growth in TCL xenograft models treated with CFI‐402257 and Duvelisib
   (n  =  5 per group). L) Tumor volumes of xenografts taken from TCL
   mouse models treated with CFI‐402257 and Duvelisib (n  =  5 per group).
   M) Tumor weights of xenografts taken from TCL mouse models treated with
   CFI‐402257 and Duvelisib (n  =  5 per group). Data are shown as the
   mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

   As Duvelisib is the PI3K δ/γ inhibitor, we further examined the
   regulatory effect of TTK on PI3K/AKT pathway, and found that the
   expression of p‐AKT in TCL was reduced after TTK knockdown
   (Figure [166]7E). While the relative expression level of p‐PI3K showed
   no remarkable change. The membrane aggregation and activation of AKT
   play an important role in the regulation of downstream molecules, for
   which IF staining was utilized to observe the effect of TTK knockdown
   on p‐AKT expression and cellular localization. It was found that TTK
   knockdown also reduced the intensity of p‐AKT on membrane, indicating
   the downregulation of p‐AKT activity in TCL after TTK knockdown
   (Figure [167]7F). In order to further explore molecular mechanism for
   synergetic effects, HDOCK was utilized to identify potential
   interactions between TTK and key molecules in the PI3K/AKT pathway. The
   potential interaction between TTK and AKT was disclosed with a
   Confidence Score of 0.7720 in the best model (Figure [168]7G, Docking
   score = −210.98, Ligand rmsd = 59.36). Co‐IP results further revealed
   the interaction between TTK and AKT in TCL cells (Figure [169]7H). The
   co‐localization of TTK and AKT in TCL cells was found using IF
   co‐staining (Figure [170]7I).

   Furthermore, in‐vivo results revealed that both CFI‐402257 and
   Duvelisib significantly reduced the tumor growth rate, size and weight
   in TCL xenograft mouse models, and the combinational therapy of two
   drugs exerted enhanced inhibitory effect compared with single drug
   (Figure [171]7J–M). Overall, the above results supported that
   CFI‐402257 had synergistic anti‐TCL effect with Duvelisib in vitro and
   in vivo, and the synergistic effect may be mediated by the interaction
   and regulation of TTK on AKT.

3. Discussion

   Our study illuminated the regulatory role and mechanism of TTK in the
   development of TCL. Association between high TTK expression and worse
   prognosis in TCL patients was revealed. Inhibition of TTK, by either
   knockdown or specific inhibitor CFI‐402257, exerted anti‐tumor
   effects in vitro and in vivo. Mechanically, targeting TTK induced
   autophagy through dephosphorylating p38α at Thr180/Tyr182, which
   further promoted the activation of AMPK/mTOR pathway.

   It was recently reported that high expression level of TTK was
   correlated with the progression, clinical features and prognosis in
   various tumors.^[ [172]^31 , [173]^32 ^] TTK exhibited significant high
   expression in lung cancer and ovarian cancer, and was associated with
   Cisplatin resistance.^[ [174]^27 , [175]^33 ^] Meanwhile, TTK was also
   found to be up‐regulated in mantle cell lymphoma and diffuse large
   B‐cell lymphoma (DLBCL), and was associated with higher Ki‐67 index.^[
   [176]^34 ^] However, the expression level and clinical prognostic value
   of TTK have not been reported in TCL. Here, our study found that the
   expression level of TTK was elevated in TCL patients, and high
   expression of TTK was associated with poor OS and PFS, suggesting that
   TTK could serve as a potential prognostic biomarker for TCL patients.
   Studies containing more patients and longer follow‐up time are still
   needed to further verify the prognostic role and clinical value of TTK
   in TCL.

   Previous studies have demonstrated the oncogenic role of TTK in several
   solid tumors. Inhibition of TTK has been found to suppress the
   proliferation of tumor cells in lung cancer, colon cancer and breast
   cancer.^[ [177]^35 , [178]^36 , [179]^37 , [180]^38 , [181]^39 ^]
   Moreover, TTK inhibition could induce mitotic errors and enhance
   apoptosis in breast and pancreatic cancer.^[ [182]^36 , [183]^39 ^] In
   HCC, targeted inhibition of TTK reduced tumor aggressiveness, and
   induced DNA damage, micronuclei formation, autophagy, and cellular
   senescence.^[ [184]^16 ^] TTK has also been reported to be associated
   with drug resistance of tumor cells. Downregulation of TTK has been
   found to increase the sensitivity of tumor cells to Temozolomide,
   Cisplatin and Sorafenib in Glioblastoma (GBM) and colon cancer.^[
   [185]^27 , [186]^40 ^] Furthermore, recent studies have reported the
   relevance of TTK to tumor immune microenvironment, where TTK inhibition
   could promote T cell‐mediated anti‐tumor immunity and enhance the
   sensitivity to PD‐1 blockade therapy.^[ [187]^41 , [188]^42 ^] In terms
   of hematologic malignancy, TTK inhibition was found to induce
   chromosome segregation errors, DNA damage, and micronuclei formation in
   acute myeloid leukemia (AML).^[ [189]^43 ^] However, the biological
   role of TTK in TCL has not yet been reported. Our study found that TTK
   knockdown exerted inhibitory effect on TCL progression by suppressing
   cell viability and proliferation, inducing G2/M cell cycle arrest,
   promoting DNA damage and activating apoptosis. These results suggested
   that TTK might play a pro‐tumorigenic role in TCL, indicating its
   potential as therapeutic target in TCL treatment.

   As a specific inhibitor of TTK, CFI‐402257 has been found to exert
   significant anti‐tumor effects in solid tumors, such as GBM, malignant
   mesothelioma, and lung cancer, by competitively inhibiting the kinase
   activity of TTK.^[ [190]^40 , [191]^44 ^] Previous studies have
   reported that CFI‐402257 activated the cGAS‐STING pathway by promoting
   micronuclei formation, and enhanced the efficacy of anti‐PD‐1 treatment
   in KRAS‐LKB1 mutant lung cancer.^[ [192]^45 ^] CFI‐402257 exhibited
   anti‐tumor effects in in vivo and in vitro models of HCC and induced
   senescence‐associated secretory phenotype through activating the
   DDX41‐STING pathway.^[ [193]^16 ^] Moreover, CFI‐402257 was found to
   display enhanced anti‐tumor effects in ER^+ breast cancer with
   resistance to CDK4/6 inhibitors.^[ [194]^15 ^] Clinical trials
   assessing CFI‐402257 included monotherapy in advanced cancer patients
   ([195]NCT02792465, [196]NCT05251714), in combination with fulvestrant
   in ER and/or PR‐positive breast cancer patients ([197]NCT03568422), and
   in combination with paclitaxel in ER+/HER2‐advanced breast cancer
   patients ([198]NCT05251714). According to the interim results of
   [199]NCT03568422, 47% (8/17) of patients who received CFI‐402257 in
   combination with paclitaxel achieved clinical benefit (complete
   response or partial response or stable disease). Notably, CFI‐402257
   has received FDA fast‐track designation, indicating the clinical
   application potential of CFI‐402257 for tumor treatment. Here, our
   results demonstrated that CFI‐402257 exerted anti‐tumor effects in TCL
   cells and xenograft mouse models, providing a promising strategy for
   the treatment of TCL.

   TTK, as a bispecific protein kinase, has been reported to be involved
   in cell proliferation and division processes during tumor
   progression.^[ [200]^46 ^] For example, TTK was found to phosphorylate
   tyrosine kinase c‐Abl at Thr735, and the knockdown of TTK suppressed
   the growth of breast cancer cells by downregulating the phosphorylation
   of c‐Abl.^[ [201]^7 ^] Besides, TTK also promoted the phosphorylation
   of E3 ubiquitin ligase MDM2, thereby promoting DNA oxidative damage
   repair and cell survival.^[ [202]^8 ^] In our study, we identified that
   p38α was a downstream kinase substrate of TTK, and TTK could directly
   interact with p38α in TCL cells. p38α, the central component of
   p38‐MAPK pathway, has been found to regulate various cellular
   functions, including cell proliferation, differentiation, stress
   response, apoptosis, cell migration and autophagy, through
   phosphorylating downstream transcription factors.^[ [203]^47 ^] In
   breast cancer, p38α knockout induced the DNA damage response, increased
   replication stress, and chromosomal instability.^[ [204]^48 ^] In
   osteosarcoma, p38α activation triggered the formation of autophagosomes
   and enhanced basal autophagy flux, which in turn induced tumor cells to
   enter senescence instead of apoptosis.^[ [205]^30 ^] Several studies
   have shown that p38α exerts regulating effect on ubiquitin ligases. As
   previously reported, phosphorylation of p38α promoted its K48‐linked
   polyubiquitination mediated by the E3 ubiquitin ligase Nedd4.^[
   [206]^49 ^] P38α has been reported to inhibit the polyubiquitination
   activity of Ube3c in response to progesterone by phosphorylating serine
   741 of Ube3c.^[ [207]^24 ^] In differentiated muscle cells, activation
   of p38α is involved in the regulation of EZH2 levels via the E3
   ubiquitin ligase Praja1.^[ [208]^25 ^] In our study, we found that TTK
   could regulate the expression and activation of p38α in TCL by
   phosphorylating it at Thr180/Tyr182 and then inhibiting its
   ubiquitin‐proteasomal degradation coupled with dephosphorylation.
   Further results indicated that TTK modulated the autophagy of TCL cells
   through phosphorylating p38α. Moreover, further exploration is still
   needed to investigate the relevant enzymes and target sites involved in
   ubiquitination regulation of p38α by TTK in TCL.

   Additionally, AMPK and its key downstream molecule mTOR play a
   significant role in maintaining cellular energy homeostasis and cell
   growth.^[ [209]^50 , [210]^51 ^] Previous studies have shown that
   p‐p38α regulated gluconeogenesis as a negative regulator of AMPK
   signaling.^[ [211]^52 ^] The dysregulation of the AMPK/mTOR pathway was
   also found in lymphoma,^[ [212]^53 ^] which has been considered as a
   potential target for lymphoma treatment. In T‐ALL, metformin could
   suppress tumor growth by activating AMPK,^[ [213]^54 ^] and the AMPK
   inhibitor compound C could induce apoptosis in HTLV‐1‐infected T cell
   lines through promoting DNA damage.^[ [214]^55 ^] Meanwhile, it has
   been proposed that genomic instability generated micronuclear
   structures could trigger autophagy degradation,^[ [215]^56 ^]
   Activation of Ataxia‐telangiectasia mutated proteins (ATM) by DNA
   damage induced the activation of AMPK and further abrogated the
   inhibitory effect of TORC1 on autophagy.^[ [216]^57 ^] In addition, the
   HDACi valproic acid (VPA) has been found to induce autophagy in T‐ALL
   through the activation of AMPK/mTOR pathway.^[ [217]^58 ^] Recent
   research found that malate metabolizing enzyme ME2 stimulated mTOR
   complex 1 activity to promote the development of TCL, while mTOR
   inhibitor Rapamycin effectively inhibited the growth of TCL in vivo.^[
   [218]^59 ^] The mTOR inhibitor Rapamycin has also shown clinical
   activity in mantle cell lymphoma.^[ [219]^60 ^] However, limited
   research on its clinical effects in TCL has been reported. Here, our
   results revealed that TTK regulated the AMPK/mTOR pathway through p38α
   during the development of TCL. Besides, we disclosed that Rapamycin had
   inhibitory effect on TCL proliferation, which could be enhanced by the
   TTK inhibitor CFI‐402257. The precise regulatory mechanism of TTK in
   the regulation of AMPK/mTOR pathway and the potential of combining
   CFI‐402257 and Rapamycin in TCL treatment remain to be further
   explored.

   Moreover, given the important role of PI3K in promoting tumor
   progression, Duvelisib, a dual inhibitor targeting PI3Kγ/δ, has been
   indicated to exhibit anti‐tumor effects in tumors with predominant
   expression of PI3Kγ/δ, especially in B‐cell and T‐cell lymphoma.^[
   [220]^61 ^] The results of phase 2 clinical trial for Duvelisib showed
   that the objective response rate (ORR) of monotherapy for refractory
   PTCL was 49%, indicating the promising application prospect of
   Duvelisib in TCL.^[ [221]^62 ^] Here, the potential combined anti‐TCL
   effect of Duvelisib with CFI‐402257 was discovered through drug library
   screening and further in vitro and in vivo study found that CFI‐402257
   exerted stable synergistic effect with Duvelisib, which might be
   mediated by the interaction between AKT and TTK. Overall, our finding
   might provide a promising novel combination therapy strategy for TCL
   patients, and further research on key problems, such as the optimal
   dosage, timing of administration and adverse reactions, is still needed
   to evaluate the clinical potential of Duvelisib and CFI‐402257 combined
   therapy strategy for TCL treatment.

   Considering the limitations of this study, our functional experiments
   incorporated TCL cells including T‐ALL, anaplastic large cell lymphoma
   and cutaneous T‐cell lymphoma. Due to the heterogeneity of molecular
   features and clinical manifestations among TCL subtypes, the
   variability of the biological function and regulatory mechanisms for
   TTK in cells of different TCL subtypes still needs to be further
   explored. Meanwhile, recent studies have shown that the promoter of TTK
   is regulated by H3K18 lactylation in pancreatic cancer,^[ [222]^63 ^]
   the regulatory mechanisms of upstream transcription and activation of
   TTK in TCL remains to be further investigated.

   Collectively, our results revealed the elevated expression, prognostic
   value and oncogenic role of TTK in TCL, and highlighted the therapeutic
   potential of CFI‐402257 in TCL treatment. Notably, inhibiting TTK
   exerted anti‐TCL effects in vitro and in vivo through downregulating
   the phosphorylation and upregulating the ubiquitination of p38α, which
   further promoted autophagy and the activation of the AMPK/mTOR pathway.
   Overall, these findings identified TTK as a novel target for TCL
   treatment and underscored the potential of CFI‐402257 as promising
   therapeutic strategy for TCL, which might contribute to the targeted
   therapy and prognosis improvement in TCL patients.

4. Experimental Section

Sex as a Biological Variable

   In this study, sex was not considered as a biological variable.

Patient Samples

   This study was approved by the Medical Ethical Committee of Shandong
   Provincial Hospital and written informed consent in accordance with the
   Declaration of Helsinki was obtained from each patient.
   Paraffin‐embedded archived samples were collected from 92 newly
   diagnosed TCL patients and 40 reactive hyperplasia patients from 2012
   to 2023. Histological diagnoses were established according to the 2022
   WHO classification.^[ [223]^64 ^] All enrolled TCL patients have
   received standard treatment according to international guidelines.
   Details of patients were provided in Table [224]S1 (Supporting
   Information). Normal T cells were purified from freshly isolated
   Peripheral blood mononuclear cells (PBMCs) from healthy donors by
   double‐rosetting with 2‐aminoethylosothiouronium bromide‐treated sheep
   red blood cells (SRBCs).^[ [225]^65 ^]

Cell Lines

   Karpas‐299, Jurkat, Molt‐3 and Myla 3676 cell lines were purchased from
   ATCC, cultured in IMDM (Gibco, CA, USA) supplemented with 10% fetal
   bovine serum (HyClone, UT, USA), 1% penicillin/streptomycin mixture,
   and 2mM glutamine, and incubated at 37 °C in humidified air containing
   5% CO[2]. All cells were examined for short tandem repeat (STR) and
   mycoplasma infection periodically.

Reagents

   CFI‐402257 (GC18491) and Dorsomorphin (GC17243) was obtained from
   GlpBio (GlpBio, CA, USA). Duvelisib (T1988), MHY1485 (T1908) were
   bought from Topscience (Topscience, Shanghai, China). FDA Anti‐tumor
   Drug Library was purchased from Selleck Chemicals (L8000, Selleck
   Chemicals, TX, USA). MG132 (HY‐13259), CHX (HY‐12320), and Chloroquine
   (HY‐17589A) were obtained from MedChemExpress (MedChemExpress, NJ,
   USA).

Bioinformatics Analysis

   The mRNA expression microarray and prognosis data of TCL patients and
   healthy donors were derived from the public database GEO
   ([226]https://ww.ncbinlm.nih.gov/geo). The data from [227]GSE45712
   (n = 117) and [228]GSE6338 (n = 51) were used for differentially
   expressed genes (DEGs) analysis. The sva package in R was utilized to
   eliminate potential batch effects across different datasets. The limma
   package in R was used to identify DEGs in TCL clinical samples and
   normal T cells from healthy donors (P < 0.05, fold change>1.2).
   Functional enrichment was performed based on the KEGG and GO databases
   using clusterProfiler in R. CRISPR Screen data of TCL cell lines was
   obtained from public database BioGRID ORCS
   ([229]https://orcs.thebiogrid.org/).

Cell Transfection

   Lentivirus vectors encoding short hairpin TTK (Sh‐TTK), TTK
   overexpression (LV‐TTK) or control were from Genechem (Shanghai,
   China), and plasmids, including TTK‐Flag/His, TTK‐Flag/His (K553A),
   TTK‐Flag/His (1‐192aa), TTK‐Flag/His (193‐524aa), TTK‐Flag/His
   (525‐857aa), p38α‐HA, p38α‐HA (T180E/Y182E), p38α‐HA (T180A/Y182A) were
   synthesized by Biosune (Shanghai, China).

IHC and Hematoxylin–Eosin (HE) Staining

   IHC and HE staining were performed according to standard methods.^[
   [230]^66 ^] Reactive hyperplasia of lymph nodes was considered as
   control group. Staining results were evaluated by two independent
   observers who were blinded to patients’ clinical data at two different
   time points. IHC score was calculated by the accumulation of
   multiplying proportion score (0, none; 1, 1–25%; 2, 26–50%; 3, 51–75%;
   and 4, 76–100%) and intensity score (1, negative; 2, weak; 3, moderate;
   and 4, strong). Scores of 0–5 were defined as negative expression, 6–16
   as positive expression. The primary antibodies included TTK
   (10381‐1‐AP, Proteintech, Wuhan, China) and Ki‐67 (27309‐1‐AP,
   Proteintech).

Western Blotting

   Cell lysates were extracted using RIPA buffer (Pierce) together with
   inhibitors for proteases and phosphatases (PhosSTOP, Roche, Basel,
   Switzerland). Following the previous description,^[ [231]^67 ^] WB
   analysis was conducted. The primary antibodies were as follows, TTK
   (A2500, Abclonal, Wuhan, China), p38α (66234‐1‐Ig, Proteintech),
   Caspase‐3 (ab184787, Abcam, MA, USA), cleaved‐Capase‐3 (68773‐1‐Ig,
   Proteintech), Caspase‐9 (10380‐1‐AP, Proteintech), cleaved‐Caspase‐9
   (9509, Cell Signaling Technology (CST), MA, USA), PARP (13371‐1‐AP,
   Proteintech), LC3B (ab192890, Abcam), phospho‐p38α (Thr180/Tyr182)
   (4511, CST), AKT (60203‐2‐Ig, Proteintech), phospho‐AKT (2535, CST),
   AMPK (ab32047, Abcam), phospho‐AMPK (2535, CST), mTOR (2983, CST),
   phospho‐mTOR (5536, CST), GAPDH (10381‐1‐AP, Proteintech), p62
   (ab207305, Abcam), Beclin (ab207612, Abcam), c‐myc (18583, CST), p‐H2AX
   (9718, CST), Ubiquitin (3936, CST), HA (3724, CST), TSC2 (24601‐1‐AP,
   Proteintech), p‐TSC2 (5584, CST), Raptor (20984‐1‐AP, Proteintech),
   phospho‐Raptor (2083, CST). Following overnight incubation with primary
   antibodies at 4 °C, membranes were washed with TBS‐T and then treated
   with HRP‐conjugated secondary antibody (Zhongshan Goldenbridge). Images
   were captured with the Amersham Imager 680 imaging system (General
   Electric, USA).

Cell Proliferation and Viability Assay

   Cell proliferation was assessed using the Cell Counting Kit‐8 (CCK8)
   assay kit (CK04, DOJINDO, Japan) and Multiskan GO Microplate Reader
   (Thermo Scientific, IL, USA). Cell viability was evaluated using CTG
   assays (G7570, Promega Corporation, WI, USA) and luminescence was
   recorded with a microplate luminometer (Centro XS3 LB960, Berthold
   Technologies, Stuttgart, Germany).

Flow Cytometry Analysis

   For apoptosis analysis, TCL cells were harvested and washed twice with
   cold PBS and resuspended cells in 1× binding buffer, followed by adding
   5 µL of Annexin V‐PE and 5 µL of 7‐AAD (559763, BD Biosciences, USA).
   For cell cycle analysis, the 70% ethanol fixation at −20 °C overnight
   was followed by washing with PBS and staining with PI/RNAse stain
   (550825, BD Biosciences) for 15 min. Flow cytometry was used to analyze
   cells after incubation in the dark for 15 min. Cell cycle and apoptosis
   were determined by flow cytometric analysis on a Navios Flow Cytometer
   (Beckman Coulter, CA, USA).

Quantitative Proteomic Analysis of Phosphorylation Modification

   TTK knockdown and control Jurkat cells were centrifuged at 1000 rpm for
   5 min, then washed three times with pre‐cooled PBS solution. Cells were
   collected after centrifuging at 1000 rpm for 5 min. Phosphoproteomic
   quantification was performed by Novogene (Beijing, China).

Molecular Docking

   Protein structures were obtained from the PDB database
   ([232]https://www.rcsb.org/). The HDOCK online server
   ([233]http://hdock.phys.hust.edu.cn/) was used for molecular docking.
   HDOCK was also utilized to analyze protein‐protein docking with
   different conformations, binding activities under various
   conformations, and amino acid residues interacting within 5Å. The PyMOL
   (version 4.6.0) software was employed to illustrate the amino acid
   residues involved in the interaction between the two proteins and to
   measure hydrogen bond distances.

Co‐IP

   Protein extraction and purification were performed using Pierce
   Co‐Immunoprecipitation Kit (26149, Thermo Fisher Scientific). The
   primary antibodies included TTK (A2500, Abclonal), p38α (66234‐1‐Ig,
   Proteintech), AKT (60203‐2‐Ig, Proteintech), Flag (66008‐4‐Ig,
   Proteintech), Rabbit Control IgG (AC005, Abclonal), Mouse Control IgG
   (AC011, Abclonal).

CHX Chase Assay

   TCL cells were incubated with 20µM MG132 (proteasomal inhibitor), or
   left untreated. After the treatment of 50 µg mL^−1 CHX for different
   time, cells were harvested and prepared for WB.

IF Assay and Confocal Microscopy

   Confocal microscopy was performed using the Leica TCS SP8 MP confocal
   microscope system (Germany). The primary antibodies included TTK
   (10381‐1‐AP, Proteintech), p38α (66234‐1‐Ig, Proteintech), AKT
   (60203‐2‐Ig, Proteintech), phospho‐AKT (2535, CST), p‐H2AX (9718, CST),
   LC3B (14600‐1‐AP, Proteintech), α‐Tubulin (11224‐1‐AP, Proteintech).

In Vivo Xenograft Mouse Models

   This study was approved by the Animal Care and Research Advisory
   Committee of Shandong Provincial Hospital and guidelines of it were
   strictly followed in all animal experiments. No blinding was performed.
   The 4‐week‐old SCID beige female mouse were randomized (simple
   randomization) into groups and 1 × 10^7 Karpas‐299 cells (Blank,
   Sh‐Control, Sh‐TTK) were subcutaneously injected into their right hind
   legs. CFI‐402257 and Duvelisib were dissolved in 90% PEG400 and 10%
   water, and daily administered by gavage for a week at a concentration
   of 6 and 10mg kg^−1, respectively. The animals were imaged using an in
   vivo small animal imaging system (PerkinElmer, USA).

Statistical Analysis

   All calculations were performed using SPSS 23.0 (SPSS Inc., USA) and R.
   Each in vitro experiment was repeated three times and experimental data
   were depicted as mean ± standard deviation (SD). Data were tested for
   homogeneity of variances and normality. Quantitative variables were
   analyzed using Students t‐test and non‐parametric tests while
   categorical variables were analyzed by χ2‐tests. Kaplan–Meier analysis
   was performed for survival curves and the difference between survival
   curves was compared using the Log‐rank test. There was no statistical
   method used to determine the sample size in our study. Differences were
   considered statistically significant at p < 0.05 (*p < 0.05, **p <
   0.01, ***p < 0.001).

Ethics Approval and Consent to Participate

   This study was approved by the Medical Ethical Committee of Shandong
   Provincial Hospital and written informed consent in accordance with the
   Declaration of Helsinki was obtained from each patient.

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   B.Y.L. collected clinical data. B.Y.L., T.G.L., M.F.D. and X.L.Z. wrote
   and edited this manuscript and created figures and tables. B.Y.L.,
   Y.Y.J., J.J.S., W.Y.S. performed research and analyzed the data.
   X.X.Z., X.W. and S.F.H. reviewed and revised the manuscript. All
   authors read and approved the final manuscript.

Supporting information

   Supporting Information
   [234]ADVS-12-2413990-s001.pdf^ (2MB, pdf)

Acknowledgements