Exploiting TLK1 and Cisplatin Synergy for Synthetic Lethality in Androgen-Insensitive Prostate Cancer.

Cellular organisms possess intricate DNA damage repair and tolerance pathways to manage various DNA lesions arising from endogenous or exogenous sources. The dysregulation of these pathways is associated with cancer development and progression. Synthetic lethality (SL), a promising cancer therapy concept, involves exploiting the simultaneous functional loss of two genes for selective cell death. PARP inhibitors (PARPis) have demonstrated success in BRCA-deficient tumors. Cisplatin (CPT), a widely used chemotherapy agent, forms DNA adducts and crosslinks, rendering it effective against various cancers, but less so for prostate cancer (PCa) due to resistance and toxicity. Here, we explore the therapeutic potential of TLK1, a kinase upregulated in androgen-insensitive PCa cells, as a target for enhancing CPT-based therapy. TLK1 phosphorylates key homologous recombination repair (HRR) proteins, RAD54L and RAD54B, which are critical for HRR alongside RAD51. The combination of CPT with TLK1 inhibitor J54 exhibits SL in androgen-insensitive PCa cells. The formation of double-strand break intermediates during inter-strand crosslink processing necessitates HRR for effective repair. Therefore, targeting TLK1 with J54 enhances the SL of CPT by impeding HRR, leading to increased sensitivity in PCa cells. These findings suggest a promising approach for improving CPT-based therapies in PCa, particularly in androgen-insensitive cases. By elucidating the role of TLK1 in CPT resistance, this study provides valuable insights into potential therapeutic targets to overcome PCa resistance to CPT chemotherapy. Further investigations into TLK1 inhibition in combination with other DNA-damaging agents may pave the way for more effective and targeted treatments for PCa and other cancers that exhibit resistance to traditional chemotherapy agents.

Untousling the Role of Tousled-like Kinase 1 in DNA Damage Repair.

DNA damage repair lies at the core of all cells' survival strategy, including the survival strategy of cancerous cells. Therefore, targeting such repair mechanisms forms the major goal of cancer therapeutics. The mechanism of DNA repair has been tousled with the discovery of multiple kinases. Recent studies on tousled-like kinases have brought significant clarity on the effectors of these kinases which stand to regulate DSB repair. In addition to their well-established role in DDR and cell cycle checkpoint mediation after DNA damage or inhibitors of replication, evidence of their suspected involvement in the actual DSB repair process has more recently been strengthened by the important finding that TLK1 phosphorylates RAD54 and regulates some of its activities in HRR and localization in the cell. Earlier findings of its regulation of RAD9 during checkpoint deactivation, as well as defined steps during NHEJ end processing, were earlier hints of its broadly important involvement in DSB repair. All this has opened up new avenues to target cancer cells in combination therapy with genotoxins and TLK inhibitors.

Targeting Prostate Cancer, the 'Tousled Way'.

Androgen deprivation therapy (ADT) has been the mainstay of prostate cancer (PCa) treatment, with success in developing more effective inhibitors of androgen synthesis and antiandrogens in clinical practice. However, hormone deprivation and AR ablation have caused an increase in ADT-insensitive PCas associated with a poor prognosis. Resistance to ADT arises through various mechanisms, and most castration-resistant PCas still rely on the androgen axis, while others become truly androgen receptor (AR)-independent. Our research identified the human tousled-like kinase 1 (TLK1) as a crucial early mediator of PCa cell adaptation to ADT, promoting androgen-independent growth, inhibiting apoptosis, and facilitating cell motility and metastasis. Although explicit, the growing role of TLK1 biology in PCa has remained underrepresented and elusive. In this review, we aim to highlight the diverse functions of TLK1 in PCa, shed light on the molecular mechanisms underlying the transition from androgen-sensitive (AS) to an androgen-insensitive (AI) disease mediated by TLK1, and explore potential strategies to counteract this process. Targeting TLK1 and its associated signaling could prevent PCa progression to the incurable metastatic castration-resistant PCa (mCRPC) stage and provide a promising approach to treating PCa.

TLK1-mediated RAD54 phosphorylation spatio-temporally regulates Homologous Recombination Repair.

Environmental agents like ionizing radiation (IR) and chemotherapeutic drugs can cause severe damage to the DNA, often in the form of double-strand breaks (DSBs). Remaining unrepaired, DSBs can lead to chromosomal rearrangements, and cell death. One major error-free pathway to repair DSBs is homologous recombination repair (HRR). Tousled-like kinase 1 (TLK1), a Ser/Thr kinase that regulates the DNA damage checkpoint, has been found to interact with RAD54, a central DNA translocase in HRR. To determine how TLK1 regulates RAD54, we inhibited or depleted TLK1 and tested how this impacts HRR in human cells using a ISce-I-GR-DsRed fused reporter endonuclease. Our results show that TLK1 phosphorylates RAD54 at three threonines (T41, T59 and T700), two of which are located within its N-terminal domain (NTD) and one is located within its C-terminal domain (CTD). Phosphorylation at both T41 and T59 supports HRR and protects cells from DNA DSB damage. In contrast, phosphorylation of T700 leads to impaired HRR and engenders no protection to cells from cytotoxicity and rather results in repair delay. Further, our work enlightens the effect of RAD54-T700 (RAD54-CTD) phosphorylation by TLK1 in mammalian system and reveals a new site of interaction with RAD51.

SARS-CoV-2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions.

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

NEK1-Mediated Phosphorylation of YAP1 Is Key to Prostate Cancer Progression.

The key to preventing mCRPC progression is understanding how androgen-dependent PCa cells progress to independence and modify their transcriptional repertoire accordingly. We recently identified a novel axis of the Hippo pathway characterized by the sequential kinase cascade induced by androgen deprivation, AR->TLK1B>NEK1>pYAP1-Y407, leading to CRPC adaptation. Phosphorylation of YAP1-Y407 increases upon ADT or induction of DNA damage, correlated with the known increase in NEK1 expression/activity, and this is suppressed in the Y407F mutant. Dominant expression of YAP1-Y407F in Hek293 cells reprograms the YAP1-mediated transcriptome to reduce TEAD- and p73-regulated gene expression and mediates sensitivity to MMC. NEK1 haploinsufficient TRAMP mice display reduced YAP1 expression and, if castrated, fail to progress to overt prostate carcinomas, even while displaying reduced E-Cadherin (E-Cad) expression in hyperplastic ductules. YAP1 overexpression, but not the Y407F mutant, transforms LNCaP cells to androgen-independent growth with a mesenchymal morphology. Immunohistochemical examination of prostate cancer biopsies revealed that the pYAP1-Y407 nuclear signal is low in samples of low-grade cancer but elevated in high GS specimens. We also found that J54, a pharmacological inhibitor of the TLK1>NEK1>YAP1 nexus leading to degradation of YAP1, can suppress the transcriptional reprogramming of LNCaP cells to androgen-independent growth and EMT progression, even when YAP1-WT is overexpressed.

Loss of tumor suppressor menin expression in high grade cholangiocarcinomas.

BACKGROUND: MEN1, which codes for the protein menin, is a tumor suppressor in neuroendocrine tissue. In cholangiocarcinoma (CCA) cell lines the overexpression of menin decreased proliferation, angiogenesis, migration, and invasion in vitro and in xenografts, but its expression in CCA tumor tissue samples is not established. OBJECTIVE: Determine whether the expression of menin correlates with disease progression in patient samples of CCA in a tissue microarray (TMA) by immunohistochemical (IHC) staining. RESULTS: IHC analysis of 97 biopsies revealed that low-grade tumors (Grade I) exhibited intense, diffuse, finely granular nuclear menin immunoreactivity with a pronounced linear perinuclear pattern (mean IHC score = 2.00), whereas high-grade tumors (Grade III) mostly lacked such staining (mean IHC score = 0.35). Collectively, there was a significant inverse association between tumor grade and menin staining (P = 0.0005). We also found a significant association between fibrosis status and menin staining, in that, 81.2% (56/69) of patients without fibrosis had no menin staining, whereas 92.9% (26/28) patients with fibrosis exhibited menin staining (P < 0.0001). No association was found between fibrosis status and grade. Overall, menin expression is inversely associated with tumor grade and positively associated with fibrosis status.

Synthesis, kinetics and cellular studies of new phenothiazine analogs as potent human-TLK inhibitors.

The alterations in the expression patterns of protein kinases often implicate human cancer initiation and progression. Human tousled-like kinases (TLKs), both TLK1/1B and TLK2, are evolutionary kinases found in cell signaling pathways and are involved in DNA repair, replication, and chromosomal integrity. Several reports have demonstrated the numerous roles of TLK1B in the development and progression of cancer via its interactions with different partners, and this direct association has made them viable molecular targets for cancer therapy. Previous studies have shown phenothiazines to be potent TLK1B inhibitors. Herein, we report the design and synthesis of a class of phenothiazine molecules and their biological inhibitory effect on hTLK1B/KD through in vitro kinase assays, cellular assays, and in silico studies. We identified a few inhibitors with better inhibition and physio-chemical properties than the reported TLK1B inhibitors using a recombinant human tousled-like kinase 1B-kinase domain (hTLK1B-KD). Very interestingly, inhibitory activity with LNCap cells was found to be on the sub-nanomolar level. Our attempts to study the newly designed phenothiazine analogs, as well as generate a stable catalytically active hTLK1B-KD in high yield, represent a fundamental step towards the structure-based design of future TLK-specific inhibitors.

The TLK1-MK5 Axis Regulates Motility, Invasion, and Metastasis of Prostate Cancer Cells.

Background: Metastatic dissemination of prostate cancer (PCa) accounts for the majority of PCa-related deaths. However, the exact mechanism of PCa cell spread is still unknown. We uncovered a novel interaction between two unrelated promotility factors, tousled-like kinase 1 (TLK1) and MAPK-activated protein kinase 5 (MK5), that initiates a signaling cascade promoting metastasis. In PCa, TLK1−MK5 signaling might be crucial, as androgen deprivation therapy (ADT) leads to increased expression of both TLK1 and MK5 in metastatic patients, but in this work, we directly investigated the motility, invasive, and metastatic capacity of PCa cells following impairment of the TLK1 > MK5 axis. Results: We conducted scratch wound repair and transwell invasion assays with LNCaP and PC3 cells to determine if TLK1 and MK5 can regulate motility and invasion. Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 resulted in reduced migration and invasion through a Matrigel plug. We further elucidated the potential mechanisms underlying these effects and found that this is likely due to the reorganization of the actin fibers at lamellipodia and the focal adhesions network, in conjunction with increased expression of some MMPs that can affect penetration through the ECM. PC3, a highly metastatic cell line when assayed in xenografts, was further tested in a tail-vein injection/lung metastasis model, and we showed that, following inoculation, treatment with GLPG0259 (MK5 specific inhibitor) or J54 (TLK1 inhibitor) resulted in the lung tumor nodules being greatly diminished in number, and for J54, also in size. Conclusion: Our data support that the TLK1−MK5 axis is functionally involved in driving PCa cell metastasis and clinical aggressiveness; hence, disruption of this axis may inhibit the metastatic capacity of PCa.

Tousled-like kinase 1: a novel factor with multifaceted role in mCRPC progression and development of therapy resistance.

Standard treatment for advanced Prostate Cancer (PCa) consists of androgen deprivation therapy (ADT), but ultimately fails, resulting in the incurable phase of the disease: metastatic castration-resistant prostate cancer (mCRPC). Targeting PCa cells before their progression to mCRPC would greatly improve the outcome, if strategies could be devised selectively targeting androgen receptor (AR)-dependent and/or independent compensatory pathways which promote mCRPC development. Combination therapy by targeting the DNA damage response (DDR) along with ADT has been limited by general toxicity, and a goal of clinical trials is how to target the DDR more specifically. In recent years, our lab has identified a key role for the DDR kinase, TLK1, in mediating key aspects of adaptation to ADT, first by promoting a cell cycle arrest (through the TLK1>NEK1>ATR>Chk1 kinase cascade) under the unfavorable growth conditions (androgen deprivation), and then by reprogramming the PCa cells to adapt to androgen-independent growth via the NEK1>YAP/AR>CRPC conversion. In addition, TLK1 plays a key anti-apoptotic role via the NEK1>VDAC1 regulation on the intrinsic mitochondrial apoptotic pathway when the DDR is activated. Finally, TLK1 was recently identified as having an important role in motility and metastasis via regulation of the kinases MK5/PRAK and AKT (indirectly via AKTIP).

TLK1-mediated MK5-S354 phosphorylation drives prostate cancer cell motility and may signify distinct pathologies.

Metastases account for the majority of prostate cancer (PCa) deaths, and targeting them is a major goal of systemic therapy. We identified a novel interaction between two kinases: tousled-like kinase 1 (TLK1) and MAP kinase-activated protein kinase 5 (MK5) that promotes PCa spread. In PCa progression, TLK1-MK5 signalling appears to increase following antiandrogen treatment and in metastatic castration-resistant prostate cancer (mCRPC) patients. Determinations of motility rates (2D and 3D) of different TLK1- and MK5-perturbed cells, including knockout (KO) and knockdown (KD), as well as the use of specific inhibitors, showed the importance of these two proteins for in vitro dissemination. We established that TLK1 phosphorylates MK5 on three residues (S160, S354 and S386), resulting in MK5 activation, and additionally, mobility shifts of MK5 also supported its phosphorylation by TLK1 in transfected HEK 293 cells. Expression of MK5-S354A or kinase-dead MK5 in MK5-depleted mouse embryonic fibroblast (MEF) cells failed to restore their motility compared with that of wild-type (WT) MK5-rescued MK5-/- MEF cells. A pMK5-S354 antiserum was used to establish this site as an authentic TLK1 target in androgen-sensitive human prostate adenocarcinoma (LNCaP) cells, and was used in immunohistochemistry (IHC) studies of age-related PCa sections from TRAMP (transgenic adenocarcinoma of the mouse prostate) mice and to probe a human tissue microarray (TMA), which revealed pMK5-S354 level is correlated with disease progression (Gleason score and nodal metastases). In addition, The Cancer Genome Atlas (TCGA) analyses of PCa expression and genome-wide association study (GWAS) relations identify TLK1 and MK5 as potential drivers of advanced PCa and as markers of mCRPC. Our work suggests that TLK1-MK5 signalling is functionally involved in driving PCa cell motility and clinical features of aggressiveness; hence, disruption of this axis may inhibit the metastatic spread of PCa.

Interaction of TLK1 and AKTIP as a Potential Regulator of AKT Activation in Castration-Resistant Prostate Cancer Progression.

Prostate cancer (PCa) progression is characterized by the emergence of resistance to androgen deprivation therapy (ADT). AKT/PKB has been directly implicated in PCa progression, often due to the loss of PTEN and activation of PI3K>PDK1>AKT signaling. However, the regulatory network of AKT remains incompletely defined. Here, we describe the functional significance of AKTIP in PCa cell growth. AKTIP, identified in an interactome analysis as a substrate of TLK1B (that itself is elevated following ADT), enhances the association of AKT with PDK1 and its phosphorylation at T308 and S473. The interaction between TLK1 and AKTIP led to AKTIP phosphorylation at T22 and S237. The inactivation of TLK1 led to reduced AKT phosphorylation, which was potentiated with AKTIP knockdown. The TLK1 inhibitor J54 inhibited the growth of the LNCaP cells attributed to reduced AKT activation. However, LNCaP cells that expressed constitutively active, membrane-enriched Myr-AKT (which is expected to be active, even in the absence of AKTIP) were also growth-inhibited with J54. This suggested that other pathways (like TLK1>NEK1>YAP) regulating proliferation are also suppressed and can mediate growth inhibition, despite compensation by Myr-AKT. Nonetheless, further investigation of the potential role of TLK1>AKTIP>AKT in suppressing apoptosis, and conversely its reversal with J54, is warranted.

NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output.

Most prostate cancer (PCa) deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa (mCRPC); however, the mechanism and key players leading to this are not fully understood. While studying the role of tousled-like kinase 1 (TLK1) and never in mitosis gene A (NIMA)-related kinase 1 (NEK1) in a DNA damage response (DDR)-mediated cell cycle arrest in LNCaP cells treated with bicalutamide, we uncovered that overexpression of wt-NEK1 resulted in a rapid conversion to androgen-independent (AI) growth, analogous to what has been observed when YAP1 is overexpressed. We now report that overexpression of wt-NEK1 results in accumulation of YAP1, suggesting the existence of a TLK1>NEK1>YAP1 axis that leads to adaptation to AI growth. Further, YAP1 is co-immunoprecipitated with NEK1. Importantly, NEK1 was able to phosphorylate YAP1 on six residues in vitro, which we believe are important for stabilization of the protein, possibly by increasing its interaction with transcriptional partners. In fact, knockout (KO) of NEK1 in NT1 PCa cells resulted in a parallel decrease of YAP1 level and reduced expression of typical YAP-regulated target genes. In terms of cancer potential implications, the expression of NEK1 and YAP1 proteins was found to be increased and correlated in several cancers. These include PCa stages according to Gleason score, head and neck squamous cell carcinoma, and glioblastoma, suggesting that this co-regulation is imparted by increased YAP1 stability when NEK1 is overexpressed or activated by TLK1, and not through transcriptional co-expression. We propose that the TLK1>NEK1>YAP1 axis is a key determinant for cancer progression, particularly during the process of androgen-sensitive to -independent conversion during progression to mCRPC.

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy.

Through in vitro kinase assays and docking studies, we report the synthesis and biological evaluation of a phenothiazine analog J54 with potent TLK1 inhibitory activity for prostate cancer (PCa) therapy. Most PCa deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa. Treatments that can suppress the conversion to mCRPC have high potential to be rapidly implemented in the clinics. ADT results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DNA damage response that typically results in temporary cell-cycle arrest of androgen-responsive PCa cells, whereas its abrogation leads to apoptosis. We studied J54 as a potent inhibitor of this axis and as a mediator of apoptosis in vitro and in LNCaP xenografts, which has potential for clinical investigation in combination with ADT. J54 has low affinity for the dopamine receptor in modeling and competition studies and weak detrimental behavioral effects in mice and C. elegans.

The TLK1/Nek1 axis contributes to mitochondrial integrity and apoptosis prevention via phosphorylation of VDAC1.

The TLK1/Nek1 axis contributes to cell cycle arrest and implementation of the DDR to mediate survival upon DNA damage. However, when the damage is too severe, the cells typically are forced into apoptosis, and the contribution of TLKs in this process has not been investigated. In contrast, it is known that Nek1 may play a role by phosphorylating VDAC1 maintaining proper opening and closure of the channel and thus mitochondrial integrity. We now show that the activating phosphorylation of Nek1-T141 by TLK1 contributes to the phosphorylation and stability of VDAC1 and thereby to mitochondrial permeability and integrity. Treatment of three different cell lines model that overexpress Nek1-T141A mutant with doxorubicin showed exquisite sensitivity to the drug, with implementation of rapid accumulation of cells with subG1 DNA content (apoptotic) and other alterations in the cell cycle. In addition, these cells displayed reduced oxygen consumption under normal conditions and less reliance on mitochondria and more dependence on glycolysis for energy production. Consistent with greater apoptosis, upon treatment with low doses of doxorubicin, cells overexpressing Nek1-T141A displayed leakage of Cyt-C into the cytoplasmic fraction. This suggests that inhibiting the TLK1/Nek1/VDAC1 nexus could sensitize cancer cells to apoptotic killing in combination with an appropriate DNA damaging agent. We in fact have previously reported that Nek1 expression is elevated in advanced Prostate Cancer (PCa) and we now report that VDAC1 expression is elevated and correlated with disease stage, thereby making the TLK1/Nek1/VDAC1 nexus a very attractive target for PCa.

The TLK1-Nek1 axis promotes prostate cancer progression.

We recently uncovered the critical TLK1>NEK1>ATR > Chk1 axis in mediating the DDR and cell cycle checkpoint while transiting from Androgen Sensitive to Insensitive growth for LNCaP and TRAMP-C2 cells. However, we did not know the generality of this pathway in PCa progression since there are few cell lines where the transition has been studied. Furthermore, the identification of Nek1, and more importantly the TLK-mediated phosphorylation of T141, has never been studied in PCa biopsies. We now report the first study of a PCa TMA of p-Nek1-T141 and correlation to the Gleason score. In addition we found that TRAMP mice treated with the TLK inhibitor, thioridazine (THD), following castration did not recover cancerous growth of their prostates. Moreover, we recapitulated the process of translational increase in TLK1B expression in a naïve PDX model that was established from an AR + adenocarcinoma. Therefore, we believe that this TLK1-Nek1 mediated DDR axis is likely to be a common adaptive response during the transition of PCa cells toward androgen-insensitive growth, and hence CRPC progression, which has the potential to be targeted with THD and other TLK or Nek1 inhibitors.

Targeting the TLK1/NEK1 DDR axis with Thioridazine suppresses outgrowth of androgen independent prostate tumors.

Standard therapy for advanced Prostate Cancer (PCa) consists of antiandrogens, which provide respite from disease progression, but ultimately fail resulting in the incurable phase of the disease: mCRPC. Targeting PCa cells before their progression to mCRPC would greatly improve the outcome. Combination therapy targeting the DNA Damage Response (DDR) has been limited by general toxicity, and a goal of clinical trials is how to target the DDR more specifically. We now show that androgen deprivation therapy (ADT) of LNCaP cells results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DDR that typically results in a temporary cell cycle arrest of androgen responsive PCa cells. Following DNA damage, addition of the TLK specific inhibitor, thioridazine (THD), impairs ATR and Chk1 activation, establishing the existence of a ADT > TLK1 > NEK1 > ATR > Chk1, DDR pathway, while its abrogation leads to apoptosis. Treatment with THD suppressed the outgrowth of androgen-independent (AI) colonies of LNCaP and TRAMP-C2 cells cultured with bicalutamide. Moreover, THD significantly inhibited the growth of several PCa cells in vitro (including AI lines). Administration of THD or bicalutamide was not effective at inhibiting long-term tumor growth of LNCaP xenografts. In contrast, combination therapy remarkably inhibited tumor growth via bypass of the DDR. Moreover, xenografts of LNCaP cells overexpressing a NEK1-T141A mutant were durably suppressed with bicalutamide. Collectively, these results suggest that targeting the TLK1/NEK1 axis might be a novel therapy for PCa in combination with standard of care (ADT).

Identification of the proteome complement of humanTLK1 reveals it binds and phosphorylates NEK1 regulating its activity.

The Tousled Like kinases (TLKs) are involved in numerous cellular functions, including the DNA Damage Response (DDR), but only a handful of substrates have been identified thus far. Through a novel proteomic screen, we have now identified 165 human proteins interacting with TLK1, and we have focused this work on NEK1 because of its known role in the DDR, upstream of ATR and Chk1. TLK1 and NEK1 were found to interact by coIP, and their binding is strengthened following exposure of cells to H2O2. Following incubation with doxorubicin, TLK1 and NEK1 relocalize with nuclear repair foci along with γH2AX. TLK1 phosphorylated NEK1 at T141, which lies in the kinase domain, and caused an increase in its activity. Following DNA damage, addition of the TLK1 inhibitor, THD, or overexpression of NEK1-T141A mutant impaired ATR and Chk1 activation, indicating the existence of a TLK1>NEK1>ATR>Chk1 pathway. Indeed, overexpression of the NEK1-T141A mutant resulted in an altered cell cycle response after exposure of cells to oxidative stress, including bypass of G1 arrest and implementation of an intra S-phase checkpoint.

TLK1B mediated phosphorylation of Rad9 regulates its nuclear/cytoplasmic localization and cell cycle checkpoint.

BACKGROUND: The Tousled like kinase 1B (TLK1B) is critical for DNA repair and survival of cells. Upon DNA damage, Chk1 phosphorylates TLK1B at S457 leading to its transient inhibition. Once TLK1B regains its kinase activity it phosphorylates Rad9 at S328. In this work we investigated the significance of this mechanism by overexpressing mutant TLK1B in which the inhibitory phosphorylation site was eliminated. RESULTS AND DISCUSSION: These cells expressing TLK1B resistant to DNA damage showed constitutive phosphorylation of Rad9 S328 that occurred even in the presence of hydroxyurea (HU), and this resulted in a delayed checkpoint recovery. One possible explanation was that premature phosphorylation of Rad9 caused its dissociation from 9-1-1 at stalled replication forks, resulting in their collapse and prolonged activation of the S-phase checkpoint. We found that phosphorylation of Rad9 at S328 results in its dissociation from chromatin and redistribution to the cytoplasm. This results in double stranded breaks formation with concomitant activation of ATM and phosphorylation of H2AX. Furthermore, a Rad9 (S328D) phosphomimic mutant was exclusively localized to the cytoplasm and not the chromatin. Another Rad9 phosphomimic mutant (T355D), which is also a site phosphorylated by TLK1, localized normally. In cells expressing the mutant TLK1B treated with HU, Rad9 association with Hus1 and WRN was greatly reduced, suggesting again that its phosphorylation causes its premature release from stalled forks. CONCLUSIONS: We propose that normally, the inactivation of TLK1B following replication arrest and genotoxic stress functions to allow the retention of 9-1-1 at the sites of damage or stalled forks. Following reactivation of TLK1B, whose synthesis is concomitantly induced by genotoxins, Rad9 is hyperphosphorylated at S328, resulting in its dissociation and inactivation of the checkpoint that occurs once repair is complete.

Tousled kinase activator, gallic acid, promotes homologous recombinational repair and suppresses radiation cytotoxicity in salivary gland cells.

Accidental or medical radiation exposure of the salivary glands can gravely impact oral health. Previous studies have shown the importance of Tousled-like kinase 1 (TLK1) and its alternate start variant TLK1B in cell survival against genotoxic stresses. Through a high-throughput library screening of natural compounds, the phenolic phytochemical, gallic acid (GA), was identified as a modulator of TLK1/1B. This small molecule possesses anti-oxidant and free radical scavenging properties, but in this study, we report that in vitro it promotes survival of human salivary acinar cells, NS-SV-AC, through repair of ionizing radiation damage. Irradiated cells treated with GA show improved clonogenic survival compared to untreated controls. And, analyses of DNA repair kinetics by alkaline single-cell gel electrophoresis and γ-H2AX foci immunofluorescence indicate rapid resolution of DNA breaks in drug-treated cells. Study of DR-GFP transgene repair indicates GA facilitates homologous recombinational repair to establish a functional GFP gene. In contrast, inactivation of TLK1 or its shRNA knockdown suppressed resolution of radiation-induced DNA tails in NS-SV-AC, and homology directed repair in DR-GFP cells. Consistent with our results in culture, animals treated with GA after exposure to fractionated radiation showed better preservation of salivary function compared to saline-treated animals. Our results suggest that GA-mediated transient modulation of TLK1 activity promotes DNA repair and suppresses radiation cytoxicity in salivary gland cells.