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.

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.

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.

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.

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.

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.