Inhibition of PLK1 Destabilizes EGFR and Sensitizes EGFR-Mutated Lung Cancer Cells to Small Molecule Inhibitor Osimertinib.

Tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) have significantly prolonged survival in EGFR-mutant non-small cell lung cancer patients. However, the development of resistance mechanisms prohibits the curative potential of EGFR TKIs. Combination therapies emerge as a valuable approach to preventing or delaying disease progression. Here, we investigated the combined inhibition of polo-like kinase 1 (PLK1) and EGFR in TKI-sensitive EGFR-mutant NSCLC cells. The pharmacological inhibition of PLK1 destabilized EGFR levels and sensitized NSCLC cells to Osimertinib through induction of apoptosis. In addition, we found that c-Cbl, a ubiquitin ligase of EGFR, is a direct phosphorylation target of PLK1 and PLK1 impacts the stability of c-Cbl in a kinase-dependent manner. In conclusion, we describe a novel interaction between mutant EGFR and PLK1 that may be exploited in the clinic. Co-targeting PLK1 and EGFR may improve and prolong the clinical response to EGFR TKI in patients with an EGFR-mutated NSCLC.

The EGFR-STYK1-FGF1 axis sustains functional drug tolerance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) patients harboring activating mutations in epidermal growth factor receptor (EGFR) are sensitive to therapy with EGFR tyrosine kinase inhibitors (TKI). Despite remarkable clinical responses using EGFR TKI, surviving drug tolerant cells serve as a reservoir from which drug resistant tumors may emerge. This study addresses the need for improved efficacy of EGFR TKI by identifying targets involved in functional drug tolerance against them. To this aim, a high-throughput siRNA kinome screen was performed using two EGFR TKI-sensitive EGFR-mutant NSCLC cell lines in the presence/absence of the second-generation EGFR TKI afatinib. From the screen, Serine/Threonine/Tyrosine Kinase 1 (STYK1) was identified as a target that when downregulated potentiates the effects of EGFR inhibition in vitro. We found that chemical inhibition of EGFR combined with the siRNA-mediated knockdown of STYK1 led to a significant decrease in cancer cell viability and anchorage-independent cell growth. Further, we show that STYK1 selectively interacts with mutant EGFR and that the interaction is disrupted upon EGFR inhibition. Finally, we identified fibroblast growth factor 1 (FGF1) as a downstream effector of STYK1 in NSCLC cells. Accordingly, downregulation of STYK1 counteracted the afatinib-induced upregulation of FGF1. Altogether, we unveil STYK1 as a valuable target to repress the pool of surviving drug tolerant cells arising upon EGFR inhibition. Co-targeting of EGFR and STYK1 could lead to a better overall outcome for NSCLC patients.

USP13 modulates the stability of the APC/C adaptor CDH1.

BACKGROUND: The cell division cycle is a process that is exquisitely controlled by a complex interplay between E3 ubiquitin ligases and deubiquitinating enzymes (DUBs). We have previously reported that the DUB USP13 regulates Aurora B levels along the cell cycle. That observation prompted us to explore any possible connection between USP13 and the APC/CCDH1, the major E3 controlling Aurora B levels in cells. METHODS: We performed immunoprecipitation assays followed by western-blotting to assess the interaction between USP13 and CDH1. The cellular effects of USP13 gain or loss of function were analyzed by transfection of FLAG-tagged USP13 plasmid or small interfering RNAs and short hairpin RNAs directed against USP13. The levels of CDH1 and other proteins were quantified in cell extracts by western-blotting. RESULTS: We found that USP13 binds to the APC/C adaptor CDH1. In addition, we report for the first time that USP13 controls CDH1 protein levels in cells: overexpression of USP13 increased CDH1 levels, whereas depletion of USP13 decreased CDH1 levels. CONCLUSIONS: We unveil the existing interplay between USP13 and CDH1: USP13 is capable of stabilizing CDH1 levels. We previously reported that USP13 stabilizes Aurora B in cells, a known substrate of the APC/CCDH1 E3 ubiquitin ligase, before their entry into mitosis. Altogether, our data identify and establish the USP13-CDH1-Aurora B axis as a new regulatory module required for flawless cell cycle progression in mammalian cells, whose misfunction may be involved in the rewiring of cell cycle pathways linked to cancer development.

Structure-Activity Relationship (SAR) Study of Spautin-1 to Entail the Discovery of Novel NEK4 Inhibitors.

Lung cancer is one of the most frequently diagnosed cancers accounting for the highest number of cancer-related deaths in the world. Despite significant progress including targeted therapies and immunotherapy, the treatment of advanced lung cancer remains challenging. Targeted therapies are highly efficacious at prolonging life, but not curative. In prior work we have identified Ubiquitin Specific Protease 13 (USP13) as a potential target to significantly enhance the efficacy of mutant EGFR inhibition. The current study aimed to develop lead molecules for the treatment of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) by developing potent USP13 inhibitors initially starting from Spautin-1, the only available USP13 inhibitor. A SAR study was performed which revealed that increasing the chain length between the secondary amine and phenyl group and introducing a halogen capable of inducing a halogen bond at position 4' of the phenyl group, dramatically increased the activity. However, we could not confirm the binding between Spautin-1 (or its analogues) and USP13 using isothermal titration calorimetry (ITC) or thermal shift assay (TSA) but do not exclude binding under physiological conditions. Nevertheless, we found that the anti-proliferative activity displayed by Spautin-1 towards EGFR-mutant NSCLC cells in vitro was at least partially associated with kinase inhibition. In this work, we present N-[2-(substituted-phenyl)ethyl]-6-fluoro-4-quinazolinamines as promising lead compounds for the treatment of NSCLC. These analogues are significantly more effective towards EGFR-mutant NSCLC cells than Spautin-1 and act as potent never in mitosis A related kinase 4 (NEK4) inhibitors (IC50~1 µM) with moderate selectivity over other kinases.

Targeting USP13-mediated drug tolerance increases the efficacy of EGFR inhibition of mutant EGFR in non-small cell lung cancer.

In non-small cell lung cancer (NSCLC), activating mutations in the epidermal growth factor receptor (EGFR) induce sensitivity to EGFR tyrosine kinase inhibitors. Despite impressive clinical responses, patients ultimately relapse as a reservoir of drug-tolerant cells persist, which ultimately leads to acquired resistance mechanisms. We performed an unbiased high-throughput siRNA screen to identify proteins that abrogate the response of EGFR-mutant NSCLC to EGFR-targeted therapy. The deubiquitinase USP13 was a top hit resulting from this screen. Targeting USP13 increases the sensitivity to EGFR inhibition with small molecules in vitro and in vivo. USP13 selectively stabilizes mutant EGFR in a peptidase-independent manner by counteracting the action of members of the Cbl family of E3 ubiquitin ligases. We conclude that USP13 is a strong mutant EGFR-specific cotarget that could improve the treatment efficacy of EGFR-targeted therapies.

USP13 controls the stability of Aurora B impacting progression through the cell cycle.

Aurora B kinase plays essential roles in mitosis. Its protein levels increase before the onset of mitosis and sharply decrease during mitosis exit. The latter decrease is due to a balance between the actions of the E3 ubiquitin ligase anaphase-promoting complex or cyclosome (activated by the Cdh1 adapter), and the deubiquitinating enzyme USP35. Aurora B also executes important functions in interphase. Abnormal modulation of Aurora B in interphase leads to cell cycle defects often linked to aberrant chromosomal condensation and segregation. Very little is however known about how Aurora B levels are regulated in interphase. Here we found that USP13-associates with and stabilizes Aurora B in cells, especially before their entry into mitosis. In order for USP13 to exert its stabilizing effect on Aurora B, their association is promoted by the Aurora B-mediated phosphorylation of USP13 at Serine 114. We also present evidence that USP13 instigates Aurora B deubiquitination and/or protect it from degradation in a non-catalytic manner. In addition, we report that genetic or chemical modulation of the cellular levels/activity of USP13 affects unperturbed cell-cycle progression. Overall our study unveils the molecular and cellular connections of the USP13-Aurora B axis, which potentially participates in the rewiring of the cell cycle happening in cancer cells.

Degradation of newly synthesized polypeptides by ribosome-associated RACK1/c-Jun N-terminal kinase/eukaryotic elongation factor 1A2 complex.

Folding of newly synthesized polypeptides (NSPs) into functional proteins is a highly regulated process. Rigorous quality control ensures that NSPs attain their native fold during or shortly after completion of translation. Nonetheless, signaling pathways that govern the degradation of NSPs in mammals remain elusive. We demonstrate that the stress-induced c-Jun N-terminal kinase (JNK) is recruited to ribosomes by the receptor for activated protein C kinase 1 (RACK1). RACK1 is an integral component of the 40S ribosome and an adaptor for protein kinases. Ribosome-associated JNK phosphorylates the eukaryotic translation elongation factor 1A isoform 2 (eEF1A2) on serines 205 and 358 to promote degradation of NSPs by the proteasome. These findings establish a role for a RACK1/JNK/eEF1A2 complex in the quality control of NSPs in response to stress.

JNK-mediated phosphorylation of Cdc25C regulates cell cycle entry and G(2)/M DNA damage checkpoint.

c-Jun NH(2)-terminal Kinases (JNKs) play a central role in the cellular response to a wide variety of stress signals. After their activation, JNKs induce phosphorylation of substrates, which control proliferation, migration, survival, and differentiation. Recent studies suggest that JNKs may also play a role in cell cycle control, although the underlying mechanisms are largely unexplored. Here we show that JNK directly phosphorylates Cdc25C at serine 168 during G(2) phase of the cell cycle. Cdc25C phosphorylation by JNK negatively regulates its phosphatase activity and thereby Cdk1 activation, enabling a timely control of mitosis onset. Unrestrained phosphorylation by JNK, as obtained by a cell cycle-stabilized form of JNK or as seen in some human tumors, results in aberrant cell cycle progression. Additionally, UV irradiation-induced G(2)/M checkpoint requires inactivation of Cdc25C by JNK phosphorylation. JNK phosphorylation of Cdc25C as well as Cdc25A establishes a novel link between stress signaling and unperturbed cell cycle and checkpoint pathways.

Control of p53 multimerization by Ubc13 is JNK-regulated.

The p53 tumor suppressor protein is a key regulator of cellular proliferation and survival whose function is tightly regulated at the levels of transcription and protein stability. Here, we unveil the fine control of p53 on translationally active polysomes. We have previously reported that Ubc13, an E2 ubiquitin-conjugating enzyme, directly regulates p53 localization and transcriptional activity. We now demonstrate that the association of p53 and Ubc13 on polysomes requires ongoing translation and results in p53 ubiquitination that interferes with its tetramerization. JNK phosphorylation of p53 at Threonine 81 occurring on polysomes is required for the dissociation of Ubc13 from p53, leading to p53 multimerization and transcriptional activation. Inhibition of JNK activity or expression of a nonphosphorylatable mutant of p53 maintains an Ubc13-p53 complex that inhibits p53 multimerization. Our findings reveal a layer in the regulation of p53 multimerization that requires the concerted action of JNK and Ubc13 on polysome-bound p53.

Autophosphorylation properties of inactive and active JNK2.

The c-Jun N-terminal kinases (JNKs) are ubiquitous proteins that phosphorylate their substrates, such as transcription factors, in response to physical stress, cytokines or UV radiation. This leads to changes in gene expression, ensuing either cell cycle progression or apoptosis. Active phospho JNK1 is the main in vivo kinase component of the JNK cascade, whereas JNK2 is presumed not to participate as a kinase during JNK signalling. However, there is evidence that JNK isoforms interact functionally in vivo. Also, a recent chemical genetics investigation has confirmed that JNK transient activation leads to cellular proliferation, whereas a sustained one is pro-apoptotic. Here we investigate the phosphorylation pattern of JNK2, with protein biochemistry tools and tandem mass spectrometry. We choose to focus on JNK2 because of its reported constitutive activity in glioma cells. Our results indicate that purified JNK2 from transfected nonstressed 293T cells is a mixture of the mono-sites pThr183 and pTyr185 of its activation loop and of pThr386 along its unique C-terminal region. Upon UV stimulation, its phosphorylation stoichiometry is upregulated on the activation loop, generating a mixture of mono-pTyr185 and the expected dual-pThr183/pTyr185 species, with the pThr386 specie present but unaltered respect to the basal conditions.

Meiotic regulation of the CDK activator RINGO/Speedy by ubiquitin-proteasome-mediated processing and degradation.

Xenopus RINGO/Speedy (XRINGO) is a potent inducer of oocyte meiotic maturation that can directly activate Cdk1 and Cdk2. Here, we show that endogenous XRINGO protein accumulates transiently during meiosis I entry and then is downregulated. This tight regulation of XRINGO expression is the consequence of two interconnected mechanisms: processing and degradation. XRINGO processing involves recognition of at least three distinct phosphorylated recognition motifs by the SCF(betaTrCP) ubiquitin ligase, followed by proteasome-mediated limited degradation, resulting in an amino-terminal XRINGO fragment. XRINGO processing is directly stimulated by several kinases, including protein kinase A and glycogen synthase kinase-3beta, and may contribute to the maintenance of G2 arrest. On the other hand, XRINGO degradation after meiosis I is mediated by the ubiquitin ligase Siah-2, which probably requires phosphorylation of XRINGO on Ser 243 and may be important for the omission of S phase at the meiosis-I-meiosis-II transition in Xenopus oocytes.

Identification and initial characterization of three novel cyclin-related proteins of the human malaria parasite Plasmodium falciparum.

The molecular mechanisms regulating cell proliferation and development during the life cycle of malaria parasites remain to be elucidated. The peculiarities of the cell cycle organization during Plasmodium falciparum schizogony suggest that the modalities of cell cycle control in this organism may differ from those in other eukaryotes. Indeed, existing data concerning Plasmodium cell cycle regulators such as cyclin-dependent kinases reveal structural and functional properties that are divergent from those of their homologues in other systems. The work presented here lies in the context of the exploitation of the recently available P. falciparum genome sequence toward the characterization of putative cell cycle regulators. We describe the in silico identification of three open reading frames encoding proteins with maximal homology to various members of the cyclin family and demonstrate that the corresponding polypeptides are expressed in the erythrocytic stages of the infection. We present evidence that these proteins possess cyclin activity by demonstrating either their association with histone H1 kinase activity in parasite extracts or their ability to activate PfPK5, a P. falciparum cyclin-dependent kinase homologue, in vitro. Furthermore, we show that RINGO, a protein with no sequence homology to cyclins but that is nevertheless a strong activator of mammalian CDK1/2, is also a strong activator of PfPK5 in vitro. This raises the possibility that "cryptic" cell cycle regulators may be found among the 50% of the open reading frames in the P. falciparum genome that display no homology to any known proteins.

Histone H3 phosphorylation during Xenopus oocyte maturation: regulation by the MAP kinase/p90Rsk pathway and uncoupling from DNA condensation.

Here we show that during the meiotic maturation of Xenopus oocytes, histone H3 becomes phosphorylated on serine-10 at about the time of maturation promoting factor activation and meiosis I entry. However, overexpression of cAMP-dependent protein kinase that blocks entry into M phase, also leads to massive serine-10 phosphorylation of histone H3 in intact Xenopus oocytes but does not cause chromosome condensation. We also show that the phosphorylation of histone H3 during oocyte maturation requires the activation of the mitogen-activated protein kinase/p90Rsk pathway. Our results indicate that in G2-arrested oocytes, which are about to enter M phase, histone H3 phosphorylation is not sufficient for chromosome condensation.

Par-1 regulates stability of the posterior determinant Oskar by phosphorylation.

Par-1 kinase is critical for polarization of the Drosophila melanogaster oocyte and the one-cell Caenorhabditis elegans embryo. Although Par-1 localizes specifically to the posterior pole in both cells, neither its targets nor its function at the posterior pole have been elucidated. Here we show that Drosophila Par-1 phosphorylates the posterior determinant Oskar (Osk) and demonstrate genetically that Par-1 is required for accumulation of Osk protein. We show in cell-free extracts that Osk protein is intrinsically unstable and that it is stabilized after phosphorylation by Par-1. Our data indicate that posteriorly localized Par-1 regulates posterior patterning by stabilizing Osk.