Tubulin Acetylation Research Articles

HDAC6 inhibition attenuates renal injury by reducing IL-1β secretion and RIP kinase mediated necroptosis in acute oxalate nephropathy

BackgroundPathogenesis of acute kidney injury is driven by necro-inflammation, which is comprised of IL-1β mediated inflammation and RIP-1 mediated tubular necroptosis. HDAC6 is reported to regulate both inflammation and cell death. In the present study, we explored the role of HDAC6 in the lysosomal exocytosis of IL-1β and RIP-1 mediated necroptosis in the context of oxalate nephropathy. MethodsRaw 264.7 macrophages and NRK52E stimulated with oxalate crystals and LPS with or without HDAC6 inhibitor for in vitro experiments. Acute oxalate nephropathy was induced in C57BL/6 mice by injecting sodium oxalate (75 mg/kg). For the drug intervention study, Tubastain A (TSA) was given an hour before injection of sodium oxalate. Mice were sacrificed 24 hrs after the oxalate injection, blood and kidney were harvested. Blood samples were analyzed for BUN and IL-1β levels. Renal tissues were analyzed for histology, immunohistochemistry, RNA, and protein expression. ResultsHDAC6 and IL-1β upregulated in crystal stimulated macrophages and acute oxalate nephropathy. Pre-treatment of macrophages with TSA reduced IL-1β in supernatant without affecting the expression of pro-IL-1β and mature IL-1β in cell lysate. The effect of TSA on IL-1β secretion was influenced by tubulin acetylation. Renal epithelial cell NRK52E stimulated with crystals showed upregulation of necroptosis pathway markers and concentration-dependent cell death. TSA inhibited RIP-1, RIP3, and MLKL expression along with p-MLKL in stimulated epithelial cells. TSA treatment of oxalate nephropathy mice showed decreased inflammation and tubular cell death by regulating IL-1β and necroptosis and reduced renal injury. ConclusionThis study highlights the role of HDAC6 in regulating the tubulin-mediated secretion of IL-1β and RIP kinase mediated necroptosis in acute oxalate nephropathy.

Abstract 270: The novel control mechanism of the tubulin code and vesicular trafficking in breast cancer bone metastatic cells

Abstract Metastasis of breast cancer (BC) to bone results in severe bone loss, fractures, and death. The crosstalk between BC cells and bone resident cells dramatically increases osteoclast activity, resulting in the release of growth factors from the bone matrix that causes aggressive tumor growth and bone loss. A potentially important aspect of this process is vesicular trafficking on microtubules (MTs) which can affect the output of signaling pathways and secretory activity of metastatic bone cells. MTs are cytoskeletal filaments composed of heterodimers, α- and β-tubulin. The tubulin isotypes and their variety of post-translational modifications (PTMs) control the properties and functions of MT filaments, a concept known as the ‘tubulin code’. Recent studies show an emerging link between alterations of the tubulin code with poor prognosis of breast cancer. However, the regulation of the code in metastatic bone cells is currently unknown. Tubulin acetylation occurs via α-tubulin N-acetyl transferase-1 (αTAT1) and can be reversed by histone deacetylase-6 (HDAC6). MTs lacking acetylation lose flexural rigidity and are prone to breaks following repetitive bending during vesicular trafficking. We found that HDAC6 interaction with α-tubulin is inhibited by a Runt-related factor (Runx2). Our biochemical, mass spectrometry and IPs analyses of MTs revealed that loss of Runx2 can reduce (i) acetylation and stability of MTs, (ii) interaction of HSP90 with α-tubulin, and (iii) levels of β 2a-tubulin. Our studies with Runx2 mutants indicate that the C-terminal of Runx2 serves a scaffolding function by interacting with MTs and HDAC6. Confocal microscopy revealed reduced puncta and altered distribution of endosomal vesicles and autophagosomes with Runx2 silencing. As MT targeting agents are often used as chemotherapeutics, we found that loss of Runx2 sensitizes breast cancer cells to docetaxel and vinblastine and reduces the secretion of IL-6. We found MDA-MB-231 cells metastasized to the bone show significant differences in microtubule isotype expression, with 10 of 19 studied showing 1.3 to 4.1 fold increases. Further analysis of whether Runx2 knockdown affects isotype expression, we found the majority of the 19 studied remain unchanged. Immunohistochemistry for Ac-α-Tub levels in bone metastatic patient samples shows significantly more Ac-α-Tub positive cells in metastatic bone tumors than primary tumors. These findings suggest a novel control mechanism of MTs stability via Runx2-HDAC6 interactions that can impact trafficking and cellular activity. Our results indicate that inhibition of Runx2 may sensitize metastatic tumors to MT targeting agents, and Runx2/HDAC6/Ac-α-Tub levels may serve as markers for metastatic tumors to help stratify patients for optimal treatment for metastatic bone disease. Citation Format: Shreya Patel, Marcus Winogradzki, Ahmad Othman, Waddell Holmes, Jitesh Pratap. The novel control mechanism of the tubulin code and vesicular trafficking in breast cancer bone metastatic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 270.

Target Design of Novel Histone Deacetylase 6 Selective Inhibitors with 2-Mercaptoquinazolinone as the Cap Moiety.

Epigenetic alterations found in all human cancers are promising targets for anticancer therapy. In this sense, histone deacetylase inhibitors (HDACIs) are interesting anticancer agents that play an important role in the epigenetic regulation of cancer cells. Here, we report 15 novel hydroxamic acid-based histone deacetylase inhibitors with quinazolinone core structures. Five compounds exhibited antiproliferative activity with IC50 values of 3.4–37.8 µM. Compound 8 with a 2-mercaptoquinazolinone cap moiety displayed the highest antiproliferative efficacy against MCF-7 cells. For the HDAC6 target selectivity study, compound 8 displayed an IC50 value of 2.3 µM, which is 29.3 times higher than those of HDAC3, HDAC4, HDAC8, and HDAC11. Western blot assay proved that compound 8 strongly inhibited tubulin acetylation, a substrate of HDAC6. Compound 8 also displayed stronger inhibition activity against HDAC11 than the control drug Belinostat. The inhibitory mechanism of action of compound 8 on HDAC enzymes was then explored using molecular docking study. The data revealed a high binding affinity (−7.92 kcal/mol) of compound 8 toward HDAC6. In addition, dock pose analysis also proved that compound 8 might serve as a potent inhibitor of HDAC11.

Essential role of hyperacetylated microtubules in innate immunity escape orchestrated by the EBV-encoded BHRF1 protein.

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This "mito-aggresome" is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.

Kinesin motor KIFC1 is required for tubulin acetylation and actin-dependent spindle migration in mouse oocyte meiosis.

Mammalian oocyte maturation is a unique asymmetric division, which is mainly because of actin-based spindle migration to the cortex. In the present study, we report that a kinesin motor KIFC1, which is associated with microtubules for the maintenance of spindle poles in mitosis, is also involved in actin dynamics in murine oocyte meiosis, co-localizing with microtubules during mouse oocyte maturation. Depletion of KIFC1 caused the failure of polar body extrusion, and we found that meiotic spindle formation and chromosome alignment were disrupted. This might be because of the effects of KIFC1 on HDAC6 and NAT10-based tubulin acetylation, which further affected microtubule stability. Mass spectroscopy analysis revealed that KIFC1 also associated with several actin nucleation factors and we found that KIFC1 was essential for the distribution of actin filaments, which further affected spindle migration. Depletion of KIFC1 leaded to aberrant expression of formin 2 and the ARP2/3 complex, and endoplasmic reticulum distribution was also disturbed. Exogenous KIFC1 mRNA supplement could rescue these defects. Taken together, as well as its roles in tubulin acetylation, our study reported a previously undescribed role of kinesin KIFC1 on the regulation of actin dynamics for spindle migration in mouse oocytes.

A Polytherapy Strategy Using Vincristine and ALK Inhibitors to Sensitise EML4-ALK-Positive NSCLC.

Simple SummaryLung cancer is the third most common cancer worldwide with poor survival after initial diagnosis. A large proportion, approximately 85%, of patients with lung cancer are classified as non-small cell lung cancer (NSCLC). The most common genetic alterations in NSCLCs are KRAS, EGFR and ALK mutations. Patients carrying the EML4-ALK mutation are treated with potent ALK drugs. Up to date, there are 15 different EML4-ALK mutations found in NSCLC patients. Of note, patients carrying the EML4-ALK V3 mutation respond poorly to ALK chemotherapeutic regime and acquire resistance. The aim of the present study was to assess the potential of a combination chemotherapy using vincristine, a traditional chemotherapeutic drug, and potent ALK drugs. We demonstrated that vincristine sensitises cells carrying EML4-ALK V1 mutation but not V3. Cells carrying the EML4-ALK V3 mutation confer low response to drug combination due to high levels of tubulin acetylation and active proliferation pathways compared to V1 cells.The oncogenic fusion of EML4-ALK is present in about 4–6% of non-small cell lung cancer (NSCLC). A targeted approach with ALK tyrosine kinase inhibitors (TKIs) has been proven highly effective in ALK-positive NSCLC patients. However, despite the initial responses, the outcome of the treatment is variable. Previous studies have shown that the differential response depends in part on the type of EML4-ALK variant. Here, we examined the combination of ALK inhibitors and microtubule poison, vincristine, in cells expressing EML4-ALK V1 and V3, the two most common variants in NSCLC. We showed that combination therapy of ALK-TKIs with vincristine had anti-proliferative effects and blocked RAS/MAPK, PI3K/AKT and JAK/STAT3 signalling pathways in EML4-ALK V1 but not V3 cells. Our results demonstrate that high levels of tubulin acetylation are associated with poor response to vincristine in EML4-ALK V3 cells. Additionally, we demonstrated differences in microtubule stability between the two EML4-ALK fusions. EML4-ALK V3 cells exhibited dynamic microtubules that confer poor response to vincristine compared to V1 cells. Hence, we suggested that the portion of EML4 in the fusion has an important role for the outcome of the combination treatment.

Comparison of Cellular Target Engagement Methods for the Tubulin Deacetylases Sirt2 and HDAC6: NanoBRET, CETSA, Tubulin Acetylation, and PROTACs.

The tubulin deacetylases Sirt2 and HDAC6 have been associated with the development of various diseases. Herein, we discuss recent approaches that enable cellular target engagement studies for these deacetylases and thus play a critical role in the evaluation of small molecule inhibitors of Sirt2 or HDAC6 as potential therapeutic agents.

Acetylated α-tubulin K394 regulates microtubule stability to shape the growth of axon terminals

Microtubules are essential to neuron shape and function. Acetylation of tubulin has the potential to directly tune the behavior and function of microtubules in cells. Although proteomic studies have identified several acetylation sites in α-tubulin, the effects of acetylation at these sites remains largely unknown. This includes the highly conserved residue lysine 394 (K394), which is located at the αβ-tubulin dimer interface. Using a fly model, we show that α-tubulin K394 is acetylated in the nervous system and is an essential residue. We found that an acetylation-blocking mutation in endogenous α-tubulin, K394R, perturbs the synaptic morphogenesis of motoneurons and reduces microtubule stability. Intriguingly, the K394R mutation has opposite effects on the growth of two functionally and morphologically distinct motoneurons, revealing neuron-type-specific responses when microtubule stability is altered. Eliminating the deacetylase HDAC6 increases K394 acetylation, and the over-expression of HDAC6 reduces microtubule stability similar to the K394R mutant. Thus, our findings implicate α-tubulin K394 and its acetylation in the regulation of microtubule stability and suggest that HDAC6 regulates K394 acetylation during synaptic morphogenesis.

Nivalenol affects spindle formation and organelle functions during mouse oocyte maturation

Oocyte maturation is essential for fertilization and early embryo development, and proper organelle functions guarantee this process to maintain high-quality oocytes. The type B trichothecene nivalenol (NIV) is a mycotoxin produced by Fusarium oxysporum and is commonly found in contaminated food. NIV intake affect growth, the immune system, and the female reproductive system. Here, we investigated NIV toxicity on mouse oocyte quality. Transcriptome analysis results showed that NIV exposure altered the expression of multiple genes involved in spindle formation and organelle function in mouse oocytes, indicating its toxicity on mouse oocyte maturation. Further analysis indicated that NIV exposure disrupted spindle structure and chromosome alignment, possibly through tubulin acetylation. NIV exposure induced aberrant mitochondria distribution and reduced mitochondria number, mitochondria membrane potential (MMP), and ATP levels. In addition, NIV caused the abnormal distribution of the Golgi apparatus and altered the expression of the vesicle trafficking protein Rab11. ER distribution was also disturbed under NIV exposure, indicating the effects of NIV on protein modification and transport in oocytes. Thus, our results demonstrated that NIV exposure affected spindle structure and organelles function in mouse oocytes.

Phase Ib Study of the Histone Deacetylase 6 Inhibitor Citarinostat in Combination With Paclitaxel in Patients With Advanced Solid Tumors.

BackgroundCitarinostat (CC-96241; previously ACY-241), an oral inhibitor of histone deacetylases (HDACs) with selectivity for HDAC6, has demonstrated synergistic anticancer activity with paclitaxel in multiple solid tumor models. Combination therapy using citarinostat with paclitaxel was evaluated in this phase Ib 3 + 3 dose-escalation study in patients with advanced solid tumors.MethodsPatients with previously treated advanced solid tumors received citarinostat 180, 360, or 480 mg once daily on days 1 to 21 plus paclitaxel 80 mg/m2 on days 1, 8, and 15 of 28-day cycles until disease progression or unacceptable toxicity. The primary endpoint was determination of the maximum tolerated dose (MTD). Secondary endpoints included safety, antitumor activity, pharmacokinetics, and pharmacodynamics.ResultsTwenty patients were enrolled and received study treatment; 15 had received prior taxane therapy. No dose-limiting toxicities were reported at any dose; therefore, the MTD was not identified. Citarinostat 360 vs 480 mg was associated with reduced incidence and severity of neutropenia. Three patients experienced a confirmed partial response and 13 achieved stable disease. Pharmacokinetic parameters were linear up to citarinostat 360 mg, the dose at which the highest levels of histone and tubulin acetylation were observed in peripheral blood mononuclear cells.ConclusionsThe combination of citarinostat plus paclitaxel showed an acceptable safety profile, with no unexpected or dose-limiting toxicities and potential evidence of antitumor activity in patients with heavily pretreated advanced solid tumors. Citarinostat 360 mg once daily is considered the recommended phase II dose for use in combination with paclitaxel 80 mg/m2 every 3 of 4 weeks. This trial is registered on ClinicalTrials.gov (NCT02551185).

αTAT1-induced tubulin acetylation promotes ameloblastoma migration and invasion

AbstractAmeloblastoma (AB) is the most common benign epithelial odontogenic tumor occurring in the jawbone. AB is a slowly growing tumor but sometimes shows a locally invasive and an aggressive growth pattern with a marked bone resorption. In addition, the local recurrence and distant metastasis of AB also sometimes occurs, which resembles one of the typical malignant potentials. From these points of view, to understand better the mechanisms of AB cell migration or invasion is necessary for the better clinical therapy and improvements of the patients' quality of life. Microtubules in eukaryotic cells reveal the shape of hollow cylinders made up of polymerized alpha (α)- and beta (β)-tubulin dimers and form the cytoskeleton together with microfilaments and intermediate filaments. Microtubules play important roles in cell migration by undergoing assembly and disassembly with post-translational modifications. Stability of microtubules caused by their acetylation is involved in cell migration. In this study, we investigated the expression and distribution of acetylated α-tubulin and alpha-tubulin N-acetyltransferase 1 (αTAT1), an enzyme which acetylates Lys-40 in α-tubulin, in AB specimens, and analyzed how tubulin was acetylated by αTAT1 activation in a human AB cell line, AM-1. Finally, we clarified that TGF-β-activated kinase1 (TAK1) was phosphorylated by TGF-β stimulation, then, induced tubulin acetylation via αTAT1 activation, which subsequently activated the migration and invasion of AB cells.

Tubulin posttranslational modifications modify the atypical spermatozoon centriole.

Sperm cells are transcriptionally and translationally silent. Therefore, they may use one of the remaining mechanisms to respond to stimuli in their environment, the post-translational modification of their proteins. Here we examined three post-translational modifications, acetylation, glutamylation, and glycylation of the protein tubulin in human and cattle sperm. Tubulin is the monomer that makes up microtubules, and microtubules constitute the core component of both the sperm centrioles and the axoneme. We found that the sperm of both species were labeled by antibodies against acetylated tubulin and glutamylated tubulin.

Acetyl-CoA Carboxylase Inhibitor CP640.186 Increases Tubulin Acetylation and Impairs Thrombin-Induced Platelet Aggregation.

Acetyl-CoA carboxylase (ACC) is the first enzyme regulating de novo lipid synthesis via the carboxylation of acetyl-CoA into malonyl-CoA. The inhibition of its activity decreases lipogenesis and, in parallel, increases the acetyl-CoA content, which serves as a substrate for protein acetylation. Several findings support a role for acetylation signaling in coordinating signaling systems that drive platelet cytoskeletal changes and aggregation. Therefore, we investigated the impact of ACC inhibition on tubulin acetylation and platelet functions. Human platelets were incubated 2 h with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. We have herein demonstrated that CP640.186 treatment does not affect overall platelet lipid content, yet it is associated with increased tubulin acetylation levels, both at the basal state and after thrombin stimulation. This resulted in impaired platelet aggregation. Similar results were obtained using human platelets that were pretreated with tubacin, an inhibitor of tubulin deacetylase HDAC6. In addition, both ACC and HDAC6 inhibitions block key platelet cytoskeleton signaling events, including Rac1 GTPase activation and the phosphorylation of its downstream effector, p21-activated kinase 2 (PAK2). However, neither CP640.186 nor tubacin affects thrombin-induced actin cytoskeleton remodeling, while ACC inhibition results in decreased thrombin-induced reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK) phosphorylation. We conclude that when using washed human platelets, ACC inhibition limits tubulin deacetylation upon thrombin stimulation, which in turn impairs platelet aggregation. The mechanism involves a downregulation of the Rac1/PAK2 pathway, being independent of actin cytoskeleton.

Factor quinolinone inhibitors alter cell morphology and motility by destabilizing interphase microtubules

Factor quinolinone inhibitors are promising anti-cancer compounds, initially characterized as specific inhibitors of the oncogenic transcription factor LSF (TFCP2). These compounds exert anti-proliferative activity at least in part by disrupting mitotic spindles. Herein, we report additional interphase consequences of the initial lead compound, FQI1, in two telomerase immortalized cell lines. Within minutes of FQI1 addition, the microtubule network is disrupted, resulting in a substantial, although not complete, depletion of microtubules as evidenced both by microtubule sedimentation assays and microscopy. Surprisingly, this microtubule breakdown is quickly followed by an increase in tubulin acetylation in the remaining microtubules. The sudden breakdown and partial depolymerization of the microtubule network precedes FQI1-induced morphological changes. These involve rapid reduction of cell spreading of interphase fetal hepatocytes and increase in circularity of retinal pigment epithelial cells. Microtubule depolymerization gives rise to FH-B cell compaction, as pretreatment with taxol prevents this morphological change. Finally, FQI1 decreases the rate and range of locomotion of interphase cells, supporting an impact of FQI1-induced microtubule breakdown on cell motility. Taken together, our results show that FQI1 interferes with microtubule-associated functions in interphase, specifically cell morphology and motility.

Acetyl-CoA carboxylase inhibition alters tubulin acetylation and aggregation in thrombin-stimulated platelets

Abstract Introduction Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content. Inhibition of its activity decreases lipogenesis and increases the content in acetyl-CoA which can serve as a substrate for protein acetylation. This posttranslational modification plays a key role in the regulation of platelet aggregation, via tubulin acetylation. Purpose To demonstrate that ACC inhibition may affect platelet functions via an alteration of lipid content and/or tubulin acetylation. Methods Platelets were treated 2 hours with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. Platelet functions were assessed by aggregometry and flow cytometry. Lipogenesis was measured via 14C-acetate incorporation into lipids. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Protein phosphorylation and acetylation were evaluated by western blot. Results Treatment with CP640.186 drastically decreased platelet lipogenesis. However, the quantitative lipidomics analyses showed that preincubation with the compound did not affect global platelet lipid content. Interestingly, this short-term ACC inhibition was sufficient to increase tubulin acetylation level, at basal state and after thrombin stimulation. It was associated with an impaired platelet aggregation, in response to low thrombin concentration, while granules secretion was not affected. Mechanistically, we highlighted a decrease in Rac1 activity, associated with a reduced phosphorylation of its downstream effector PAK2. Surprisingly, actin cytoskeleton was not impacted but we evidenced a significant decrease in ROS production which could result from a decreased NOX2 activity. Conclusion Pharmacological ACC inhibition decreases platelet aggregation upon thrombin stimulation. The mechanism depends on increased tubulin acetylation, with subsequent alteration of the Rac1/PAK2/NOX2 signaling pathway Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)

Ttc30a affects tubulin modifications in a model for ciliary chondrodysplasia with polycystic kidney disease

Skeletal ciliopathies (e.g., Jeune syndrome, short rib polydactyly syndrome, and Sensenbrenner syndrome) are frequently associated with nephronophthisis-like cystic kidney disease and other organ manifestations. Despite recent progress in genetic mapping of causative loci, a common molecular mechanism of cartilage defects and cystic kidneys has remained elusive. Targeting two ciliary chondrodysplasia loci (ift80 and ift172) by CRISPR/Cas9 mutagenesis, we established models for skeletal ciliopathies in Xenopus tropicalis Froglets exhibited severe limb deformities, polydactyly, and cystic kidneys, closely matching the phenotype of affected patients. A data mining-based in silico screen found ttc30a to be related to known skeletal ciliopathy genes. CRISPR/Cas9 targeting replicated limb malformations and renal cysts identical to the models of established disease genes. Loss of Ttc30a impaired embryonic renal excretion and ciliogenesis because of altered posttranslational tubulin acetylation, glycylation, and defective axoneme compartmentalization. Ttc30a/b transcripts are enriched in chondrocytes and osteocytes of single-cell RNA-sequenced embryonic mouse limbs. We identify TTC30A/B as an essential node in the network of ciliary chondrodysplasia and nephronophthisis-like disease proteins and suggest that tubulin modifications and cilia segmentation contribute to skeletal and renal ciliopathy manifestations of ciliopathies in a cell type-specific manner. These findings have implications for potential therapeutic strategies.

The cell adhesion molecule TMIGD1 binds to moesin and regulates tubulin acetylation and cell migration

BackgroundThe cell adhesion molecule transmembrane and immunoglobulin (Ig) domain containing1 (TMIGD1) is a novel tumor suppressor that plays important roles in regulating cell–cell adhesion, cell proliferation and cell cycle. However, the mechanisms of TMIGD1 signaling are not yet fully elucidated.ResultsTMIGD1 binds to the ERM family proteins moesin and ezrin, and an evolutionarily conserved RRKK motif on the carboxyl terminus of TMIGD1 mediates the interaction of TMIGD1 with the N-terminal ERM domains of moesin and ezrin. TMIGD1 governs the apical localization of moesin and ezrin, as the loss of TMIGD1 in mice altered apical localization of moesin and ezrin in epithelial cells. In cell culture, TMIGD1 inhibited moesin-induced filopodia-like protrusions and cell migration. More importantly, TMIGD1 stimulated the Lysine (K40) acetylation of α-tubulin and promoted mitotic spindle organization and CRISPR/Cas9-mediated knockout of moesin impaired the TMIGD1-mediated acetylation of α-tubulin and filamentous (F)-actin organization.ConclusionsTMIGD1 binds to moesin and ezrin, and regulates their cellular localization. Moesin plays critical roles in TMIGD1-dependent acetylation of α-tubulin, mitotic spindle organization and cell migration. Our findings offer a molecular framework for understanding the complex functional interplay between TMIGD1 and the ERM family proteins in the regulation of cell adhesion and mitotic spindle assembly, and have wide-ranging implications in physiological and pathological processes such as cancer progression.

Selective Histone Deacetylase Inhibitor ACY-241 (Citarinostat) Plus Nivolumab in Advanced Non-Small Cell Lung Cancer: Results From a Phase Ib Study.

BackgroundHistone deacetylase (HDAC) overexpression has been documented in various cancers and may be associated with worse outcomes. Data from early-phase studies of advanced non-small cell lung cancer (NSCLC) suggest encouraging antitumor activity with the combination of an HDAC inhibitor and either platinum-based chemotherapy or an EGFR inhibitor; however, toxicity is a limiting factor in the use of pan-HDAC inhibitors. Selective inhibition of HDAC6 may represent a potential therapeutic target and preclinical studies revealed immunomodulatory effects with HDAC6 inhibition, suggesting the potential for combination with immune checkpoint inhibitors. This phase Ib, multicenter, single-arm, open-label, dose-escalation study investigated the HDAC6 inhibitor ACY-241 (citarinostat) plus nivolumab in patients with previously treated advanced NSCLC who had not received a prior HDAC or immune checkpoint inhibitor.MethodsThe orally administered ACY-241 dose was escalated (180, 360, or 480 mg once daily). Nivolumab was administered at 240 mg (day 15 of cycle 1, then every 2 weeks thereafter). The primary endpoint was to determine the maximum tolerated dose (MTD) of ACY-241 plus nivolumab. Secondary endpoints included safety, tolerability, and preliminary antitumor activity. Pharmacodynamics was an exploratory endpoint.ResultsA total of 18 patients were enrolled, with 17 patients treated. No dose-limiting toxicities (DLTs) occurred with ACY-241 at 180 or 360 mg; 2 DLTs occurred at 480 mg. The MTD of ACY-241 was 360 mg. The most common grade ≥ 3 treatment-emergent adverse events were dyspnea (n = 3; 18%) and pneumonia (n = 3; 18%). At the 180-mg dose, 1 complete response and 2 partial responses (PRs) were observed. At the 360-mg dose, 3 PRs were observed; 1 patient achieved stable disease (SD) and 1 experienced progressive disease (PD). At the 480-mg dose, no responses were observed; 1 patient achieved SD and 3 experienced PD. Acetylation analyses revealed transient increases in histone and tubulin acetylation levels following treatment. An increase in infiltrating total CD3+ T cells was observed following treatment.ConclusionsThe study identified an MTD for ACY-241 plus nivolumab and the data suggest that the combination may be feasible in patients with advanced NSCLC. Responses were observed in patients with advanced NSCLC. Clinical Trial Registration https://clinicaltrials.gov/ct2/show/NCT02635061 (identifier, NCT02635061).

Pharmacodynamic, pharmacokinetic, and phase 1a study of bisthianostat, a novel histone deacetylase inhibitor, for the treatment of relapsed or refractory multiple myeloma

HDAC inhibitors (HDACis) have been intensively studied for their roles and potential as drug targets in T-cell lymphomas and other hematologic malignancies. Bisthianostat is a novel bisthiazole-based pan-HDACi evolved from natural HDACi largazole. Here, we report the preclinical study of bisthianostat alone and in combination with bortezomib in the treatment of multiple myeloma (MM), as well as preliminary first-in-human findings from an ongoing phase 1a study. Bisthianostat dose dependently induced acetylation of tubulin and H3 and increased PARP cleavage and apoptosis in RPMI-8226 cells. In RPMI-8226 and MM.1S cell xenograft mouse models, oral administration of bisthianostat (50, 75, 100 mg·kg-1·d-1, bid) for 18 days dose dependently inhibited tumor growth. Furthermore, bisthianostat in combination with bortezomib displayed synergistic antitumor effect against RPMI-8226 and MM.1S cell in vitro and in vivo. Preclinical pharmacokinetic study showed bisthianostat was quickly absorbed with moderate oral bioavailability (F% = 16.9%–35.5%). Bisthianostat tended to distribute in blood with Vss value of 0.31 L/kg. This distribution parameter might be beneficial to treat hematologic neoplasms such as MM with few side effects. In an ongoing phase 1a study, bisthianostat treatment was well tolerated and no grade 3/4 nonhematological adverse events (AEs) had occurred together with good pharmacokinetics profiles in eight patients with relapsed or refractory MM (R/R MM). The overall single-agent efficacy was modest, stable disease (SD) was identified in four (50%) patients at the end of first dosing cycle (day 28). These preliminary in-patient results suggest that bisthianostat is a promising HDACi drug with a comparable safety window in R/R MM, supporting for its further phase 1b clinical trial in combination with traditional MM therapies.

Tubulin acetylation promotes penetrative capacity of cells undergoing radial intercalation.

SUMMARYPost-translational modification of tubulin provides differential functions to microtubule networks. Here, we address the role of tubulin acetylation on the penetrative capacity of cells undergoing radial intercalation, which is the process by which cells move apically, insert between outer cells, and join an epithelium. There are opposing forces that regulate intercalation, namely, the restrictive forces of the epithelial barrier versus the penetrative forces of the intercalating cell. Positively and negatively modulating tubulin acetylation in intercalating cells alters the developmental timing such that cells with more acetylation penetrate faster. We find that intercalating cells preferentially penetrate higher-order vertices rather than the more prevalent tricellular vertices. Differential timing in the ability of cells to penetrate different vertices reveals that lower-order vertices represent more restrictive sites of insertion. We shift the accessibility of intercalating cells toward more restrictive junctions by increasing tubulin acetylation, and we provide a geometric-based mathematical model that describes our results.