Identification of cell type-specific correlations between ERK activity and cell viability upon treatment with ERK1/2 inhibitors.

Increased MAPK signaling is a hallmark of various cancers and is a central regulator of cell survival. Direct ERK1/2 inhibition is considered a promising approach to avoid ERK1/2 reactivation caused by upstream kinases BRAF, MEK1/2, and KRAS, as well as by receptor tyrosine kinase inhibitors, but the dynamics and selectivity of ERK1/2 inhibitors are much less studied compared with BRAF or MEK inhibitors. Using ERK1/2 and downstream kinase ELK1 reporter cell lines of lung cancer (H1299; NRASQ61K), colon cancer (HCT-116; KRASG13D), neuroblastoma (SH-SY5Y), and leukemia (U937), we examined the relationship between ERK inhibition and drug-induced toxicity for five ERK inhibitors: SCH772984, ravoxertinib, LY3214996, ulixertinib, and VX-11e, as well as one MEK inhibitor, PD0325901. Comparing cell viability and ERK inhibition revealed different ERK dependencies for these cell lines. We identify several drugs, such as SCH772984 and VX-11e, which induce excessive toxicity not directly related to ERK1/2 inhibition in specific cell lines. We also show that PD0325901, LY3214996, and ulixertinib are prone to ERK1/2 reactivation over time. We distinguished two types of ERK1/2 reactivation: the first could be reversed by adding a fresh dose of inhibitors, while the second persists even after additional treatments. We also showed that cells that became resistant to the MEK1/2 inhibitor PD0325901 due to ERK1/2 reactivation remained sensitive to ERK1/2 inhibitor ulixertinib. Our data indicate that correlation of ERK inhibition with drug-induced toxicity in multiple cell lines may help to find more selective and effective ERK1/2 inhibitors.

Adversary of DNA integrity: A long non-coding RNA stimulates driver oncogenic chromosomal rearrangement in human thyroid cells.

The flurry of publications devoted to the functions of long non-coding RNAs (lncRNAs) published in the last decade leaves no doubt about the exceptional importance of lncRNAs in various areas including tumor biology. However, contribution of lncRNAs to the early stages of oncogenesis remains poorly understood. In this study we explored a new role for lncRNAs: stimulation of specific chromosomal rearrangements upon DNA damage. We demonstrated that lncRNA CASTL1 (ENSG00000269945) stimulates the formation of the CCDC6-RET inversion (RET/PTC1) in human thyroid cells subjected to radiation or chemical DNA damage. Facilitation of chromosomal rearrangement requires lncRNA to contain regions complementary to the introns of both CCDC6 and RET genes as deletion of these regions deprives CASTL1 of the ability to stimulate the gene fusion. We found that CASTL1 expression is elevated in tumors with CCDC6-RET fusion which is the most frequent rearrangement in papillary thyroid carcinoma. Our results open a new venue for the studies of early oncogenesis in various tumor types, especially those associated with physical or chemical DNA damage.

A New Mild Method for Synthesis of Marine Alkaloid Fascaplysin and Its Therapeutically Promising Derivatives.

Fascaplysin is a marine alkaloid which is considered to be a lead drug candidate due to its diverse and potent biological activity. As an anticancer agent, fascaplysin holds a great potential due to the multiple targets affected by this alkaloid in cancer cells, including inhibition of cyclin-dependent kinase 4 (CDK4) and induction of intrinsic apoptosis. At the same time, the studies on structural optimization are hampered by its rather high toxicity, mainly caused by DNA intercalation. In addition, the number of methods for the syntheses of its derivatives is limited. In the current study, we report a new two-step method of synthesis of fascaplysin derivatives based on low temperature UV quaternization for the synthesis of thermolabile 9-benzyloxyfascaplysin and 6-tert-butylfascaplysin. 9-Benzyloxyfascaplysin was used as the starting compound to obtain 9-hydroxyfascaplysin. However, the latter was found to be chemically highly unstable. 6-tert-Butylfascaplysin revealed a significant decrease in DNA intercalation when compared to fascaplysin, while cytotoxicity was only slightly reduced. Therefore, the impact of DNA intercalation for the cytotoxic effects of fascaplysin and its derivatives needs to be questioned.

Principal Component Analysis of Alternative Splicing Profiles Revealed by Long-Read ONT Sequencing in Human Liver Tissue and Hepatocyte-Derived HepG2 and Huh7 Cell Lines.

The long-read RNA sequencing developed by Oxford Nanopore Technology provides a direct quantification of transcript isoforms. That makes the number of transcript isoforms per gene an intrinsically suitable metric for alternative splicing (AS) profiling in the application to this particular type of RNA sequencing. By using this simple metric and recruiting principal component analysis (PCA) as a tool to visualize the high-dimensional transcriptomic data, we were able to group biospecimens of normal human liver tissue and hepatocyte-derived malignant HepG2 and Huh7 cells into clear clusters in a 2D space. For the transcriptome-wide analysis, the clustering was observed regardless whether all genes were included in analysis or only those expressed in all biospecimens tested. However, in the application to a particular set of genes known as pharmacogenes, which are involved in drug metabolism, the clustering worsened dramatically in the latter case. Based on PCA data, the subsets of genes most contributing to biospecimens' grouping into clusters were selected and subjected to gene ontology analysis that allowed us to determine the top 20 biological processes among which translation and processes related to its regulation dominate. The suggested metrics can be a useful addition to the existing metrics for describing AS profiles, especially in application to transcriptome studies with long-read sequencing.

Study of Structure-Activity Relationships of the Marine Alkaloid Fascaplysin and Its Derivatives as Potent Anticancer Agents.

Marine alkaloid fascaplysin and its derivatives are known to exhibit promising anticancer properties in vitro and in vivo. However, toxicity of these molecules to non-cancer cells was identified as a main limitation for their clinical use. Here, for the very first time, we synthesized a library of fascaplysin derivatives covering all possible substituent introduction sites, i.e., cycles A, C and E of the 12H-pyrido[1-2-a:3,4-b']diindole system. Their selectivity towards human prostate cancer versus non-cancer cells, as well as the effects on cellular metabolism, membrane integrity, cell cycle progression, apoptosis induction and their ability to intercalate into DNA were investigated. A pronounced selectivity for cancer cells was observed for the family of di- and trisubstituted halogen derivatives (modification of cycles A and E), while a modification of cycle C resulted in a stronger activity in therapy-resistant PC-3 cells. Among others, 3,10-dibromofascaplysin exhibited the highest selectivity, presumably due to the cytostatic effects executed via the targeting of cellular metabolism. Moreover, an introduction of radical substituents at C-9, C-10 or C-10 plus C-3 resulted in a notable reduction in DNA intercalating activity and improved selectivity. Taken together, our research contributes to understanding the structure-activity relationships of fascaplysin alkaloids and defines further directions of the structural optimization.

Subtype of Neuroblastoma Cells with High KIT Expression Are Dependent on KIT and Its Knockdown Induces Compensatory Activation of Pro-Survival Signaling.

Neuroblastoma (NB) is a pediatric cancer with high clinical and molecular heterogeneity, and patients with high-risk tumors have limited treatment options. Receptor tyrosine kinase KIT has been identified as a potential marker of high-risk NB and a promising target for NB treatment. We investigated 19,145 tumor RNA expression and molecular pathway activation profiles for 20 cancer types and detected relatively high levels of KIT expression in NB. Increased KIT expression was associated with activation of cell survival pathways, downregulated apoptosis induction, and cell cycle checkpoint control pathways. KIT knockdown with shRNA encoded by lentiviral vectors in SH-SY5Y cells led to reduced cell proliferation and apoptosis induction up to 50%. Our data suggest that apoptosis induction was caused by mitotic catastrophe, and there was a 2-fold decrease in percentage of G2-M cell cycle phase after KIT knockdown. We found that KIT knockdown in NB cells leads to strong upregulation of other pro-survival growth factor signaling cascades such as EPO, NGF, IL-6, and IGF-1 pathways. NGF, IGF-1 and EPO were able to increase cell proliferation in KIT-depleted cells in an ERK1/2-dependent manner. Overall, we show that KIT is a promising therapeutic target in NB, although such therapy efficiency could be impeded by growth factor signaling activation.

Cytotoxic Marine Alkaloid 3,10-Dibromofascaplysin Induces Apoptosis and Synergizes with Cytarabine Resulting in Leukemia Cell Death.

Myeloid leukemia is a hematologic neoplasia characterized by a clonal proliferation of hematopoietic stem cell progenitors. Patient prognosis varies depending on the subtype of leukemia as well as eligibility for intensive treatment regimens and allogeneic stem cell transplantation. Although significant progress has been made in the therapy of patients including novel targeted treatment approaches, there is still an urgent need to optimize treatment outcome. The most common therapy is based on the use of chemotherapeutics cytarabine and anthrayclines. Here, we studied the effect of the recently synthesized marine alkaloid 3,10-dibromofascaplysin (DBF) in myeloid leukemia cells. Unsubstituted fascaplysin was early found to affect cell cycle via inhibiting CDK4/6, thus we compared the activity of DBF and other brominated derivatives with known CDK4/6 inhibitor palbociclib, which was earlier shown to be a promising candidate to treat leukemia. Unexpectedly, the effect DBF on cell cycle differs from palbociclib. In fact, DBF induced leukemic cells apoptosis and decreased the expression of genes responsible for cancer cell survival. Simultaneously, DBF was found to activate the E2F1 transcription factor. Using bioinformatical approaches we evaluated the possible molecular mechanisms, which may be associated with DBF-induced activation of E2F1. Finally, we found that DBF synergistically increase the cytotoxic effect of cytarabine in different myeloid leukemia cell lines. In conclusion, DBF is a promising drug candidate, which may be used in combinational therapeutics approaches to reduce leukemia cell growth.

New Insights into the Structure of Kappa/Beta-Carrageenan: A Novel Potential Inhibitor of HIV-1.

New insights into the structure of the hybrid κ/ß-carrageenan (κ/ß-CRG) of the red alga Tichocarpus crinitus have been obtained. Carrageenan oligosaccharides were prepared through the chemical and enzymatic depolymerization of κ/ß-CRG with κ-carrageenase and its the enzyme-resistant fraction. The composition and distribution of the repetition units of κ/ß- CRG were investigated by using the negative ion tandem MALDI-TOFMS and ESIMS method, which made it possible to prove and characterize the hybrid structure of this polysaccharide. An analysis revealed the blockwise distribution of the long ß-blocks along the polysaccharide chain, with the inclusion of κ/ß, µ/ν-blocks and some ι-blocks. Furthermore, the desulfated κ/ß-CRG was shown to contain of -G-D- repeating units up to 3.5 kDa. Previous studies have demonstrated that CRGs suppress the replication of several viruses. Here, we established that κ/ß-CRG and its oligosaccharides significantly inhibit the transduction efficiency of replication-defective lentiviral particles pseudotyped with the envelope proteins of three different viruses. We found that the polysaccharide and its oligosaccharides strongly reduced the transduction efficiency of lentiviral particles pseudotyped with GP160-the envelope protein of the human immunodeficiency virus HIV-1-when added to T-lymphocyte Jurkat cells. The CRG oligosaccharides displayed significantly higher antiviral activity.

Efficacy and Mechanism of Action of Marine Alkaloid 3,10-Dibromofascaplysin in Drug-Resistant Prostate Cancer Cells.

Efficacy and mechanism of action of marine alkaloid 3,10-dibromofascaplysin (DBF) were investigated in human prostate cancer (PCa) cells harboring different levels of drug resistance. Anticancer activity was observed across all cell lines examined without signs of cross-resistance to androgen receptor targeting agents (ARTA) or taxane based chemotherapy. Kinome analysis followed by functional investigation identified JNK1/2 to be one of the molecular targets of DBF in 22Rv1 cells. In contrast, no activation of p38 and ERK1/2 MAPKs was observed. Inhibition of the drug-induced JNK1/2 activation or of the basal p38 activity resulted in increased cytotoxicity of DBF, whereas an active ERK1/2 was identified to be important for anticancer activity of the alkaloid. Synergistic effects of DBF were observed in combination with PARP-inhibitor olaparib most likely due to the induction of ROS production by the marine alkaloid. In addition, DBF intensified effects of platinum-based drugs cisplatin and carboplatin, and taxane derivatives docetaxel and cabazitaxel. Finally, DBF inhibited AR-signaling and resensitized AR-V7-positive 22Rv1 prostate cancer cells to enzalutamide, presumably due to AR-V7 down-regulation. These findings propose DBF to be a promising novel drug candidate for the treatment of human PCa regardless of resistance to standard therapy.

LINC00973 Induces Proliferation Arrest of Drug-Treated Cancer Cells by Preventing p21 Degradation.

Overcoming drug resistance of cancer cells is the major challenge in molecular oncology. Here, we demonstrate that long non-coding RNA LINC00973 is up-regulated in normal and cancer cells of different origins upon treatment with different chemotherapeutics. Bioinformatics analysis shows that this is a consequence of DNA damage response pathway activation or mitotic arrest. Knockdown of LINC0973 decreases p21 levels, activates cellular proliferation of cancer cells, and suppresses apoptosis of drug-treated cells. We have found that LINC00973 strongly increases p21 protein content, possibly by blocking its degradation. Besides, we have found that ectopic over-expression of LINC00973 inhibits formation of the pro-survival p53-Ser15-P isoform, which preserves chromosome integrity. These results might open a new approach to the development of more efficient anti-cancer drugs.

Improving the power of drug toxicity measurements by quantitative nuclei imaging.

Imaging-based anticancer drug screens are becoming more prevalent due to development of automated fluorescent microscopes and imaging stations, as well as rapid advancements in image processing software. Automated cell imaging provides many benefits such as their ability to provide high-content data, modularity, dynamics recording and the fact that imaging is the most direct way to access cell viability and cell proliferation. However, currently most publicly available large-scale anticancer drugs screens, such as GDSC, CTRP and NCI-60, provide cell viability data measured by assays based on colorimetric or luminometric measurements of NADH or ATP levels. Although such datasets provide valuable data, it is unclear how well drug toxicity measurements can be integrated with imaging data. Here we explored the relations between drug toxicity data obtained by XTT assay, two quantitative nuclei imaging methods and trypan blue dye exclusion assay using a set of four cancer cell lines with different morphologies and 30 drugs with different mechanisms of action. We show that imaging-based approaches provide high accuracy and the differences between results obtained by different methods highly depend on drug mechanism of action. Selecting AUC metrics over IC50 or comparing data where significantly drugs reduced cell numbers noticeably improves consistency between methods. Using automated cell segmentation protocols we analyzed mitochondria activity in more than 11 thousand drug-treated cells and showed that XTT assay produces unreliable data for CDK4/6, Aurora A, VEGFR and PARP inhibitors due induced cell size growth and increase in individual mitochondria activity. We also explored several benefits of image-based analysis such as ability to monitor cell number dynamics, dissect changes in total and individual mitochondria activity from cell proliferation, and ability to identify chromatin remodeling drugs. Finally, we provide a web tool that allows comparing results obtained by different methods.

Multikinase inhibitors modulate non-constitutive proteasome expression in colorectal cancer cells.

Introduction: Proteasomes are multi-subunit protein complexes responsible for protein degradation in cells. Immunoproteasomes and intermediate proteasomes (together non-constitutive proteasomes) are specific forms of proteasomes frequently associated with immune response, antigen presentation, inflammation and stress. Expression of non-constitutive proteasome subunits has a prognostic value in several types of cancer. Thus, factors that modulate non-constitutive proteasome expression in tumors are of particular interest. Multikinase inhibitors (MKIs) demonstrate promising results in treatment of cancer. At the same time, their immunomodulatory properties and effects on non-constitutive proteasome expression in colorectal cancer cells are poorly investigated. Methods: Proteasome subunit expression in colorectal cancer was evaluated by bioinformatic analysis of available datasets. Two colorectal cancer cell lines, expressing fluorescent non-constitutive proteasomes were treated with multikinase inhibitors: regorafenib and sorafenib. The proteasome subunit expression was assessed by real-time PCR, Western blotting and flow cytometry. The proteasome activity was studied using proteasome activity-based probe and fluorescent substrates. Intracellular proteasome localization was revealed by confocal microscopy. Reactive oxygen species levels following treatment were determined in cells. Combined effect of proteasome inhibition and treatment with MKIs on viability of cells was estimated. Results: Expression of non-constitutive proteasomes is increased in BRAF-mutant colorectal tumors. Regorafenib and sorafenib stimulated the activity and synthesis of non-constitutive proteasomes in examined cell lines. MKIs induced oxidative stress and redistribution of proteasomes within cells. Sorafenib stimulated formation of cytoplasmic aggregates, containing proteolyticaly active non-constitutive proteasomes, while regorafenib had no such effect. MKIs caused no synergistic action when were combined with the proteasome inhibitor. Discussion: Obtained results indicate that MKIs might affect the crosstalk between cancer cells and immune cells via modulation of intracellular proteasome pool. Observed phenomenon should be considered when MKI-based therapy is applied.

Synthesis and new DNA targeting activity of 6- and 7-tert-butylfascaplysins.

Fascaplysin is a red cytotoxic pigment with anticancer properties isolated from the marine sponge Fascaplysinopsis sp. Recently, structure-activity relationship analysis reported by our group suggested that selective cytotoxicity of fascaplysin derivatives towards tumor cells negatively correlates with their ability to intercalate into DNA. To validate this hypothesis, we synthesized 6- and 7-tert-butylfascaplysins which reveal mitigated DNA-intercalating properties. These derivatives were found to be strongly cytotoxic to drug-resistant human prostate cancer cells, albeit did not demonstrate improved selectivity towards cancer cells when compared to fascaplysin. At the same time, kinome analysis suggested an activation of CHK1/ATR axis in cancer cells shortly after the drug exposure. Further experiments revealed induction of replication stress that is eventually converted to the toxic DNA double-strand breaks, resulting in caspase-independent apoptosis-like cell death. Our observations highlight new DNA-targeting effect of some fascaplysin derivatives and indicate more complex structure-activity relationships within the fascaplysin family, suggesting that cytotoxicity and selectivity of these alkaloids are influenced by multiple factors. Furthermore, combination with clinically-approved inhibitors of ATR/CHK1 as well as testing in tumors particularly sensitive to the DNA damage should be considered in further studies.

A new system for parallel drug screening against multiple-resistant HIV mutants based on lentiviral self-inactivating (SIN) vectors and multi-colour analyses.

BACKGROUND: Despite progress in the development of combined antiretroviral therapies (cART), HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed. METHODS: We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN) LeGO vector technology. RESULTS: We demonstrated the successful use of this approach for screening compounds against up to four HIV gag-pol variants (wild-type and three mutants) simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment. CONCLUSIONS: The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs.

Fucoidans as potential inhibitors of HIV-1.

The antiviral activity of different structure fucoidans (α-l-fucans and galactofucans) was studied using two model viral systems based on a lentiviral vectors and a replication competent Moloney murine leukemia virus (Mo-MuLV). It was found that investigated fucoidans have no cytotoxic effects on Jurkat and SC-1cell at the concentration range of 0.001-100 µg/mL. Fucoidans with different efficiency suppressed transduction of Jurkat cell line by pseudo-HIV-1 particles carrying the envelope protein of HIV-1 and infection of SC-1 cells by Mo-MuLV. According to our data, all natural fucoidans can be considered as potential anti-HIV agents regardless of their carbohydrate backbone and degree of sulfating, since their activity is shown at low concentrations (0.001-0.05 µg/mL). High molecular weight fucoidans isolated from Saccharina cichorioides (1.3-α-l-fucan), and S. japonica (galactofucan) were the most effective inhibitors.

Nucleoside Analog 2',3'-Isopropylidene-5-Iodouridine as Novel Efficient Inhibitor of HIV-1.

Nucleoside reverse transcriptase inhibitors are the first class of drugs to be approved by the FDA for the suppression of HIV-1 and are widely used for this purpose in combination with drugs of other classes. Despite the progress in HIV-1 treatment, there is still the need to develop novel efficient antivirals. Here the efficiency of HIV-1 inhibition by a set of original 5-substituted uridine nucleosides was studied. We used the replication deficient human immunodeficiency virus (HIV-1)-based lentiviral particles and identified that among the studied compounds, 2',3'-isopropylidene-5-iodouridine was shown to cause anti-HIV-1 activity. Importantly, no toxic action of this compound against the cells of T-cell origin was found. We determined that this compound is significantly more efficient at suppressing HIV-1 compared to Azidothymidine (AZT) when taken at the high non-toxic concentrations. We did not find any profit when using AZT in combination with 2',3'-isopropylidene-5-iodouridine. 2',3'-Isopropylidene-5-iodouridine acts synergistically to repress HIV-1 when combined with the CDK4/6 inhibitor Palbociclib in low non-toxic concentration. No synergistic antiviral action was detected when AZT was combined with Palbociclib. We suggest 2',3'-isopropylidene-5-iodouridine as a novel perspective non-toxic compound that may be used for HIV-l suppression.

Comparison of Alternative Splicing Landscapes Revealed by Long-Read Sequencing in Hepatocyte-Derived HepG2 and Huh7 Cultured Cells and Human Liver Tissue.

The long-read RNA sequencing developed by Oxford Nanopore Technologies provides a direct quantification of transcript isoforms, thereby making it possible to present alternative splicing (AS) profiles as arrays of single splice variants with different abundances. Additionally, AS profiles can be presented as arrays of genes characterized by the degree of alternative splicing (the DAS-the number of detected splice variants per gene). Here, we successfully utilized the DAS to reveal biological pathways influenced by the alterations in AS in human liver tissue and the hepatocyte-derived malignant cell lines HepG2 and Huh7, thus employing the mathematical algorithm of gene set enrichment analysis. Furthermore, analysis of the AS profiles as abundances of single splice variants by using the graded tissue specificity index τ provided the selection of the groups of genes expressing particular splice variants specifically in liver tissue, HepG2 cells, and Huh7 cells. The majority of these splice variants were translated into proteins products and appeal to be in focus regarding further insights into the mechanisms underlying cell malignization. The used metrics are intrinsically suitable for transcriptome-wide AS profiling using long-read sequencing.

Emetine in Combination with Chloroquine Induces Oncolytic Potential of HIV-1-Based Lentiviral Particles.

Chloroquine and Emetine are drugs used to treat human parasitic infections. In addition, it has been shown that these drugs have an antiviral effect. Both drugs were also found to cause a suppressive effect on the growth of cancer cells of different origins. Here, using the replication-deficient HIV-1-based lentiviral vector particles, we evaluated the ability of the combination of these drugs to reduce viral transduction efficiency. We showed that these drugs act synergistically to decrease cancer cell growth when added in combination with medium containing lentiviral particles. We found that the combination of these drugs with lentiviral particles decreases the viability of treated cells. Taken together, we state the oncolytic potential of the medium containing HIV-1-based particles provoked by the combination of Chloroquine and Emetine.

Immunoproteasome Activity and Content Determine Hematopoietic Cell Sensitivity to ONX-0914 and to the Infection of Cells with Lentiviruses.

Proteasomes are intracellular structures responsible for protein degradation. The 20S proteasome is a core catalytic element of the proteasome assembly. Variations of catalytic subunits generate different forms of 20S proteasomes including immunoproteasomes (iPs), which are present mostly in the immune cells. Certain cells of the immune system are primary targets of retroviruses. It has been shown that several viral proteins directly affect proteasome functionality, while inhibition of proteasome activity with broad specificity proteasome inhibitors stimulates viral transduction. Here we specifically addressed the role of the immunoproteasomes during early stages of viral transduction and investigated the effects of specific immunoproteasome inhibition and activation prior to infection using a panel of cell lines. Inhibition of iPs in hematopoietic cells with immunoproteasome-specific inhibitor ONX-0914 resulted in increased infection by VSV-G pseudotyped lentiviruses. Moreover, a tendency for increased infection of cloned cells with endogenously decreased proteasome activity was revealed. Conversely, activation of iPs by IFN-γ markedly reduced the viral infectivity, which was rescued upon simultaneous immunoproteasome inhibition. Our results indicate that immunoproteasome activity might be determinative for the cellular antiretroviral resistance at least for the cells with high iP content. Finally, therapeutic application of immunoproteasome inhibitors might promote retroviral infection of cells in vivo.

Selective Inhibition of HDAC Class I Sensitizes Leukemia and Neuroblastoma Cells to Anticancer Drugs.

The acquired resistance of neuroblastoma (NB) and leukemia cells to anticancer therapy remains the major challenge in the treatment of patients with these diseases. Although targeted therapy, such as receptor tyrosine kinase (RTK) inhibitors, has been introduced into clinical practice, its efficacy is limited to patients harboring mutant kinases. Through the analysis of transcriptomic data of 701 leukemia and NB patient samples and cell lines, we revealed that the expression of RTK, such as KIT, FLT3, AXL, FGFR3, and NTRK1, is linked with HDAC class I. Although HDAC inhibitors have antitumor activity, they also have high whole-body toxicity. We developed a novel belinostat derivative named hydrazostat, which targets HDAC class I with limited off-target effects. We compared the toxicity of these drugs within the panel of leukemia and NB cell lines. Next, we revealed that HDAC inhibition with hydrazostat reactivates NTRK1, FGFR3, ROR2, KIT, and FLT3 expression. Based on this finding, we tested the efficacy of hydrazostat in combination with RTK inhibitor imatinib. Additionally, we show the ability of hydrazostat to enhance venetoclax-induced apoptosis. Thus, we reveal the connection between HDACs and RTK and describe a useful strategy to overcome the complications of single-agent therapies.