Alterations in lipid metabolism accompanied by changes in protein and carotenoid content as spectroscopic markers of human T cell activation.

This work aims to understand better the mechanism of cellular processes accompanying the activation of human T cells and to develop a novel, fast, label-free approach to identify molecular biomarkers for this process. The standard methodology for confirming the activation state of T cells is based on flow cytometry and using antibodies recognizing activation markers. The method provide high specificity detection but may be susceptible to background staining or non-specific secondary antibody reactions. Here, we evaluated the potential of Raman-based molecular imaging in distinguishing non-activated and activated human T cells. Confocal Raman microscopy was performed on T cells followed by chemometrics to obtain comprehensive molecular information, while Stimulated Raman Scattering imaging was used to quickly provide high-resolution images of selected cellular components of activated and non-activated cells. For the first time, carotenoids, lipids, and proteins were shown to be important biomarkers of T-cell activation. We found that T-cell activation was accompanied by lipid accumulation and loss of carotenoid content. Our findings on the biochemical, morphological, and structural changes associated with activated mature T cells provide insights into the molecular changes that occur during therapeutic manipulation of the immune response. The methodology for identifying activated T cells is based on a novel imaging method and supervised and unsupervised chemometrics. It unambiguously identifies specific and unique molecular changes without the need for staining, fixation, or any other sample preparation.

Automatic subtyping of Diffuse Large B-cell Lymphomas (DLBCL): Raman-based genetic and metabolic classification.

Diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin's lymphoma in adults, is a genetically and metabolically heterogeneous group of aggressive malignancies. The complexity of their molecular composition and the variability in clinical presentation make clinical diagnosis and treatment selection a serious challenge. The challenge is therefore to quickly and correctly classify DLBCL cells. In this work, we show that Raman imaging is a tool with high diagnostic potential, providing unique information about the biochemical components of tumor cells and their metabolism. We present models of classification of lymphoma cells based on their Raman spectra. The models automatically and efficiently identify DLBCL cells and assign them to a given cell-of-origin (COO) subtype (activated B cell-like (ABC) or germinal center B cell-like (GCB)) or, respectively, to a comprehensive cluster classification (CCC) subtype (OxPhos/non-OxPhos). In addition, we describe each lymphoma subtype by its unique spectral profile, linking it to biochemical, genetic, or metabolic features.

Prognostic Impact of Copy Number Alterations' Profile and AID/RAG Signatures in Acute Lymphoblastic Leukemia (ALL) with BCR::ABL and without Recurrent Genetic Aberrations (NEG ALL) Treated with Intensive Chemotherapy.

Adult acute lymphoblastic leukemia (ALL) is associated with poor outcomes. ALL is initiated by primary aberrations, but secondary genetic lesions are necessary for overt ALL. In this study, we reassessed the value of primary and secondary aberrations in intensively treated ALL patients in relation to mutator enzyme expression. RT-PCR, genomic PCR, and sequencing were applied to evaluate primary aberrations, while qPCR was used to measure the expression of RAG and AID mutator enzymes in 166 adult ALL patients. Secondary copy number alterations (CNA) were studied in 94 cases by MLPA assay. Primary aberrations alone stratified 30% of the patients (27% high-risk, 3% low-risk cases). The remaining 70% intermediate-risk patients included BCR::ABL1pos subgroup and ALL lacking identified genetic markers (NEG ALL). We identified three CNA profiles: high-risk bad-CNA (CNAhigh/IKZF1pos), low-risk good-CNA (all other CNAs), and intermediate-risk CNAneg. Furthermore, based on RAG/AID expression, we report possible mechanisms underlying the CNA profiles associated with poor outcome: AID stratified outcome in CNAneg, which accompanied most likely a particular profile of single nucleotide variations, while RAG in CNApos increased the odds for CNAhigh/IKZF1pos development. Finally, we integrated primary genetic aberrations with CNA to propose a revised risk stratification code, which allowed us to stratify 75% of BCR::ABL1pos and NEG patients.

SIRT1 and HSP90α feed-forward circuit safeguards chromosome segregation integrity in diffuse large B cell lymphomas.

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma in adults, exhibiting highly heterogenous clinical behavior and complex molecular background. In addition to the genetic complexity, different DLBCL subsets exhibit phenotypic features independent of the genetic background. For example, a subset of DLBCLs is distinguished by increased oxidative phosphorylation and unique transcriptional features, including overexpression of certain mitochondrial genes and a molecular chaperone, heat shock protein HSP90α (termed "OxPhos" DLBCLs). In this study, we identified a feed-forward pathogenetic circuit linking HSP90α and SIRT1 in OxPhos DLBCLs. The expression of the inducible HSP90α isoform remains under SIRT1-mediated regulation. SIRT1 knockdown or chemical inhibition reduced HSP90α expression in a mechanism involving HSF1 transcription factor, whereas HSP90 inhibition reduced SIRT1 protein stability, indicating that HSP90 chaperones SIRT1. SIRT1-HSP90α interaction in DLBCL cells was confirmed by co-immunoprecipitation and proximity ligation assay (PLA). The number of SIRT1-HSP90α complexes in PLA was significantly higher in OxPhos- dependent than -independent cells. Importantly, SIRT1-HSP90α interactions in OxPhos DLBCLs markedly increased in mitosis, suggesting a specific role of the complex during this cell cycle phase. RNAi-mediated and chemical inhibition of SIRT1 and/or HSP90 significantly increased the number of cells with chromosome segregation errors (multipolar spindle formation, anaphase bridges and lagging chromosomes). Finally, chemical SIRT1 inhibitors induced dose-dependent cytotoxicity in OxPhos-dependent DLBCL cell lines and synergized with the HSP90 inhibitor. Taken together, our findings define a new OxPhos-DLBCL-specific pathogenetic loop involving SIRT1 and HSP90α that regulates chromosome dynamics during mitosis and may be exploited therapeutically.

Targeting arginase-1 exerts antitumor effects in multiple myeloma and mitigates bortezomib-induced cardiotoxicity.

Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here, we investigated the role of arginase 1 (ARG1) in Vκ*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in ʟ-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of Vκ*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However, arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether, these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors.

Activity and rational combinations of a novel, engineered chimeric, TRAIL-based ligand in diffuse large B-cell lymphoma.

Background: TRAIL (TNF-related apoptosis inducing ligand) exhibits selective proapoptotic activity in multiple tumor types, while sparing normal cells. This selectivity makes TRAIL an attractive therapeutic candidate. However, despite encouraging activity in preclinical models, clinical trials with TRAIL mimetics/death receptor agonists demonstrated insufficient activity, largely due to emerging resistance to these agents. Herein, we investigated the cytotoxic activity of a novel, TRAIL-based chimeric protein AD-O51.4 combining TRAIL and VEGFA-derived peptide sequences, in hematological malignancies. We characterize key molecular mechanisms leading to resistance and propose rational pharmacological combinations sensitizing cells to AD-O51.4. Methods: Sensitivity of DLBCL, classical Hodgkin lymphoma, (cHL), Burkitt lymphoma (BL) and acute myeloid leukemia (AML) to AD-O51.4 was assessed in vitro with MTS assay and apoptosis tests (Annexin V/PI staining). Markers of apoptosis were assessed using immunoblotting, flow cytometry or fluorogenic caspase cleavage assays. Resistant cell lines were obtained by incubation with increasing doses of AD-O51.4. Transcriptomic analyses were performed by RNA sequencing. Sensitizing effects of selected pathway modulators (BCL2, dynamin and HDAC inhibitors) were assessed using MTS/apoptosis assays. Results: AD-O51.4 exhibited low-nanomolar cytotoxic activity in DLBCL cells, but not in other lymphoid or AML cell lines. AD-O51.4 induced death-receptor (DR) mediated, caspase-dependent apoptosis in sensitive DLBCL cells, but not in primary resistant cells. The presence of DRs and caspase 8 in cancer cells was crucial for AD-O51.4-induced apoptosis. To understand the potential mechanisms of resistance in an unbiased way, we engineered AD-O51.4-resistant cells and evaluated resistance-associated transcriptomic changes. Resistant cells exhibited changes in the expression of multiple genes and pathways associated with apoptosis, endocytosis and HDAC-dependent epigenetic reprogramming, suggesting potential therapeutic strategies of sensitization to AD-O51.4. In subsequent analyses, we demonstrated that HDAC inhibitors, BCL2 inhibitors and endocytosis/dynamin inhibitors sensitized primary resistant DLBCL cells to AD-O51.4. Conclusions: Taken together, we identified rational pharmacologic strategies sensitizing cells to AD-O51.4, including BCL2, histone deacetylase inhibitors and dynamin modulators. Since AD-O51.4 exhibits favorable pharmacokinetics and an acceptable safety profile, its further clinical development is warranted. Identification of resistance mechanisms in a clinical setting might indicate a personalized pharmacological approach to override the resistance.

A Specific CD44lo CD25lo Subpopulation of Regulatory T Cells Inhibits Anti-Leukemic Immune Response and Promotes the Progression in a Mouse Model of Chronic Lymphocytic Leukemia.

Regulatory T cells (Tregs) are capable of inhibiting the proliferation, activation and function of T cells and play an important role in impeding the immune response to cancer. In chronic lymphocytic leukemia (CLL) a dysfunctional immune response and elevated percentage of effector-like phenotype Tregs have been described. In this study, using the Eµ-TCL1 mouse model of CLL, we evaluated the changes in the Tregs phenotype and their expansion at different stages of leukemia progression. Importantly, we show that Tregs depletion in DEREG mice triggered the expansion of new anti-leukemic cytotoxic T cell clones leading to leukemia eradication. In TCL1 leukemia-bearing mice we identified and characterized a specific Tregs subpopulation, the phenotype of which suggests its role in the formation of an immunosuppressive microenvironment, supportive for leukemia survival and proliferation. This observation was also confirmed by the gene expression profile analysis of these TCL1-specific Tregs. The obtained data on Tregs are consistent with those described so far, however, above all show that the changes in the Tregs phenotype described in CLL result from the formation of a specific, described in this study Tregs subpopulation. In addition, functional tests revealed the ability of Tregs to inhibit T cells that recognize model antigens expressed by leukemic cells. Moreover, inhibition of Tregs with a MALT1 inhibitor provided a therapeutic benefit, both as monotherapy and also when combined with an immune checkpoint inhibitor. Altogether, activation of Tregs appears to be crucial for CLL progression.

Super enhancers as master gene regulators in the pathogenesis of hematologic malignancies.

Transcriptional deregulation of multiple oncogenes, tumor suppressors and survival pathways is a cancer cell hallmark. Super enhancers (SE) are long stretches of active enhancers in close linear proximity that ensure extraordinarily high expression levels of key genes associated with cell lineage, function and survival. SE landscape is intrinsically prone to changes and reorganization during the course of normal cell differentiation. This functional plasticity is typically utilized by cancer cells, which remodel their SE landscapes to ensure oncogenic transcriptional reprogramming. Multiple recent studies highlighted structural genetic mechanisms in non-coding regions that create new SE or hijack already existing ones. In addition, alterations in abundance/activity of certain SE-associated proteins or certain viral infections can elicit new super enhancers and trigger SE-driven transcriptional changes. For these reasons, SE profiling emerged as a powerful tool for discovering the core transcriptional regulatory circuits in tumor cells. This, in turn, provides new insights into cancer cell biology, and identifies main nodes of key cellular pathways to be potentially targeted. Since SEs are susceptible to inhibition, their disruption results in exponentially amassing 'butterfly' effect on gene expression and cell function. Moreover, many of SE elements are druggable, opening new therapeutic opportunities. Indeed, SE targeting drugs have been studied preclinically in various hematologic malignancies with promising effects. Herein, we review the unique features of SEs, present different cis- and trans-acting mechanisms through which hematologic tumor cells acquire SEs, and finally, discuss the potential of SE targeting in the therapy of hematologic malignancies.

Persistent imbalance, anti-apoptotic, and anti-inflammatory signature of circulating C-C chemokines and cytokines in patients with paroxysmal nocturnal hemoglobinuria.

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal non-malignant disease in which hematopoietic cell apoptosis may play an important pathophysiological role. Previous studies of the content of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) indicated the possibility of remote transmission of anti-apoptotic signals between pathological and normal hematopoietic progenitors. METHODS: The study determined the plasma levels of beta chemokines and cytokines in N = 19 patients with PNH and 31 healthy controls. The research material was peripheral blood plasma (EDTA) stored at -80 °C until the test. Beta chemokine and cytokine concentrations were tested in duplicate with Bio-Plex Pro Human Cytokine Assay (Bio-Rad, Hercules, CA, USA) using a Luminex 200 flow cytometer and xPONENT software (Luminex Corporation, Austin, TX, USA). In peripheral blood CD34+ cells we tested the proportions of PI(3,4,5)P3+ and Annexin binding apoptotic phenotype using FC and phosflow. RESULTS: Compared to the control group, the PNH group showed a significant increase in the plasma concentration of some beta chemokines and cytokines, including MIP-1alpha/CCL3, eotaxin/CCL11, MCP1/CCL2, IL4 and G-CSF. In the group of PNH patients, a significant decrease in the concentration of some cytokines was also observed: RANTES/CCL5, MIP-1beta/CCL4, PDGF-BB and IL9. At the same time, the plasma concentrations of the chemokine IP-10/CXCL10 and the cytokines IFN-gamma, TNF, IL6 and IL10 showed no significant deviations from the values for the control group. Anti-apoptotic phenotype and phosphatidylinositol (3,4,5)-trisphosphate content in PNH clone of CD34+ cells were associated with the level of CCL3 and negatively associated with CCL5, CCL4, PDGF-BB and IL9. CONCLUSIONS: This data suggest the existence of apoptotic and PI(3,4,5)P3 imbalance in PNH CD34+ cells driven by anti-apoptotic cytokine biosignature in PNH. Plasma cytokines and intracellular enzymes that regulate the phosphoinositide pathways may become a therapeutic target in PNH.

Inhibition of PIM Kinases in DLBCL Targets MYC Transcriptional Program and Augments the Efficacy of Anti-CD20 Antibodies.

The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase-associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition-induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. SIGNIFICANCE: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.

The CRBN, CUL4A and DDB1 Expression Predicts the Response to Immunomodulatory Drugs and Survival of Multiple Myeloma Patients.

Immunomodulatory drugs (IMiDs) are effective in the treatment of multiple myeloma (MM), myelodysplastic syndrome with deletion of chromosome 5q and other haematological malignancies. Recent studies showed that IMiDs bind to cereblon (CRBN), a substrate receptor of the CRL4-CRBN complex, to induce the ubiquitination and degradation of IKZF1 and IKZF3 in MM cells, contributing to their anti-myeloma activity. We aimed to determine whether the CRL4-CRBN complex proteins' expression predicts the prognosis of MM patients treated with IMiDs. Here, we evaluated the expression of CRL4-CRBN complex proteins and their downstream targets with immunohistochemistry (IHC) staining in 130 bone marrow samples from MM patients treated with thalidomide or lenalidomide-based regimens. We found that the expression of CRBN and CUL4A was associated with the superior IMiD-based treatment response (p = 0.007 and p = 0.007, respectively). Moreover, the CUL4A expression was associated with improved PFS (HR = 0.66, 95% CI 0.44-0.99; p = 0.046) and DDB1 expression showed a negative impact on OS both in the univariate (HR = 2.75, 95% CI 1.65-4.61; p = 0.001) and the multivariate (HR 3.67; 95% CI 1.79-7.49; p < 0.001) analysis. Overall, our data suggest that the expression of DDB1, CUL4A and CRBN assessed by IHC predicts the clinical course of MM patients and identifies patients with a high probability of responding to IMiD-based therapy.

PIM Kinases Promote Survival and Immune Escape in Primary Mediastinal Large B-Cell Lymphoma through Modulation of JAK-STAT and NF-κB Activity.

Primary mediastinal large B-cell lymphoma (PMBL) cells depend on the constitutive activity of NF-κB and STAT transcription factors, which drive expression of multiple molecules essential for their survival. In a molecularly related B-cell malignant tumor (classic Hodgkin lymphoma), tumor Reed-Sternberg cells overexpress oncogenic (proviral integration site for Moloney murine leukemia virus (PIM) 1, 2, and 3 kinases in a NF-κB- and STAT-dependent manner and PIMs enhance survival and expression of immunomodulatory molecules. Given the multiple overlapping characteristics of Reed-Sternberg and PMBL cells, we hypothesized that PIM kinases may be overexpressed in PMBL and involved in PMBL pathogenesis. The expression of PIM kinases in PMBL diagnostic biopsy specimens was assessed and their role in survival and immune escape of the tumor cells was determined. PIMs were abundantly expressed in primary tumors and PMBL cell lines. Inhibition of PIM kinases was toxic to PMBL cells, attenuated protein translation, and down-regulated NF-κB- and STAT-dependent transcription of prosurvival factors BCL2A1, BCL2L1, and FCER2. Furthermore, PIM inhibition decreased expression of molecules engaged in shaping the immunosuppressive microenvironment, including programmed death ligand 1/2 and chemokine (C-C motif) ligand 17. Taken together, our data indicate that PIMs support PMBL cell survival and immune escape and identify PIMs as promising therapeutic targets for PMBL.

Predictive significance of selected gene mutations in relapsed and refractory chronic lymphocytic leukemia patients treated with ibrutinib.

BACKGROUND: Ibrutinib, an inhibitor of the Bruton's kinase (BTK), is characterized by high efficacy in the therapy of patients with relapsed and refractory chronic lymphocytic leukemia (RR-CLL). AIMS: To analyze the potential significance of the mutational status of selected 30 genes on the disease outcome in 45 patients with RR-CLL using custom-made gene panel and sequencing on Illumina MiSeq FGx platform. RESULTS: The highest rate of mutations was observed in TP53 (n = 18; 40.0%), NOTCH1 (n = 13; 28.8%), SF3B1 (n = 11; 24.4%), ATM (n = 7; 15.6%), MED12 (n = 6, 13.3%), CHD2 (n = 5; 11.1%), XPO1 (n = 5; 11.1%), NFKBIE (n = 5; 11.1%), BIRC3 (n = 4; 8.9%), SPEN (n = 4; 8.9%), POT1 (n = 4; 8.9%), EGR2 (n = 3; 6.7%), and RPS15 (n = 3; 6.7%). With a median observation time of 45.9 months, the median progression-free survival (PFS) and overall survival (OS) were not reached. The 36-month estimated rate of PFS and OS were 64% and 68.2%, respectively. The overall response rate was noted in 23 patients (51.1%), while twenty (44.4%) patients achieved stability. Progression was noted in 2 (4.5%) cases. Analyzed molecular factors had no impact on PFS and OS. CONCLUSION: Despite accumulation of several poor prognostic factors in our real-life cohort of heavily pretreated patients with CLL, ibrutinib treatment showed long-term clinical benefit.

SYK inhibition targets acute myeloid leukemia stem cells by blocking their oxidative metabolism.

Spleen tyrosine kinase (SYK) is an important oncogene and signaling mediator activated by cell surface receptors crucial for acute myeloid leukemia (AML) maintenance and progression. Genetic or pharmacologic inhibition of SYK in AML cells leads to increased differentiation, reduced proliferation, and cellular apoptosis. Herein, we addressed the consequences of SYK inhibition to leukemia stem-cell (LSC) function and assessed SYK-associated pathways in AML cell biology. Using gain-of-function MEK kinase mutant and constitutively active STAT5A, we demonstrate that R406, the active metabolite of a small-molecule SYK inhibitor fostamatinib, induces differentiation and blocks clonogenic potential of AML cells through the MEK/ERK1/2 pathway and STAT5A transcription factor, respectively. Pharmacological inhibition of SYK with R406 reduced LSC compartment defined as CD34+CD38-CD123+ and CD34+CD38-CD25+ in vitro, and decreased viability of LSCs identified by a low abundance of reactive oxygen species. Primary leukemic blasts treated ex vivo with R406 exhibited lower engraftment potential when xenotransplanted to immunodeficient NSG/J mice. Mechanistically, these effects are mediated by disturbed mitochondrial biogenesis and suppression of oxidative metabolism (OXPHOS) in LSCs. These mechanisms appear to be partially dependent on inhibition of STAT5 and its target gene MYC, a well-defined inducer of mitochondrial biogenesis. In addition, inhibition of SYK increases the sensitivity of LSCs to cytarabine (AraC), a standard of AML induction therapy. Taken together, our findings indicate that SYK fosters OXPHOS and participates in metabolic reprogramming of AML LSCs in a mechanism that at least partially involves STAT5, and that SYK inhibition targets LSCs in AML. Since active SYK is expressed in a majority of AML patients and confers inferior prognosis, the combination of SYK inhibitors with standard chemotherapeutics such as AraC constitutes a new therapeutic modality that should be evaluated in future clinical trials.

Rebelled epigenome: histone H3S10 phosphorylation and H3S10 kinases in cancer biology and therapy.

BACKGROUND: With the discovery that more than half of human cancers harbor mutations in chromatin proteins, deregulation of epigenetic mechanisms has been recognized a hallmark of malignant transformation. Post-translational modifications (PTMs) of histone proteins, as main components of epigenetic regulatory machinery, are also broadly accepted as therapeutic target. Current "epigenetic" therapies target predominantly writers, erasers and readers of histone acetylation and (to a lesser extent) methylation, leaving other types of PTMs largely unexplored. One of them is the phosphorylation of serine 10 on histone H3 (H3S10ph). MAIN BODY: H3S10ph is emerging as an important player in the initiation and propagation of cancer, as it facilitates cellular malignant transformation and participates in fundamental cellular functions. In normal cells this histone mark dictates the hierarchy of additional histone modifications involved in the formation of protein binding scaffolds, transcriptional regulation, blocking repressive epigenetic information and shielding gene regions from heterochromatin spreading. During cell division, this mark is essential for chromosome condensation and segregation. It is also involved in the function of specific DNA-RNA hybrids, called R-loops, which modulate transcription and facilitate chromosomal instability. Increase in H3S10ph is observed in numerous cancer types and its abundance has been associated with inferior prognosis. Many H3S10-kinases, including MSK1/2, PIM1, CDK8 and AURORA kinases, have been long considered targets in cancer therapy. However, since these proteins also participate in other critical processes, including signal transduction, apoptotic signaling, metabolic fitness and transcription, their chromatin functions are often neglected. CONCLUSIONS: H3S10ph and enzymes responsible for deposition of this histone modification are important for chromatin activity and oncogenesis. Epigenetic-drugs targeting this axis of modifications, potentially in combination with conventional or targeted therapy, provide a promising angle in search for knowledge-driven therapeutic strategies in oncology.

DEPTOR is a microRNA-155 target regulating migration and cytokine production in diffuse large B-cell lymphoma cells.

MicroRNA-155 (MiR-155) is involved in normal B-cell development and lymphomagenesis, affecting cell differentiation, motility, and intracellular signaling. In this study, we searched for new targets of MiR-155 potentially involved in deregulation of the B-cell receptor pathway (BCR) in diffuse large B-cell lymphoma (DLBCL). We report that MiR-155 represses DEPTOR (an mTOR phosphatase) and c-CBL (SYK ubiquitin E3 ligase) through direct 3'-untranslated region interactions. In primary DLBCLs, MiR-155 exhibits a reciprocal expression pattern with DEPTOR and c-CBL. Inhibition of MiR-155 decreased expression of NFκB target genes and sensitized DLBCL cells to ibrutinib, confirming the role of MiR-155 in the modulation of BCR signaling. As the function of DEPTOR in DLBCLs has never been addressed, we first evaluated its expression in a series of 76 newly diagnosed DLBCL patients. DEPTOR protein expression was markedly lower in more aggressive nongerminal center-like (non-GCB) DLBCLs than in GCB tumors. In cell line models, inhibition of DEPTOR expression favored the migration of DLBCL cells toward the CXCL12 gradient. Finally, loss or gain of DEPTOR modulated the expression of specific pro-inflammatory cytokines and chemokines. We thus identified DEPTOR as a new MiR-155 target that is differentially expressed between GCB- and non-GCB-type DLBCLs and modulates cell migration and cytokine expression in DLBCL cells.

BRAF inhibition curtails IFN-gamma-inducible PD-L1 expression and upregulates the immunoregulatory protein galectin-1 in melanoma cells.

Although melanoma is considered one of the most immunogenic malignancies, spontaneous T-cell responses to melanoma antigens are ineffective due to tumor cell-intrinsic or microenvironment-driven immune evasion mechanisms. For example, oncogenic BRAF V600E mutation in melanoma cells fosters tumor immune escape by modulating cell immunogenicity and microenvironment composition. BRAF inhibition has been shown to increase melanoma cell immunogenicity, but these effects are transient and long-term responses are uncommon. For these reasons, we aimed to further characterize the role of BRAF-V600E mutation in the modulation of PD-L1, a known immunoregulatory molecule, and galectin-1 (Gal-1), a potent immunoregulatory lectin involved in melanoma immune privilege. We report herein that vemurafenib downregulates IFN-γ-induced PD-L1 expression by interfering with STAT1 activity and by decreasing PD-L1 protein translation. Surprisingly, melanoma cells exposed to vemurafenib expressed higher levels of Gal-1. In coculture experiments, A375 melanoma cells pretreated with vemurafenib induced apoptosis of interacting Jurkat T cells, whereas genetic inhibition of Gal-1 in these cells restored the viability of cocultured T lymphocytes, indicating that Gal-1 contributes to tumor immune escape. Importantly, Gal-1 plasma concentration increased in patients progressing on BRAF/MEK inhibitor treatment, but remained stable in responding patients. Taken together, these results suggest a two-faceted nature of BRAF inhibition-associated immunomodulatory effects: an early immunostimulatory activity, mediated at least in part by decreased PD-L1 expression, and a delayed immunosuppressive effect associated with Gal-1 induction. Importantly, our observations suggest that Gal-1 might be utilized as a potential biomarker and a putative therapeutic target in melanoma patients.

Serine Biosynthesis Pathway Supports MYC-miR-494-EZH2 Feed-Forward Circuit Necessary to Maintain Metabolic and Epigenetic Reprogramming of Burkitt Lymphoma Cells.

Burkitt lymphoma (BL) is a rapidly growing tumor, characterized by high anabolic requirements. The MYC oncogene plays a central role in the pathogenesis of this malignancy, controlling genes involved in apoptosis, proliferation, and cellular metabolism. Serine biosynthesis pathway (SBP) couples glycolysis to folate and methionine cycles, supporting biosynthesis of certain amino acids, nucleotides, glutathione, and a methyl group donor, S-adenosylmethionine (SAM). We report that BLs overexpress SBP enzymes, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1). Both genes are controlled by the MYC-dependent ATF4 transcription factor. Genetic ablation of PHGDH/PSAT1 or chemical PHGDH inhibition with NCT-503 decreased BL cell lines proliferation and clonogenicity. NCT-503 reduced glutathione level, increased reactive oxygen species abundance, and induced apoptosis. Consistent with the role of SAM as a methyl donor, NCT-503 decreased DNA and histone methylation, and led to the re-expression of ID4, KLF4, CDKN2B and TXNIP tumor suppressors. High H3K27me3 level is known to repress the MYC negative regulator miR-494. NCT-503 decreased H3K27me3 abundance, increased the miR-494 level, and reduced the expression of MYC and MYC-dependent histone methyltransferase, EZH2. Surprisingly, chemical/genetic disruption of SBP did not delay BL and breast cancer xenografts growth, suggesting the existence of mechanisms compensating the PHGDH/PSAT1 absence in vivo.

CXCR4 upregulation is an indicator of sensitivity to B-cell receptor/PI3K blockade and a potential resistance mechanism in B-cell receptor-dependent diffuse large B-cell lymphomas.

B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell malignancies including diffuse large B-cell lymphoma (DLBCL). In in vitro and in vivo model systems, a subset of DLBCLs depend upon BCR survival signals and respond to proximal BCR/phosphoinositide 3 kinase (PI3K) blockade. However, single-agent BCR pathway inhibitors have had more limited activity in patients with DLBCL, underscoring the need for indicators of sensitivity to BCR blockade and insights into potential resistance mechanisms. Here, we report highly significant transcriptional upregulation of C-X-C chemokine receptor 4 (CXCR4) in BCR-dependent DLBCL cell lines and primary tumors following chemical spleen tyrosine kinase (SYK) inhibition, molecular SYK depletion or chemical PI3K blockade. SYK or PI3K inhibition also selectively upregulated cell surface CXCR4 protein expression in BCR-dependent DLBCLs. CXCR4 expression was directly modulated by fork-head box O1 via the PI3K/protein kinase B/forkhead box O1 signaling axis. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased stromal cell-derived factor-1α (SDF-1α) induced chemotaxis, consistent with the role of CXCR4 signaling in B-cell migration. Select PI3K isoform inhibitors also augmented SDF-1α induced chemotaxis. These data define CXCR4 upregulation as an indicator of sensitivity to BCR/PI3K blockade and identify CXCR4 signaling as a potential resistance mechanism in BCR-dependent DLBCLs.

Targeting the thioredoxin system as a novel strategy against B-cell acute lymphoblastic leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogeneous blood cancer characterized by abnormal expansion of immature B cells. Although intensive chemotherapy provides high cure rates in a majority of patients, subtypes harboring certain genetic lesions, such as MLL rearrangements or BCR-ABL1 fusion, remain clinically challenging, necessitating a search for other therapeutic approaches. Herein, we aimed to validate antioxidant enzymes of the thioredoxin system as potential therapeutic targets in BCP-ALL. We observed oxidative stress along with aberrant expression of the enzymes associated with the activity of thioredoxin antioxidant system in BCP-ALL cells. Moreover, we found that auranofin and adenanthin, inhibitors of the thioredoxin system antioxidant enzymes, effectively kill BCP-ALL cell lines and pediatric and adult BCP-ALL primary cells, including primary cells cocultured with bone marrow-derived stem cells. Furthermore, auranofin delayed the progression of leukemia in MLL-rearranged patient-derived xenograft model and prolonged the survival of leukemic NSG mice. Our results unveil the thioredoxin system as a novel target for BCP-ALL therapy, and indicate that further studies assessing the anticancer efficacy of combinations of thioredoxin system inhibitors with conventional anti-BCP-ALL drugs should be continued.