mTOR hyperactivity and RICTOR amplification as targets for personalized treatments in malignancies.

The increasing knowledge of molecular alterations in malignancies, including mutations and regulatory failures in the mTOR (mechanistic target of rapamycin) signaling pathway, highlights the importance of mTOR hyperactivity as a validated target in common and rare malignancies. This review summarises recent findings on the characterization and prognostic role of mTOR kinase complexes (mTORC1 and mTORC2) activity regarding differences in their function, structure, regulatory mechanisms, and inhibitor sensitivity. We have recently identified new tumor types with RICTOR (rapamycin-insensitive companion of mTOR) amplification and associated mTORC2 hyperactivity as useful potential targets for developing targeted therapies in lung cancer and other newly described malignancies. The activity of mTOR complexes is recommended to be assessed and considered in cancers before mTOR inhibitor therapy, as current first-generation mTOR inhibitors (rapamycin and analogs) can be ineffective in the presence of mTORC2 hyperactivity. We have introduced and proposed a marker panel to determine tissue characteristics of mTOR activity in biopsy specimens, patient materials, and cell lines. Ongoing phase trials of new inhibitors and combination therapies are promising in advanced-stage patients selected by genetic alterations, molecular markers, and/or protein expression changes in the mTOR signaling pathway. Hopefully, the summarized results, our findings, and the suggested characterization of mTOR activity will support therapeutic decisions.

Remodeling of the ribosomal quality control and integrated stress response by viral ubiquitin deconjugases.

The strategies adopted by viruses to reprogram the translation and protein quality control machinery and promote infection are poorly understood. Here, we report that the viral ubiquitin deconjugase (vDUB)-encoded in the large tegument protein of Epstein-Barr virus (EBV BPLF1)-regulates the ribosomal quality control (RQC) and integrated stress responses (ISR). The vDUB participates in protein complexes that include the RQC ubiquitin ligases ZNF598 and LTN1. Upon ribosomal stalling, the vDUB counteracts the ubiquitination of the 40 S particle and inhibits the degradation of translation-stalled polypeptides by the proteasome. Impairment of the RQC correlates with the readthrough of stall-inducing mRNAs and with activation of a GCN2-dependent ISR that redirects translation towards upstream open reading frames (uORFs)- and internal ribosome entry sites (IRES)-containing transcripts. Physiological levels of active BPLF1 promote the translation of the EBV Nuclear Antigen (EBNA)1 mRNA in productively infected cells and enhance the release of progeny virus, pointing to a pivotal role of the vDUB in the translation reprogramming that enables efficient virus production.

Novel RICTOR amplification harbouring entities: FISH validation of RICTOR amplification in tumour tissue after next-generation sequencing.

Alterations in mTOR signalling molecules, including RICTOR amplification, have been previously described in many cancers, particularly associated with poor prognosis. In this study, RICTOR copy number variation (CNV) results of diagnostic next-generation sequencing (NGS) were analysed in 420 various human malignant tissues. RICTOR amplification was tested by Droplet Digital PCR (ddPCR) and validated using the "gold standard" fluorescence in situ hybridisation (FISH). Additionally, the consequences of Rictor protein expression were also studied by immunohistochemistry. RICTOR amplification was presumed in 37 cases with CNV ≥ 3 by NGS, among these, 16 cases (16/420; 3.8%) could be validated by FISH, however, ddPCR confirmed only 11 RICTOR-amplified cases with lower sensitivity. Based on these, neither NGS nor ddPCR could replace traditional FISH in proof of RICTOR amplification. However, NGS could be beneficial to highlight potential RICTOR-amplified cases. The obtained results of the 14 different tumour types with FISH-validated RICTOR amplification demonstrate the importance of RICTOR amplification in a broad spectrum of tumours. The newly described RICTOR-amplified entities could initiate further collaborative studies with larger cohorts to analyse the prevalence of RICTOR amplification in rare diseases. Finally, our and further work could help to improve and expand future therapeutic opportunities for mTOR-targeted therapies.

Self-affirmation increases reemployment success for the unemployed.

Losing a job is one of life's most stressful events. Furthermore, maladaptive reactions to unemployment can trap people in a vicious cycle that derails their reemployment efforts. The current research tested whether a brief values-based self-affirmation intervention increases the odds of reemployment after a job loss and during unemployment, which presumably breaks this vicious cycle. Two field experiments, including one with a governmental employment agency, found that a 15-min self-affirmation exercise-i.e., reflecting on one's most important values-increased key employment-related outcomes after 4 wk, including the probability and speed of reemployment and the number of job offers. Because the ordeal of job loss and the probability of reemployment may be particularly challenging for individuals above the age of 50 y, we also explored whether the intervention was equally effective for those above and below 50 y of age. Demonstrating the generality of this effect, the efficacy of the intervention did not differ between individuals below and above the age of 50, and it was also effective for both recently unemployed and chronically unemployed individuals. Because self-affirmations have more typically been tested in educational contexts, the current research demonstrates the wide-ranging value of this intervention. By diminishing the vicious cycle of unemployment, the present studies show how a simple self-affirmation intervention can help individuals succeed in the labor market.

Droplet Digital PCR Is a Novel Screening Method Identifying Potential Cardiac G-Protein-Coupled Receptors as Candidate Pharmacological Targets in a Rat Model of Pressure-Overload-Induced Cardiac Dysfunction.

The identification of novel drug targets is needed to improve the outcomes of heart failure (HF). G-protein-coupled receptors (GPCRs) represent the largest family of targets for already approved drugs, thus providing an opportunity for drug repurposing. Here, we aimed (i) to investigate the differential expressions of 288 cardiac GPCRs via droplet digital PCR (ddPCR) and bulk RNA sequencing (RNAseq) in a rat model of left ventricular pressure-overload; (ii) to compare RNAseq findings with those of ddPCR; and (iii) to screen and test for novel, translatable GPCR drug targets in HF. Male Wistar rats subjected to transverse aortic constriction (TAC, n = 5) showed significant systolic dysfunction vs. sham operated animals (SHAM, n = 5) via echocardiography. In TAC vs. SHAM hearts, RNAseq identified 69, and ddPCR identified 27 significantly differentially expressed GPCR mRNAs, 8 of which were identified using both methods, thus showing a correlation between the two methods. Of these, Prostaglandin-F2α-receptor (Ptgfr) was further investigated and localized on cardiomyocytes and fibroblasts in murine hearts via RNA-Scope. Antagonizing Ptgfr via AL-8810 reverted angiotensin-II-induced cardiomyocyte hypertrophy in vitro. In conclusion, using ddPCR as a novel screening method, we were able to identify GPCR targets in HF. We also show that the antagonism of Ptgfr could be a novel target in HF by alleviating cardiomyocyte hypertrophy.

[he importance of mTOR hyperactivity and RICTOR amplification, and the associated targeted therapy possibilities in malignant tumours]. / Az mTOR-hiperaktivitás és a RICTORamplifikáció jelentosége, és az ezzel összefüggo célzott terápiás lehetoségek rosszindulatú daganatokban.

Failures of anti-tumour therapies and drug resistance initiate difficulties in cancer treatments often caused by alterations in signalling network activity, including PI3K/Akt/mTOR hyperactivity due to oncogenic mutations. In this review, we summarise the relevance of mTOR (mechanistic target of rapamycin) dysregulation identified decades ago, which is now known to be characteristic of many tumours. In this context, we present differences in activity, function and testability of mTOR kinase complexes (mTORC1 and mTORC2) differing in structure, regulatory mechanisms and inhibitor sensitivity. We highlight that genetic alterations, including RICTOR amplification and associated mTOR hyperactivity, are relevant in targeted therapy development. It is recommended to investigate mTOR profile activity in patients for whom mTOR inhibitor therapies are considered since the current first-generation mTOR inhibitors (rapamycin and analogues) may be ineffective in case of mTORC2 hyperactivity. Ongoing phase trials of new inhibitors and combination therapies are promising in advanced stage patients selected by molecular markers.

Chlorotoxin binds to both matrix metalloproteinase 2 and neuropilin 1.

Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated KD values of 0.5 to 0.7 µM. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries.

Autopsy Study Defines Composition and Dynamics of the HIV-1 Reservoir after Allogeneic Hematopoietic Stem Cell Transplantation with CCR5Δ32/Δ32 Donor Cells.

Allo-HSCT with CCR5Δ32/Δ32 donor cells is the only curative HIV-1 intervention. We investigated the impact of allo-HSCT on the viral reservoir in PBMCs and post-mortem tissue in two patients. IciS-05 and IciS-11 both received a CCR5Δ32/Δ32 allo-HSCT. Before allo-HSCT, ultrasensitive HIV-1 RNA quantification; HIV-1-DNA quantification; co-receptor tropism analysis; deep-sequencing and viral characterization in PBMCs and bone marrow; and post-allo-HSCT, ultrasensitive RNA and HIV-1-DNA quantification were performed. Proviral quantification, deep sequencing, and viral characterization were done in post-mortem tissue samples. Both patients harbored subtype B CCR5-tropic HIV-1 as determined genotypically and functionally by virus culture. Pre-allo-HSCT, HIV-1-DNA could be detected in both patients in bone marrow, PBMCs, and T-cell subsets. Chimerism correlated with detectable HIV-1-DNA LTR copies in cells and tissues. Post-mortem analysis of IciS-05 revealed proviral DNA in all tissue biopsies, but not in PBMCs. In patient IciS-11, who was transplanted twice, no HIV-1-DNA could be detected in PBMCs at the time of death, whereas HIV-1-DNA was detectable in the lymph node. In conclusion, shortly after CCR5Δ32/Δ32, allo-HSCT HIV-1-DNA became undetectable in PBMCs. However, HIV-1-DNA variants identical to those present before transplantation persisted in post-mortem-obtained tissues, indicating that these tissues play an important role as viral reservoirs.

IFNγ directly counteracts imatinib-induced apoptosis of primary human CD34+ CML stem/progenitor cells potentially through the upregulation of multiple key survival factors.

Tyrosine kinase inhibitors (TKIs) have dramatically improved the survival in chronic myeloid leukemia (CML), but residual disease typically persists even after prolonged treatment. Several lines of evidence suggest that TKIs administered to CML patients upregulate interferon γ (IFNγ) production, which may counteract the anti-tumorigenic effects of the therapy. We now show that activated T cell-conditioned medium (TCM) enhanced proliferation and counteracted imatinib-induced apoptosis of CML cells, and addition of a neutralizing anti-IFNγ antibody at least partially inhibited the anti-apoptotic effect. Likewise, recombinant IFNγ also reduced imatinib-induced apoptosis of CML cells. This anti-apoptotic effect of IFNγ was independent of alternative IFNγ signaling pathways, but could be notably diminished by STAT1-knockdown. Furthermore, IFNγ upregulated the expression of several anti-apoptotic proteins, including MCL1, PARP9, and PARP14, both in untreated and imatinib-treated primary human CD34+ CML stem/progenitor cells. Our results suggest that activated T cells in imatinib-treated CML patients can directly rescue CML cells from imatinib-induced apoptosis at least partially through the secretion of IFNγ, which exerts a rapid, STAT1-dependent anti-apoptotic effect potentially through the simultaneous upregulation of several key hematopoietic survival factors. These mechanisms may have a major clinical impact, when targeting residual leukemic stem/progenitor cells in CML.

The Use of a Staggered Herringbone Micromixer for the Preparation of Rigid Liposomal Formulations Allows Efficient Encapsulation of Antigen and Adjuvant.

Anionic liposomal formulations have previously shown to have intrinsic tolerogenic capacity and these properties have been related to the rigidity of the particles. The combination of highly rigid anionic liposomes to deliver tolerogenic adjuvants and antigen peptides has potential applications for the treatment of autoimmune and inflammatory diseases. However, the preparation of these highly rigid anionic liposomes using traditional methods such as lipid film hydration presents problems in terms of scalability and loading efficiency of some costly tolerogenic adjuvants like 1-α,25-dihydroxyvitaminD3. Here we propose the use of an off-the-shelf staggered herringbone micromixer for the preparation of these formulations and performed a systematic study on the effect of temperature and flow conditions on the size and polydispersity index of the formulations. Furthermore, we show that the system allows for the encapsulation of a wide variety of peptides and significantly higher loading efficiency of 1-α,25-dihydroxyvitaminD3 compared to the traditional lipid film hydration method, without compromising their non-inflammatory interaction with dendritic cells. Therefore, the microfluidics method presented here is a valuable tool for the preparation of highly rigid tolerogenic liposomes in a fast, size-tuneable and scalable manner.

Novel loss-of-function variant in DENND5A impedes melanosomal cargo transport and predisposes to familial cutaneous melanoma.

PURPOSE: More than half of the familial cutaneous melanomas have unknown genetic predisposition. This study aims at characterizing a novel melanoma susceptibility gene. METHODS: We performed exome and targeted sequencing in melanoma-prone families without any known melanoma susceptibility genes. We analyzed the expression of candidate gene DENND5A in melanoma samples in relation to pigmentation and UV signature. Functional studies were carried out using microscopic approaches and zebrafish model. RESULTS: We identified a novel DENND5A truncating variant that segregated with melanoma in a Swedish family and 2 additional rare DENND5A variants, 1 of which segregated with the disease in an American family. We found that DENND5A is significantly enriched in pigmented melanoma tissue. Our functional studies show that loss of DENND5A function leads to decrease in melanin content in vitro and pigmentation defects in vivo. Mechanistically, harboring the truncating variant or being suppressed leads to DENND5A losing its interaction with SNX1 and its ability to transport the SNX1-associated vesicles from melanosomes. Consequently, untethered SNX1-premelanosome protein and redundant tyrosinase are redirected to lysosomal degradation by default, causing decrease in melanin content. CONCLUSION: Our findings provide evidence of a physiological role of DENND5A in the skin context and link its variants to melanoma susceptibility.

Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells.

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.

Efficacy of carboplatin chemotherapy in a metastatic, castration-resistant BRCA2 mutation positive prostate cancer patient / A karboplatin-kemoterápia hatékonysága egy áttétes, kasztrációrezisztens, BRCA2-mutáció-pozitív prosztatarákos betegben.

Összefoglaló. A sérült BRCA1/2 gént hordozó prosztatadaganatok klinikai szempontból elkülönülo, agresszív altípust képviselnek. Ugyanakkor a BRCA1/2 gén sérülése a DNS-támadáspontú kemoterápiákkal szemben érzékennyé teszi a daganatot, ami terápiás szempontból kihasználható. A platinaalapú kemoterápia hatékonysága prosztatarákban klinikai vizsgálatokkal nincs alátámasztva, ezért annak alkalmazására igen ritkán kerül sor. Közleményünkben egy elorehaladott stádiumú, agresszív prosztata adenocarcinomával diagnosztizált beteg esetét mutatjuk be, akinél a BRCA2-gén patogén mutációját találtuk, és akinél az elozoleg alkalmazott androgénmegvonásos, valamint docetaxelkezelések sikertelensége miatt karboplatinkezelést alkalmaztunk - ez a beteg állapotának, valamint radiológiai és biokémiai paramétereinek látványos javulásához vezetett. Ez az eset rámutat a DNS-hiba-javító mechanizmusban szerepet játszó gének terápiás szempontból történo felhasználásának potenciális elonyeire prosztatarákban. Orv Hetil. 2021; 162(25): 1004-1008. Summary. BRCA1/2 deficient prostate cancers represent a clinically distinct aggressive subtype. However, the presence of BRCA1/2 alterations enhance the sensitivity to platinum-based chemotherapies. The efficacy of platinum-based chemotherapies in prostate cancer has not been proven in prospective clinical studies and therefore these treatments are rarely used in prostate adenocarcinomas. Here we present a case of BRCA2 mutant prostate cancer, which was diagnosed at a metastatic stage and showed no or only little response to androgen deprivation and docetaxel therapies. Therefore, we started carboplatin chemotherapy which resulted in an exceptional response regarding biochemical, radiographic parameters accompanied by significant improvement of patients' physical condition. This case underlines the potential therapeutic benefits of testing for genes involved in the DNA repair mechanism. Orv Hetil. 2021; 162(25): 1004-1008.

Therapeutic Liposomal Vaccines for Dendritic Cell Activation or Tolerance.

Dendritic cells (DCs) are paramount in initiating and guiding immunity towards a state of activation or tolerance. This bidirectional capacity of DCs sets them at the center stage for treatment of cancer and autoimmune or allergic conditions. Accordingly, many clinical studies use ex vivo DC vaccination as a strategy to boost anti-tumor immunity or to suppress immunity by including vitamin D3, NF-κB inhibitors or retinoic acid to create tolerogenic DCs. As harvesting DCs from patients and differentiating these cells in vitro is a costly and cumbersome process, in vivo targeting of DCs has huge potential as nanoparticulate platforms equipped with activating or tolerogenic adjuvants can modulate DCs in their natural environment. There is a rapid expansion of the choices of nanoparticles and activation- or tolerance-promoting adjuvants for a therapeutic vaccine platform. In this review we highlight the most recent nanomedical approaches aimed at inducing immune activation or tolerance via targeting DCs, together with novel fundamental insights into the mechanisms inherent to fostering anti-tumor or tolerogenic immunity.

Immune cells lacking Y chromosome show dysregulation of autosomal gene expression.

Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer's disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a "genetic wasteland", and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.

Identification of a novel resistance mechanism in venetoclax treatment and its prediction in chronic lymphocytic leukemia.

BACKGROUND: The Bcl-2 inhibitor venetoclax has been recently introduced into the treatment of chronic lymphocytic leukemia. Venetoclax is a highly effective drug, however acquired resistance may make long-term treatment challenging. In our study, we present potential novel resistance mechanisms and prognostic markers that are potentially able to predict the early appearance of the resistance. MATERIAL AND METHODS: Repeated complete blood counts, flow cytometric measurements, and physical examinations were performed during the patient follow-up. Clinical and laboratory parameters showed that the patient developed clinical resistance to venetoclax on day 450 of therapy. Resistance mutation analysis (D103Y) and apoptosis arrays from samples at the time of resistance were done. RESULTS: We were able to identify the resistance mutations just a very low variant allele frequency level from the resistant samples. Furthermore we detected increased Bcl-2 expression in peripheral blood (PB), and XIAP overexpression in bone marrow (BM) that could lead to venetoclax resistance. We examined the immunophenotype of CLL cells and recognized that while the expression of CD86 did not change until day 270 of the treatment, since then its expression steadily increased. Moreover, we compared the expression of CD86 in the resistant PB and BM samples and did not find a notable difference between the compartments. CONCLUSION: Our results imply that CLL cells may try to avoid the apoptotic effect of venetoclax through increased CD86 expression by activating antiapoptotic mechanisms. Confirmatory experiments are still required to unequivocally prove that CD86 is a prognostic marker, however, its predictive property during the venetoclax treatment is promising.

Quantitative Analysis and Monitoring of EZH2 Mutations Using Liquid Biopsy in Follicular Lymphoma.

Recent advances in molecular technologies enable sensitive and quantitative assessment of circulating tumor DNA, offering a noninvasive disease monitoring tool for patients with malignant disorders. Here, we demonstrated on four follicular lymphoma cases that circulating tumor DNA based EZH2 mutation analysis performed by a highly sensitive droplet digital PCR method may be a valuable treatment monitoring approach in EZH2 mutant follicular lymphoma. EZH2 variant allele frequencies changed in parallel with the volume of metabolically active tumor sites observed on 18F-fluorodeoxyglucose positron emission tomography combined with computer tomography (PET-CT) scans. Variant allele frequencies of EZH2 mutations decreased or were eliminated rapidly upon successful treatment, with treatment failure being associated with elevated EZH2 variant allele frequencies. We also demonstrated spatial heterogeneity in a patient with two different EZH2 mutations in distinct anatomical sites, with both mutations simultaneously detected in the liquid biopsy specimen. In summary, circulating tumor DNA based EZH2 mutation analysis offers a rapid, real-time, radiation-free monitoring tool for sensitive detection of EZH2 mutations deriving from different anatomical sites in follicular lymphoma patients receiving immunochemotherapy.

Characterization of mTOR Activity and Metabolic Profile in Pediatric Rhabdomyosarcoma.

mTOR activation has been observed in rhabdomyosarcoma (RMS); however, mTOR complex (mTORC) 1 inhibition has had limited success thus far. mTOR activation alters the metabolic pathways, which is linked to survival and metastasis. These pathways have not been thoroughly analyzed in RMSs. We performed immunohistochemistry on 65 samples to analyze the expression of mTOR complexes (pmTOR, pS6, Rictor), and several metabolic enzymes (phosphofructokinase, lactate dehydrogenase-A, ß-F1-ATPase, glucose-6-phosphate dehydrogenase, glutaminase). RICTOR amplification, as a potential mechanism of Rictor overexpression, was analyzed by FISH and digital droplet PCR. In total, 64% of the studied primary samples showed mTOR activity with an mTORC2 dominance (82%). Chemotherapy did not cause any relevant change in mTOR activity. Elevated mTOR activity was associated with a worse prognosis in relapsed cases. RICTOR amplification was not confirmed in any of the cases. Our findings suggest the importance of the Warburg effect and the pentose-phosphate pathway beside a glutamine demand in RMS cells. The expression pattern of the studied mTOR markers can explain the inefficacy of mTORC1 inhibitor therapy. Therefore, we suggest performing a detailed investigation of the mTOR profile before administering mTORC1 inhibitor therapy. Furthermore, our findings highlight that targeting the metabolic plasticity could be an alternative therapeutic approach.

Various Tastes of Sugar: The Potential of Glycosylation in Targeting and Modulating Human Immunity via C-Type Lectin Receptors.

C-type lectin receptors (CLRs) are important in several immune regulatory processes. These receptors recognize glycans expressed by host cells or by pathogens. Whereas pathogens are recognized through their glycans, which leads to protective immunity, aberrant cellular glycans are now increasingly recognized as disease-driving factors in cancer, auto-immunity, and allergy. The vast variety of glycan structures translates into a wide spectrum of effects on the immune system ranging from immune suppression to hyper-inflammatory responses. CLRs have distinct expression patterns on antigen presenting cells (APCs) controlling their role in immunity. CLRs can also be exploited to selectively target specific APCs, modulate immune responses and enhance antigen presentation. Here we will discuss the role of glycans and their receptors in immunity as well as potential strategies for immune modulation. A special focus will be given to different dendritic cell subsets as these APCs are crucial orchestrators of immune responses in infections, cancer, auto-immunity and allergies. Furthermore, we will highlight the potential use of nanoscale lipid bi-layer structures (liposomes) in targeted immunotherapy.

Dissection of subclonal evolution by temporal mutation profiling in chronic lymphocytic leukemia patients treated with ibrutinib.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is inducing durable responses in chronic lymphocytic leukemia (CLL) patients with refractory/relapsed disease or with TP53 defect, with BTK and phospholipase C gamma 2 (PLCG2) mutations representing the predominant mechanisms conferring secondary ibrutinib resistance. To understand the landscape of genomic changes and the dynamics of subclonal architecture associated with ibrutinib treatment, an ultra-deep next-generation sequencing analysis of 30 recurrently mutated genes was performed on sequential samples of 20 patients, collected before and during single-agent ibrutinib treatment. Mutations in the SF3B1, MGAand BIRC3 genes were enriched during ibrutinib treatment, while aberrations in the BTK, PLCG2, RIPK1, NFKBIE and XPO1 genes were exclusively detected in posttreatment samples. Besides the canonical mutations, four novel BTK mutations and three previously unreported PLCG2 variants were identified. BTK and PLCG2 mutations were backtracked in five patients using digital droplet PCR and were detectable on average 10.5 months before clinical relapse. With a median follow-up time of 36.5 months, 7/9 patients harboring BTK mutations showed disease progression based on clinical and/or laboratory features. In conclusion, subclonal heterogeneity, dynamic clonal selection and various patterns of clonal variegation were identified with novel resistance-associated BTK mutations in individual patients treated with ibrutinib.