DNMT and HDAC inhibition induces immunogenic neoantigens from human endogenous retroviral element-derived transcripts.

Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies

CK2b regulates thrombopoiesis and Ca21-Triggered platelet activation in arterial thrombosis

© 2017 by The American Society of Hematology. Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-) physiology has remained elusive. The present study explored the impact of the CK2 regulatory b-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β (ck2β-/-) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2β-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2β-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-Triphosphate receptor-dependent intracellular calcium (Ca21) release. Presumably due to a blunted increase in the concentration of cytosolic Ca21, activation-dependent increases of a and dense-granule secretion and integrin aIIbb3 activation, and aggregation were abrogated in ck2β-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo wassignificantly blunted in ck2β-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2b-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2βfl/fl mice. The present observations reveal CK2b as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo

Analyse des Transkriptionsfaktors NFAT2 in der Pathogenese der CLL

Die Chronische Lymphatische Leukämie (CLL) ist mit über 4000 Neuerkrankungen pro Jahr die häufigste hämatologische Erkrankung in Deutschland. In den allermeisten Fällen ist die CLL nicht vollständig heilbar. Durch konventionelle Chemotherapeutika und Antikörper-basierte Immuntherapien gelingt es zwar oft die Patienten möglichst lange in Remission zu halten, trotzdem steigt das Bedürfnis nach neuen Therapieansätzen stetig. In dieser Arbeit wurde zum einen der Einfluss des Transkriptionsfaktors NFAT2 auf die Entwicklung physiologischer B-Zellen untersucht und zum anderen seine Rolle in der Pathogenese der CLL detailliert charakterisiert. Diese Analysen wurden in einem konditionellen NFAT2-Knockout Mausmodell in Kombination mit dem transgenen Eµ-TCL1-Modell der CLL durchgeführt. Während im physiologischen B-Zell-Modell der Verlust von NFAT2 zu einer Hemmung der B-Zell-Entwicklung und einer Reduktion der B1 B-Zell-Subpopulation führt, ergaben die Analysen im CLL-Modell ein differentes Ergebnis. Hier führte der Verlust von NFAT2 zu einer Akzeleration der CLL-Erkrankung. Dies führte zu einem drastisch reduzierten Überleben dieser Tiere und einer verstärkten Akkumulation der CLL-Zellen in der Peripherie. Als Ursache konnte eine gesteigerte Proliferation in der Leukämie-Kohorte mit NFAT2-Knockout beobachtet werden. Da die Proliferation der B-Zellen stark B-Zell-Rezeptor-abhängig reguliert wird, wurde die Funktionalität des BCRs untersucht. In der normalen TCL1-Kohorte zeigte sich ein anerger Phänotyp, wodurch die CLL-Zellen unfähig waren, auf αIgM-Stimulation durch Ca2+-Mobilisation zu reagieren. Ferner konnte eine konstitutive Aktivierung des AKT- und ERK-Signalwegs und eine starke Expression der in dieser Arbeit vorgeschlagenen Anergie-Gene Cbl, Grail, Pacsin1 und Lck beobachtet werden. Diese Beobachtungen entsprechen den bekannten Daten aus einer Subpopulation der CLL-Patienten, die einen sehr milden Verlauf der Erkrankung mit guter Langzeitprognose zeigen. Der Verlust von NFAT2 in den CLL-Zellen führte hingegen zu einer Aufhebung der Anergie und damit zu einem hochresponsiven Phänotyp. Die molekulare Analyse der proximalen BCR-Signalkaskaden zeigten im Vergleich zu der meist konstitutiven Aktivierung in der TCL1-Kohorte eine induzierte Aktivierung der Kinasen SYK, LYN, ERK und AKT. Interessanterweise zeigte die Tyrosinkinase LCK als einzige eine Aktivierung nach BCR-Stimulation in der anergen TCL1-Kohorte. Aufgrund dieser Tatsache werden NFAT2 und die LCK-Kinase, welche vermutlich auch durch NFAT2 aktiviert wird, als maßgebliche Regulatoren der Anergie bei der CLL postuliert

Märklin et al_RMA File Microarray

Analysis of gene expressions in which were analyzed with the Affymetrix MoGene-1.0-ST-v1 Gene Chip. Starting material were FACS-sorted CD19⁺CD5⁺ CLL cells from TCL1 NFAT2^+/+and TCL1 NFAT2^-/- mice (n=3 per group). Normalized and log₂-transformed signal intensities of each gene transcript from 6 different mice were shown

Märklin et al_Genelists_Microarray.xlsx

Statistical analysis of all transcripts which were analyzed with the Affymetrix MoGene-1.0-ST-v1 Gene Chip. Starting material were FACS-sorted CD19⁺CD5⁺ CLL cells from TCL1 NFAT2^+/+and TCL1 NFAT2^-/- mice (n=3 per group)

Asciminib Maintains Antibody-Dependent Cellular Cytotoxicity against Leukemic Blasts

B cell acute lymphoblastic leukemia (B-ALL) is characterized by an accumulation of malignant precursor cells. Treatment consists of multiagent chemotherapy followed by allogeneic stem cell transplantation in high-risk patients. In addition, patients bearing the BCR-ABL1 fusion gene receive concomitant tyrosine kinase inhibitor (TKI) therapy. On the other hand, monoclonal antibody therapy is increasingly used in both clinical trials and real-world settings. The introduction of rituximab has improved the outcomes in CD20 positive cases. Other monoclonal antibodies, such as tafasitamab (anti-CD19), obinutuzumab (anti-CD20) and epratuzumab (anti-CD22) have been tested in trials (NCT05366218, NCT04920968, NCT00098839). The efficacy of monoclonal antibodies is based, at least in part, on their ability to induce antibody-dependent cellular cytotoxicity (ADCC). Combination treatments, e.g., chemotherapy and TKI, should therefore be screened for potential interference with ADCC. Here, we report on in vitro data using BCR-ABL1 positive and negative B-ALL cell lines treated with rituximab and TKI. NK cell activation, proliferation, degranulation, cytokine release and tumor cell lysis were analyzed. In contrast to ATP site inhibitors such as dasatinib and ponatinib, the novel first-in-class selective allosteric ABL myristoyl pocket (STAMP) inhibitor asciminib did not significantly impact ADCC in our settings. Our results suggest that asciminib should be considered in clinical trials

Expression of the immune checkpoint modulator OX40 indicates poor survival in acute myeloid leukemia

Despite therapeutic advances, mortality of Acute Myeloid Leukemia (AML) is still high. Currently, the determination of prognosis which guides treatment decisions mainly relies on genetic markers. Besides molecular mechanisms, the ability of malignant cells to evade immune surveillance influences the disease outcome and, among others, the expression of checkpoints modulators contributes to this. In AML, functional expression of the checkpoint molecule OX40 was reported, but the prognostic relevance of OX40 and its ligand OX40L axis has so far not been investigated. Here we described expression and prognostic relevance of the checkpoint modulators OX40 and OX40L, analyzed on primary AML cells obtained from 92 therapy naïve patients. Substantial expression of OX40 and OX40L on AML blasts was detected in 29% and 32% of the investigated subjects, respectively, without correlation between the expression of the receptor and its ligand. Whereas OX40L expression was not associated with different survival, patients with high expression levels of the receptor (OX40(high)) on AML blasts survived significantly shorter than OX40(low) patients (p = 0.009, HR 0.46, 95% CI 0.24–0.86), which identifies OX40 as novel prognostic marker and a potential therapeutic target in AML patients