Publications by authors named "Irmela Jeremias"

56 Publications

Loss-of-function mutations in the histone methyltransferase EZH2 promote chemotherapy resistance in AML.

Sci Rep 2021 Mar 12;11(1):5838. Epub 2021 Mar 12.

Department of Medicine III, University Hospital, LMU Munich, Munich, Germany.

Chemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine. We identify seven distinct EZH2 mutations leading to loss of H3K27 trimethylation via multiple mechanisms. Analysis of matched diagnosis and relapse samples reveal a heterogenous regulation of EZH2 and a loss of EZH2 in 50% of patients. We confirm that loss of EZH2 induces resistance against cytarabine in the cell lines HEK293T and K562 as well as in a patient-derived xenograft model. Proteomics and transcriptomics analysis reveal that resistance is conferred by upregulation of multiple direct and indirect EZH2 target genes that are involved in apoptosis evasion, augmentation of proliferation and alteration of transmembrane transporter function. Our data indicate that loss of EZH2 results in upregulation of its target genes, providing the cell with a selective growth advantage, which mediates chemotherapy resistance.
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http://dx.doi.org/10.1038/s41598-021-84708-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955088PMC
March 2021

A JAK/STAT-MEDIATED INFLAMMATORY SIGNALING CASCADE DRIVES ONCOGENESIS IN AF10-REARRANGED AML.

Blood 2021 Mar 9. Epub 2021 Mar 9.

Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, United States.

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of AML driven by the most common AF10 fusion proteins, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a detailed map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent anti-oncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.
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http://dx.doi.org/10.1182/blood.2020009023DOI Listing
March 2021

Enforced sialyl-Lewis-X (sLeX) display in E-selectin ligands by exofucosylation is dispensable for CD19-CAR T-cell activity and bone marrow homing.

Clin Transl Med 2021 Feb;11(2):e280

Department of Biomedicine, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Barcelona, Spain.

CD19-directed chimeric antigen receptors (CAR) T cells induce impressive rates of complete response in advanced B-cell malignancies, specially in B-cell acute lymphoblastic leukemia (B-ALL). However, CAR T-cell-treated patients eventually progress due to poor CAR T-cell persistence and/or disease relapse. The bone marrow (BM) is the primary location for acute leukemia. The rapid/efficient colonization of the BM by systemically infused CD19-CAR T cells might enhance CAR T-cell activity and persistence, thus, offering clinical benefits. Circulating cells traffic to BM upon binding of tetrasaccharide sialyl-Lewis X (sLeX)-decorated E-selectin ligands (sialofucosylated) to the E-selectin receptor expressed in the vascular endothelium. sLeX-installation in E-selectin ligands is achieved through an ex vivo fucosylation reaction. Here, we sought to characterize the basal and cell-autonomous display of sLeX in CAR T-cells activated using different cytokines, and to assess whether exofucosylation of E-selectin ligands improves CD19-CAR T-cell activity and BM homing. We report that cell-autonomous sialofucosylation (sLeX display) steadily increases in culture- and in vivo-expanded CAR T cells, and that, the cytokines used during T-cell activation influence both the degree of such endogenous sialofucosylation and the CD19-CAR T-cell efficacy and persistence in vivo. However, glycoengineered enforced sialofucosylation of E-selectin ligands was dispensable for CD19-CAR T-cell activity and BM homing in multiple xenograft models regardless the cytokines employed for T-cell expansion, thus, representing a dispensable strategy for CD19-CAR T-cell therapy.
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http://dx.doi.org/10.1002/ctm2.280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901721PMC
February 2021

CD79a promotes CNS-infiltration and leukemia engraftment in pediatric B-cell precursor acute lymphoblastic leukemia.

Commun Biol 2021 Jan 15;4(1):73. Epub 2021 Jan 15.

Department of Pediatrics I, ALL-BFM Study Group, Christian-Albrechts University Kiel and University Medical Center Schleswig-Holstein, Arnold-Heller-Str. 3, Haus C, 24105, Kiel, Germany.

Central nervous system (CNS) involvement remains a challenge in the diagnosis and treatment of acute lymphoblastic leukemia (ALL). In this study, we identify CD79a (also known as Igα), a signaling component of the preB cell receptor (preBCR), to be associated with CNS-infiltration and -relapse in B-cell precursor (BCP)-ALL patients. Furthermore, we show that downregulation of CD79a hampers the engraftment of leukemia cells in different murine xenograft models, particularly in the CNS.
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http://dx.doi.org/10.1038/s42003-020-01591-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810877PMC
January 2021

Inducible transgene expression in PDX models in vivo identifies KLF4 as a therapeutic target for B-ALL.

Biomark Res 2020 16;8:46. Epub 2020 Sep 16.

Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), Marchioninistraße 25, 81377 Munich, Germany.

Background: Clinically relevant methods are not available that prioritize and validate potential therapeutic targets for individual tumors, from the vast amount of tumor descriptive expression data.

Methods: We established inducible transgene expression in clinically relevant patient-derived xenograft (PDX) models in vivo to fill this gap.

Results: With this technique at hand, we analyzed the role of the transcription factor Krüppel-like factor 4 (KLF4) in B-cell acute lymphoblastic leukemia (B-ALL) PDX models at different disease stages. In competitive preclinical in vivo trials, we found that re-expression of wild type KLF4 reduced the leukemia load in PDX models of B-ALL, with the strongest effects being observed after conventional chemotherapy in minimal residual disease (MRD). A nonfunctional KLF4 mutant had no effect on this model. The re-expression of KLF4 sensitized tumor cells in the PDX model towards systemic chemotherapy in vivo. It is of major translational relevance that azacitidine upregulated KLF4 levels in the PDX model and a KLF4 knockout reduced azacitidine-induced cell death, suggesting that azacitidine can regulate KLF4 re-expression. These results support the application of azacitidine in patients with B-ALL as a therapeutic option to regulate KLF4.

Conclusion: Genetic engineering of PDX models allows the examination of the function of dysregulated genes like KLF4 in a highly clinically relevant translational context, and it also enables the selection of therapeutic targets in individual tumors and links their functions to clinically available drugs, which will facilitate personalized treatment in the future.
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http://dx.doi.org/10.1186/s40364-020-00226-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493381PMC
September 2020

Requirement for LIM kinases in acute myeloid leukemia.

Leukemia 2020 12 26;34(12):3173-3185. Epub 2020 Jun 26.

Division of Translational Medical Oncology, National Center for Tumor Diseases (NCT) Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany.

Acute myeloid leukemia (AML) is an aggressive disease for which only few targeted therapies are available. Using high-throughput RNA interference (RNAi) screening in AML cell lines, we identified LIM kinase 1 (LIMK1) as a potential novel target for AML treatment. High LIMK1 expression was significantly correlated with shorter survival of AML patients and coincided with FLT3 mutations, KMT2A rearrangements, and elevated HOX gene expression. RNAi- and CRISPR-Cas9-mediated suppression as well as pharmacologic inhibition of LIMK1 and its close homolog LIMK2 reduced colony formation and decreased proliferation due to slowed cell-cycle progression of KMT2A-rearranged AML cell lines and patient-derived xenograft (PDX) samples. This was accompanied by morphologic changes indicative of myeloid differentiation. Transcriptome analysis showed upregulation of several tumor suppressor genes as well as downregulation of HOXA9 targets and mitosis-associated genes in response to LIMK1 suppression, providing a potential mechanistic basis for the anti-leukemic phenotype. Finally, we observed a reciprocal regulation between LIM kinases (LIMK) and CDK6, a kinase known to be involved in the differentiation block of KMT2A-rearranged AML, and addition of the CDK6 inhibitor palbociclib further enhanced the anti-proliferative effect of LIMK inhibition. Together, these data suggest that LIMK are promising targets for AML therapy.
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http://dx.doi.org/10.1038/s41375-020-0943-5DOI Listing
December 2020

Targeting RSPO3-LGR4 Signaling for Leukemia Stem Cell Eradication in Acute Myeloid Leukemia.

Cancer Cell 2020 08 18;38(2):263-278.e6. Epub 2020 Jun 18.

Cancer and Stem Cell Biology Group, Children's Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address:

Signals driving aberrant self-renewal in the heterogeneous leukemia stem cell (LSC) pool determine aggressiveness of acute myeloid leukemia (AML). We report that a positive modulator of canonical WNT signaling pathway, RSPO-LGR4, upregulates key self-renewal genes and is essential for LSC self-renewal in a subset of AML. RSPO2/3 serve as stem cell growth factors to block differentiation and promote proliferation of primary AML patient blasts. RSPO receptor, LGR4, is epigenetically upregulated and works through cooperation with HOXA9, a poor prognostic predictor. Blocking the RSPO3-LGR4 interaction by clinical-grade anti-RSPO3 antibody (OMP-131R10/rosmantuzumab) impairs self-renewal and induces differentiation in AML patient-derived xenografts but does not affect normal hematopoietic stem cells, providing a therapeutic opportunity for HOXA9-dependent leukemia.
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http://dx.doi.org/10.1016/j.ccell.2020.05.014DOI Listing
August 2020

41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo.

J Immunother Cancer 2020 06;8(1)

Biomedicine, Research Institute Against Leukemia Josep Carreras, Barcelona, Catalunya, Spain

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative.

Methods: We set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs.

Results: Here, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs.

Conclusions: This study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML.
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http://dx.doi.org/10.1136/jitc-2020-000845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292050PMC
June 2020

Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR.

Blood 2020 09;136(12):1407-1418

Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Dr. von Hauner Children's Hospital, University Hospital, Ludwig Maximilians University Munich, Munich, Germany.

Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.
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http://dx.doi.org/10.1182/blood.2020005185DOI Listing
September 2020

CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries.

Nucleic Acids Res 2020 07;48(13):e78

Department of Pediatrics, Dr. von Hauner Children's Hospital, University Hospital, Ludwig Maximilians University of Munich (LMU), 80337 Munich, Germany.

The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences.
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http://dx.doi.org/10.1093/nar/gkaa459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367185PMC
July 2020

TET1 promotes growth of T-cell acute lymphoblastic leukemia and can be antagonized via PARP inhibition.

Leukemia 2021 02 15;35(2):389-403. Epub 2020 May 15.

Institute of Experimental Cancer Research, University Hospital of Ulm, Ulm, Germany.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer characterized by skewed epigenetic patterns, raising the possibility of therapeutically targeting epigenetic factors in this disease. Here we report that among different cancer types, epigenetic factor TET1 is highly expressed in T-ALL and is crucial for human T-ALL cell growth in vivo. Knockout of TET1 in mice and knockdown in human T cell did not perturb normal T-cell proliferation, indicating that TET1 expression is dispensable for normal T-cell growth. The promotion of leukemic growth by TET1 was dependent on its catalytic property to maintain global 5-hydroxymethylcytosine (5hmC) marks, thereby regulate cell cycle, DNA repair genes, and T-ALL associated oncogenes. Furthermore, overexpression of the Tet1-catalytic domain was sufficient to augment global 5hmC levels and leukemic growth of T-ALL cells in vivo. We demonstrate that PARP enzymes, which are highly expressed in T-ALL patients, participate in establishing H3K4me3 marks at the TET1 promoter and that PARP1 interacts with the TET1 protein. Importantly, the growth related role of TET1 in T-ALL could be antagonized by the clinically approved PARP inhibitor Olaparib, which abrogated TET1 expression, induced loss of 5hmC marks, and antagonized leukemic growth of T-ALL cells, opening a therapeutic avenue for this disease.
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http://dx.doi.org/10.1038/s41375-020-0864-3DOI Listing
February 2021

ZBTB7A prevents RUNX1-RUNX1T1-dependent clonal expansion of human hematopoietic stem and progenitor cells.

Oncogene 2020 04 2;39(15):3195-3205. Epub 2020 Mar 2.

Department of Medicine III, University Hospital, LMU Munich, 81377, Munich, Germany.

ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1-RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-D-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.
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http://dx.doi.org/10.1038/s41388-020-1209-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142018PMC
April 2020

Deep Learning Reveals Cancer Metastasis and Therapeutic Antibody Targeting in the Entire Body.

Cell 2019 Dec;179(7):1661-1676.e19

Institute for Tissue Engineering and Regenerative Medicine (iTERM), Helmholtz Zentrum München, 85764 Neuherberg, Germany; Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig Maximilian University of Munich (LMU), 81377 Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), 81377 Munich, Germany. Electronic address:

Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2019.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591821PMC
December 2019

RIG-I-based immunotherapy enhances survival in preclinical AML models and sensitizes AML cells to checkpoint blockade.

Leukemia 2020 04 18;34(4):1017-1026. Epub 2019 Nov 18.

Center of Integrated Protein Science Munich (CIPS-M) and Division of Clinical Pharmacology, University Hospital, LMU Munich, Munich, Germany.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5'-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4 and CD8 T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3 T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.
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http://dx.doi.org/10.1038/s41375-019-0639-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214254PMC
April 2020

The Cytotoxic Natural Product Vioprolide A Targets Nucleolar Protein 14, Which Is Essential for Ribosome Biogenesis.

Angew Chem Int Ed Engl 2020 01 12;59(4):1595-1600. Epub 2019 Dec 12.

Center for Integrated Protein Science (CIPSM), Department Chemie, Technische Universität München (TUM), Lichtenbergstrasse 4, 85747, Garching, Germany.

Novel targets are needed for treatment of devastating diseases such as cancer. For decades, natural products have guided innovative therapies by addressing diverse pathways. Inspired by the potent cytotoxic bioactivity of myxobacterial vioprolides A-D, we performed in-depth studies on their mode of action. Based on its prominent potency against human acute lymphoblastic leukemia (ALL) cells, we conducted thermal proteome profiling (TPP) and deciphered the target proteins of the most active derivative vioprolide A (VioA) in Jurkat cells. Nucleolar protein 14 (NOP14), which is essential in ribosome biogenesis, was confirmed as a specific target of VioA by a suite of proteomic and biological follow-up experiments. Given its activity against ALL cells compared to healthy lymphocytes, VioA exhibits unique therapeutic potential for anticancer therapy through a novel mode of action.
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http://dx.doi.org/10.1002/anie.201911158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004033PMC
January 2020

Loss of KDM6A confers drug resistance in acute myeloid leukemia.

Leukemia 2020 01 14;34(1):50-62. Epub 2019 Jun 14.

Department of Medicine III, University Hospital, LMU Munich, Munich, Germany.

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy leading to initial treatment responses, relapse caused by intrinsic or acquired drug resistance represents a major challenge. Here, we report that histone 3 lysine 27 demethylase KDM6A (UTX) is targeted by inactivating mutations and mutation-independent regulation in relapsed AML. Analyses of matched diagnosis and relapse specimens from individuals with KDM6A mutations showed an outgrowth of the KDM6A mutated tumor population at relapse. KDM6A expression is heterogeneously regulated and relapse-specific loss of KDM6A was observed in 45.7% of CN-AML patients. KDM6A-null myeloid leukemia cells were more resistant to treatment with the chemotherapeutic agents cytarabine (AraC) and daunorubicin. Inducible re-expression of KDM6A in KDM6A-null cell lines suppressed proliferation and sensitized cells again to AraC treatment. RNA expression analysis and functional studies revealed that resistance to AraC was conferred by downregulation of the nucleoside membrane transporter ENT1 (SLC29A1) by reduced H3K27 acetylation at the ENT1 locus. Our results show that loss of KDM6A provides cells with a selective advantage during chemotherapy, which ultimately leads to the observed outgrowth of clones with KDM6A mutations or reduced KDM6A expression at relapse.
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http://dx.doi.org/10.1038/s41375-019-0497-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214274PMC
January 2020

Hepatic leukemia factor is a novel leukemic stem cell regulator in DNMT3A, NPM1, and FLT3-ITD triple-mutated AML.

Blood 2019 07 10;134(3):263-276. Epub 2019 May 10.

Department of Medicine V, Hematology, Oncology and Rheumatology, University Hospital Heidelberg, Heidelberg, Germany.

, and are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific CD34 immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from double-mutated subclones. Moreover, in R882-mutated patients, CpG hypomethylation at the transcription start site correlated with high mRNA expression, which was itself associated with poor survival. Loss of via CRISPR/Cas9 significantly reduced the CD34GPR56 LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 () and the cyclin-dependent kinase inhibitor as novel targets of HLF, potentially mediating these effects. Overall, our data establish as a novel LSC regulator in this genetically defined high-risk AML subgroup.
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http://dx.doi.org/10.1182/blood.2018862383DOI Listing
July 2019

Tumor Cell Dormancy-Triggered by the Niche.

Dev Cell 2019 05;49(3):311-312

Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), Munich, Germany; German Cancer Consortium (DKTK), Partner Site, Munich, Germany; Department of Pediatrics, Dr. von Hauner Childrens Hospital, Ludwig Maximilian University, Munich, Germany. Electronic address:

Dormant cancer cells often survive treatment and increase the risk for tumor relapse, associated with dismal prognosis. Two recent papers describe mechanisms used by the bone marrow niche to regulate leukemia dormancy. The findings provide a molecular basis for niche-targeting therapies that may enable elimination of dormant tumor cells.
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http://dx.doi.org/10.1016/j.devcel.2019.04.022DOI Listing
May 2019

JMJD1C-mediated metabolic dysregulation contributes to HOXA9-dependent leukemogenesis.

Leukemia 2019 06 8;33(6):1400-1410. Epub 2019 Jan 8.

Cancer and Stem Cell Biology Group, Children's Cancer Institute, University of New South Wales, Sydney, NSW 2052, Australia.

Abnormal metabolism is a fundamental hallmark of cancer and represents a therapeutic opportunity, yet its regulation by oncogenes remains poorly understood. Here, we uncover that JMJD1C, a jumonji C (JmjC)-containing H3K9 demethylase, is a critical regulator of aberrant metabolic processes in homeobox A9 (HOXA9)-dependent acute myeloid leukemia (AML). JMJD1C overexpression increases in vivo cell proliferation and tumorigenicity through demethylase-independent upregulation of a glycolytic and oxidative program, which sustains leukemic cell bioenergetics and contributes to an aggressive AML phenotype in vivo. Targeting JMJD1C-mediated metabolism via pharmacologic inhibition of glycolysis and oxidative phosphorylation led to ATP depletion, induced necrosis/apoptosis and decreased tumor growth in vivo in leukemias co-expressing JMJD1C and HOXA9. The anti-metabolic therapy effectively diminished AML stem/progenitor cells and reduced tumor burden in a primary AML patient-derived xenograft. Our data establish a direct link between drug responses and endogenous expression of JMJD1C and HOXA9 in human AML cell line- and patient-derived xenografts. These findings demonstrate a previously unappreciated role for JMJD1C in counteracting adverse metabolic changes and retaining the metabolic integrity during tumorigenesis, which can be exploited therapeutically.
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http://dx.doi.org/10.1038/s41375-018-0354-zDOI Listing
June 2019

SRPK1 maintains acute myeloid leukemia through effects on isoform usage of epigenetic regulators including BRD4.

Nat Commun 2018 12 19;9(1):5378. Epub 2018 Dec 19.

Haematological Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK.

We recently identified the splicing kinase gene SRPK1 as a genetic vulnerability of acute myeloid leukemia (AML). Here, we show that genetic or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemic cell differentiation and prolonged survival of mice transplanted with MLL-rearranged AML. RNA-seq analysis demonstrates that SRPK1 inhibition leads to altered isoform levels of many genes including several with established roles in leukemogenesis such as MYB, BRD4 and MED24. We focus on BRD4 as its main isoforms have distinct molecular properties and find that SRPK1 inhibition produces a significant switch from the short to the long isoform at the mRNA and protein levels. This was associated with BRD4 eviction from genomic loci involved in leukemogenesis including BCL2 and MYC. We go on to show that this switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Our findings reveal that SRPK1 represents a plausible new therapeutic target against AML.
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http://dx.doi.org/10.1038/s41467-018-07620-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300607PMC
December 2018

Characteristics and Therapeutic Targeting of Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia.

Adv Exp Med Biol 2018;1100:127-139

Department of Pediatrics I, Pediatric Hematology/Oncology, ALL-BFM Study Group, University Hospital Schleswig-Holstein, Kiel, Germany.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Early response to therapy, especially the measurement of minimal residual disease (MRD), remains the most reliable and strongest independent prognostic parameter. Intriguingly, little is known on the mechanisms sustaining MRD in that disease. Here, we summarize existing evidence on the influences of molecular genetics and clonal architecture of childhood ALL on disease persistence. Also, the impact of the leukemic niche on residual leukemia cells in the bone marrow and extramedullary compartments is reviewed. We further discuss existing in vivo models of minimal residual disease based on different cellular labelling strategies and engraftment of ALL cells in immunodeficient mouse strains. We finally draw some conclusions on potential strategies targeting residual ALL cells, with a focus on cellular and antibody-based immunotherapy.
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http://dx.doi.org/10.1007/978-3-319-97746-1_8DOI Listing
July 2019

Targeting the endoplasmic reticulum-mitochondria interface sensitizes leukemia cells to cytostatics.

Haematologica 2019 03 11;104(3):546-555. Epub 2018 Oct 11.

Department of Pharmaceutical Biology, Ludwig Maximilian University of Munich

Combination chemotherapy has proven to be a favorable strategy to treat acute leukemia. However, the introduction of novel compounds remains challenging and is hindered by a lack of understanding of their mechanistic interactions with established drugs. In the present study, we demonstrate a highly increased response of various acute leukemia cell lines, drug-resistant cells and patient-derived xenograft cells by combining the recently introduced protein disulfide isomerase inhibitor PS89 with cytostatics. In leukemic cells, a proteomics-based target fishing approach revealed that PS89 affects a whole network of endoplasmic reticulum homeostasis proteins. We elucidate that the strong induction of apoptosis in combination with cytostatics is orchestrated by the PS89 target B-cell receptor-associated protein 31, which transduces apoptosis signals at the endoplasmic reticulum -mitochondria interface. Activation of caspase-8 and cleavage of B-cell receptor-associated protein 31 stimulate a pro-apoptotic crosstalk including release of calcium from the endoplasmic reticulum and an increase in the levels of reactive oxygen species resulting in amplification of mitochondrial apoptosis. The findings of this study promote PS89 as a novel chemosensitizing agent for the treatment of acute leukemia and uncovers that targeting the endoplasmic reticulum - mitochondrial network of cell death is a promising approach in combination therapy.
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http://dx.doi.org/10.3324/haematol.2018.197368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395311PMC
March 2019

Combined inhibition of receptor tyrosine and p21-activated kinases as a therapeutic strategy in childhood ALL.

Blood Adv 2018 10;2(19):2554-2567

Research Institute Children's Cancer Center Hamburg, Hamburg, Germany.

Receptor tyrosine kinase (RTK)-dependent signaling has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) of childhood. However, the RTK-dependent signaling state and its interpretation with regard to biological behavior are often elusive. To decipher signaling circuits that link RTK activity with biological output in vivo, we established patient-derived xenograft ALL (PDX-ALL) models with dependencies on fms-like tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor β (PDGFRB), which were interrogated by phosphoproteomics using iTRAQ mass spectrometry. Signaling circuits were determined by receptor type and cellular context with few generic features, among which we identified group I p21-activated kinases (PAKs) as potential therapeutic targets. Growth factor stimulation markedly increased catalytic activities of PAK1 and PAK2. RNA interference (RNAi)-mediated or pharmacological inhibition of PAKs using allosteric or adenosine triphosphate (ATP)-competitive compounds attenuated cell growth and increased apoptosis in vitro. Notably, PAK1- or PAK2-directed RNAi enhanced the antiproliferative effects of the type III RTK and protein kinase C inhibitor midostaurin. Treatment of FLT3- or PDGFRB-dependent ALLs with ATP-competitive PAK inhibitors markedly decreased catalytic activities of both PAK isoforms. In FLT3-driven ALL, this effect was augmented by coadministration of midostaurin resulting in synergistic effects on growth inhibition and apoptosis. Finally, combined treatment of PDX-ALL with the ATP-competitive group I PAK inhibitor FRAX486 and midostaurin in vivo significantly prolonged leukemia progression-free survival compared with midostaurin monotherapy or control. Our study establishes PAKs as potential downstream targets in RTK-dependent ALL of childhood, the inhibition of which might help prevent the selection or acquisition of resistance mutations toward tyrosine kinase inhibitors.
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http://dx.doi.org/10.1182/bloodadvances.2018020693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177654PMC
October 2018

A rare subgroup of leukemia stem cells harbors relapse-inducing potential in acute lymphoblastic leukemia.

Exp Hematol 2019 01 24;69:1-10. Epub 2018 Sep 24.

Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Center Munich, German Center for Environmental Health (HMGU), Munich, Germany; Department of Pediatrics, Dr. von Hauner Childrens Hospital, Ludwig Maximilians University, Munich, Germany; German Consortium for Translational Cancer Research (DKTK), Partnering Site Munich, Munich, Germany. Electronic address:

After initially successful chemotherapy, relapse frequently jeopardizes the outcome of patients with acute leukemia. Because of their adverse characteristics of self-renewal and dormancy, leukemia stem cells have been hypothesized to play a critical role in resistance to antiproliferative chemotherapy and the development of relapse. The high abundance of stem-like cells in acute lymphoblastic leukemia (ALL), however, suggests that not all leukemia-initiating cells carry these adverse characteristics, complicating the biological characterization of relapse-inducing cells in this malignancy. Here, we review sources of therapy resistance and relapse in acute leukemias, which include tumor cell plasticity and reversible characteristics. We discuss the development of patient-derived mouse models that are genetically engineered to mimic long-term dormancy and minimal residual disease in patients. These models allow the tracking and functional characterization of patient-derived ALL blasts that combine the properties of long-term dormancy, treatment resistance, and stemness. Finally, we discuss possible therapeutic avenues to target the functional plasticity of leukemia-initiating cells in ALL.
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http://dx.doi.org/10.1016/j.exphem.2018.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542675PMC
January 2019

Frequent and reliable engraftment of certain adult primary acute lymphoblastic leukemias in mice.

Leuk Lymphoma 2019 03 20;60(3):848-851. Epub 2018 Sep 20.

a Research Unit Apoptosis in Hematopoietic Stem Cells (AHS), Helmholtz Center Munich , German Research Center for Environmental Health , Munich , Germany.

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http://dx.doi.org/10.1080/10428194.2018.1509314DOI Listing
March 2019

Spatiotemporal patterning of EpCAM is important for murine embryonic endo- and mesodermal differentiation.

Sci Rep 2018 01 29;8(1):1801. Epub 2018 Jan 29.

Department of Otorhinolaryngology, Head and Neck Surgery, Grosshadern Medical Center, Ludwig Maximilians University, Munich, Marchioninistr. 15, 81377, Munich, Germany.

Epithelial cell adhesion molecule EpCAM is expressed in pluripotent embryonic stem cells (ESC) in vitro, but is repressed in differentiated cells, except epithelia and carcinomas. Molecular functions of EpCAM, possibly imposing such repression, were primarily studied in malignant cells and might not apply to non-pathologic differentiation. Here, we comprehensively describe timing and rationale for EpCAM regulation in early murine gastrulation and ESC differentiation using single cell RNA-sequencing datasets, in vivo and in vitro models including CRISPR-Cas9-engineered ESC-mutants. We demonstrate expression of EpCAM in inner cell mass, epiblast, primitive/visceral endoderm, and strict repression in the most primitive, nascent Flk1 mesoderm progenitors at E7.0. Selective expression of EpCAM was confirmed at mid-gestation and perinatal stages. The rationale for strict patterning was studied in ESC differentiation. Gain/loss-of-function demonstrated supportive functions of EpCAM in achieving full pluripotency and guided endodermal differentiation, but repressive functions in mesodermal differentiation as exemplified with cardiomyocyte formation. We further identified embryonic Ras (ERas) as novel EpCAM interactor of EpCAM and an EpCAM/ERas/AKT axis that is instrumental in differentiation regulation. Hence, spatiotemporal patterning of EpCAM at the onset of gastrulation, resulting in early segregation of interdependent EpCAM endodermal and EpCAM/vimentin mesodermal clusters represents a novel regulatory feature during ESC differentiation.
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http://dx.doi.org/10.1038/s41598-018-20131-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789065PMC
January 2018

Azacitidine combined with the selective FLT3 kinase inhibitor crenolanib disrupts stromal protection and inhibits expansion of residual leukemia-initiating cells in -ITD AML with concurrent epigenetic mutations.

Oncotarget 2017 Dec 16;8(65):108738-108759. Epub 2017 Oct 16.

Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany.

Effectively targeting leukemia-initiating cells (LIC) in -ITD-mutated acute myeloid leukemia (AML) is crucial for cure. Tyrosine kinase inhibitors (TKI) have limited impact as single agents, failing to eradicate LIC in the bone marrow. Using primary AML samples and a patient-derived xenograft model, we investigated whether combining the FLT3-selective TKI crenolanib with the hypomethylating agent azacitidine (AZA) eliminates -ITD LIC and whether efficacy of this combination depends on co-existing mutations. Using multiparameter flow cytometry, we show -ITD occurs within the most primitive Lin/CD33/CD45/CD34CD38 LIC compartment. Crenolanib alone could not target -ITD LIC in contact with niche cells while addition of AZA overcame stromal protection resulting in dramatically reduced clonogenic capacity of LIC and severely impaired engraftment in NSG mice. Strikingly, -mutated samples harboring mutations were completely resistant to crenolanib whereas neither nor mutations influenced response. Conversely, primary AML LIC harboring either or mutations did not show increased sensitivity to AZA. In summary, resistance of -ITD LIC to TKI depends on co-existing epigenetic mutations. However, AZA + crenolanib effectively abrogates stromal protection and inhibits survival of -ITD LIC irrespective of mutations, providing evidence for this combination as a means to suppress residual LIC.
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http://dx.doi.org/10.18632/oncotarget.21877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752477PMC
December 2017

Dual Targeting of Acute Leukemia and Supporting Niche by CXCR4-Directed Theranostics.

Theranostics 2018 1;8(2):369-383. Epub 2018 Jan 1.

Internal Medicine III, Hematology and Medical Oncology, Technische Universität München, Munich, Germany.

C-X-C chemokine receptor 4 (CXCR4) is a transmembrane receptor with pivotal roles in cell homing and hematopoiesis. CXCR4 is also involved in survival, proliferation and dissemination of cancer, including acute lymphoblastic and myeloid leukemia (ALL, AML). Relapsed/refractory ALL and AML are frequently resistant to conventional therapy and novel highly active strategies are urgently needed to overcome resistance. We used patient-derived (PDX) and cell line-based xenograft mouse models of ALL and AML to evaluate the efficacy and toxicity of a CXCR4-targeted endoradiotherapy (ERT) theranostic approach. The positron emission tomography (PET) tracer Ga-Pentixafor enabled visualization of CXCR4 positive leukemic burden. In xenografts, CXCR4-directed ERT with Lu-Pentixather distributed to leukemia harboring organs and resulted in efficient reduction of leukemia. Despite a substantial cross-fire effect to the leukemia microenvironment, mesenchymal stem cells (MSCs) subjected to ERT were viable and capable of supporting the growth and differentiation of non-targeted normal hematopoietic cells . Finally, three patients with refractory AML after first allogeneic hematopoietic stem cell transplantation (alloSCT) underwent CXCR4-directed ERT resulting in leukemia clearance, second alloSCT, and successful hematopoietic engraftment. Targeting CXCR4 with ERT is feasible and provides a highly efficient means to reduce refractory acute leukemia for subsequent cellular therapies. Prospective clinical trials testing the incorporation of CXCR4 targeting into conditioning regimens for alloSCT are highly warranted.
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http://dx.doi.org/10.7150/thno.21397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743554PMC
December 2018

The target landscape of clinical kinase drugs.

Science 2017 12;358(6367)

Chair of Proteomics and Bioanalytics, Technical University of Munich (TUM), Freising, Germany.

Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.
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http://dx.doi.org/10.1126/science.aan4368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542668PMC
December 2017