Search

XLSX file 75K, This is an excel file containing 9 sheets, one for each of the 9 supplementary tables (S1 to S9) mentioned in the main text

PDF file 234K, This is a file containing the list of supplementary tables, the list of supplementary figures, as well as the 7 supplementary figures (S1 to S7) and their correspondent legends, mentioned in the main text

We discovered a novel somatic gene fusion, CD74–NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74–NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2–ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74–NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K–AKT pathway, and led to increased colony formation in soft agar. Thus, CD74–NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment.Significance: CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease. Cancer Discov; 4(4); 415–22. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 377

A growing body of data from early clinical studies supports that targeting the myeloid immune checkpoint CD47-SIRPα represents a successful strategy towards establishing effective new therapies for cancer patients. Initial studies focused on CD47 blockers and more recently also SIRPα antagonistic mAbs entered clinical studies. The latter approach is thought to be more advantageous since SIRPα expression is primarily myeloid restricted whereas CD47 is broadly expressed and -besides SIRPα- binds other ligands as well.We generated BYON4228, an antibody directed against the two existing allelic variants of human SIRPα that does not recognize the closely related T-cell expressed SIRPγ. Functional studies demonstrate that BYON4228 potentiates both macrophage- and neutrophil-mediated elimination of hematologic and solid cancer cells in vitro in the presence of a variety of tumor targeting antibodies, including rituximab, daratumumab, trastuzumab and cetuximab. In vivo, BYON4228 increases the anti-tumor activity of rituximab in a B-cell Raji xenograft model in mice and shows a favorable safety profile in cynomolgus monkeys.To position BYON4228 in the emerging field of myeloid checkpoint inhibitors, head-to-head comparison studies were performed. For these studies, previously described anti-SIRPα mAbs HEFLB, 1H9, and SIRPAB-11 were generated in house that in all probability are representative for three clinical stage SIRPα mAbs. These comparative studies focused on principal characteristics of this class of mAbs that are anticipated to affect their clinical profile, namely 1) recognition of both common allelic forms of SIRPα which determines the broadness of its clinical applicability; 2) binding to SIRPγ on T-lymphocytes which is reported to affect T-cell extravasation and activation and thereby the adaptive immune response; 3) functionality of the Fc-region of the mAb which can mediate depletion of SIRPα-positive host myeloid immune effector cells thereby compromising host defense and undermining the effect of immunotherapy. The comparative studies show that in contrast to HEFLB, BYON4228 recognizes both common allelic forms of SIRPα. Furthermore, lack of binding to T-cell expressed SIRPγ differentiates BYON4228 from 1H9 and SIRPAB-11. Finally, with its impaired Fc-region BYON4228 does not deplete SIRPα+ myeloid cells, in contrast to SIRPAB-11.Thus, BYON4228 differentiates from all three comparator SIRPα mAbs based on at least one fundamental characteristic, thereby underlining its potential to become a best-in-class drug. We therefore endorse its further development for which clinical studies are planned to start in 2023. Citation Format: Mary van Helden, Seline Zwarthoff, Roel Arends, Inge Reinieren-Beeren, Marc Parade, Lilian Driessen-Engels, Karin de Laat-Arts, Desiree Damming, Ellen Santegoeds-Lenssen, Daphne van Kuppeveld, Imke Lodewijks, Ellen Mattaar, Hugo Olsman, Hanke Matlung, Katka Franke, Marloes Stokman, Benny de Wit, Dirk Glaudemans, Danielle van Wijk, Lonnie Joosten Stoffels, Jan Schouten, Paul Boersema, Monique van der Vleuten, Jorien Sanderink, Wendy Kappers, Diels van den Dobbelsteen, Marco Timmers, Ruud Ubink, Gerard Rouwendal, Gijs Verheijden, Miranda M.C. van der Lee, Wim Dokter, Timo van den Berg. BYON4228, an antagonistic SIRPα mAb with a unique and favorable preclinical profile compared to three comparator SIRPα mAbs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2934.

[Image: see text] Protein arginine methyltransferase 5 (PRMT5) is an enzyme that can symmetrically dimethylate arginine residues in histones and nonhistone proteins by using S-adenosyl methionine (SAM) as the methyl donating cofactor. We have designed a library of SAM analogues and discovered potent, cell-active, and selective spiro diamines as inhibitors of the enzymatic function of PRMT5. Crystallographic studies confirmed a very interesting binding mode, involving protein flexibility, where both the cofactor pocket and part of substrate binding site are occupied by these inhibitors.

Background Accumulating preclinical data indicate that targeting the SIRPα/CD47 axis alone or in combination with existing targeted therapies or immune checkpoint inhibitors enhances tumor rejection. Although several CD47-targeting agents are currently in phase I clinical trials and demonstrate activity in combination therapy, high and frequent dosing was required and safety signals (acute anemia, thrombocytopenia) were recorded frequently as adverse events. Based on the restricted expression pattern of SIRPα we hypothesized that antibodies targeting SIRPα might avoid some of the concerns noted for CD47-targeting agents. Methods SIRPα-targeting antibodies were generated and characterized for binding to human SIRPα alleles and blockade of the interaction with CD47. Functional activity was established in vitro using human macrophages or neutrophils co-cultured with human Burkitt’s lymphoma cell lines. The effect of SIRPα versus CD47 targeting on human T-cell activation was studied using an allogeneic mixed lymphocyte reaction and a Staphylococcus enterotoxin B-induced T-cell proliferation assay. Potential safety concerns of the selected SIRPα-targeting antibody were addressed in vitro using a hemagglutination assay and a whole blood cytokine release assay, and in vivo in a single-dose toxicity study in cynomolgus monkeys. Results The humanized monoclonal IgG2 antibody ADU-1805 binds to all known human SIRPα alleles, showing minimal binding to SIRPβ1, while cross-reacting with SIRPγ, and potently blocking the interaction of SIRPα with CD47. Reduced FcγR binding proved critical to retaining its function towards phagocyte activation. In vitro characterization demonstrated that ADU-1805 promotes macrophage phagocytosis, with similar potency to anti-CD47 antibodies, and enhances neutrophil trogocytosis. Unlike CD47-targeting agents, ADU-1805 does not interfere with T-cell activation and is not expected to require frequent and extensive dosing due to the restricted expression of SIRPα to cells of the myeloid lineage. ADU-1805 is cross-reactive to cynomolgus monkey SIRPα and upon single-dose intravenous administration in these non-human primates (NHPs) did not show any signs of anemia, thrombocytopenia or other toxicities. Conclusions Blocking the SIRPα-CD47 interaction via SIRPα, while similarly efficacious in vitro, differentiates ADU-1805 from CD47-targeting agents with respect to safety and absence of inhibition of T-cell activation. The data presented herein support further advancement of ADU-1805 towards clinical development.

Protein kinase MELK has oncogenic properties and is highly overexpressed in some tumors. In the present study, we show that a novel MELK inhibitor causes both the inhibition and degradation of MELK, culminating in replication stress and a senescence phenotype., Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold.

PRMT5 is a type II methyltransferase that specifically adds methyl groups to arginine as a long-lasting post-translational modification. The PRMT5/MEP50 complex regulates a plethora of cellular processes, such as epigenetics and splicing, which are notable events involved in tumorigenesis. Although not frequently mutated or amplified in tumors, elevated PRMT5 protein levels in lung and hematologic cancers are correlated with poorer survival. The PRMT5 inhibitor JNJ-64619178 has been selected as a clinical candidate based on its high selectivity and potency (subnanomolar range) under different in vitro and cellular conditions, paired with favorable pharmacokinetics and safety properties. JNJ-64619178 binds into the SAM binding pocket and reaches the substrate binding pocket to inhibit PRMT5/MEP50 function in a time-dependent manner. Broad cell line panel profiling of JNJ-64619178 revealed a wide range of sensitivity, which is indicative of a genomic dependency instead of a general cytotoxic on-target consequence of PRMT5 inhibition. Further investigations indicate a synthetic lethal correlation between PRMT5 inhibition and key cancer driver pathways. JNJ-64619178, dosed orally (10 mg/kg, every day), showed selective and efficient blockage of the methylation of SMD1/3 proteins, which are crucial components of the spliceosome and substrates of PRMT5/MEP50. JNJ-64619178 also demonstrated tumor regression in a biomarker-driven human small cell lung cancer xenograft model (NCI-H1048) and prolonged tumor growth inhibition after dosing cessation. In rodent and nonrodent toxicology studies, a tolerated dose of JNJ-64619178 has been identified, with the observed toxicity consistent with on-target activity. In summary, JNJ-64619178 has a favorable preclinical package that supports clinical testing in patients diagnosed with lung cancer and hematologic malignancies. Citation Format: Dirk Brehmer, Tongfei Wu, Geert Mannens, Lijs Beke, Petra Vinken, Dana Gaffney, Weimei Sun, Vineet Pande, Jan-Willem Thuring, Hillary Millar, Italo Poggesi, Ivan Somers, An Boeckx, Marc Parade, Erika van Heerde, Thomas Nys, Carol Yanovich, Barbara Herkert, Tinne Verhulst, Marc Du Jardin, Lieven Meerpoel, Christopher Moy, Gaston Diels, Marcel Viellevoye, Wim Schepens, Alain Poncelet, Joannes T. Linders, Edward C. Lawson, James P. Edwards, Dushen Chetty, Sylvie Laquerre, Matthew V. Lorenzi. A novel PRMT5 inhibitor with potent in vitro and in vivo activity in preclinical lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT02-04. doi:10.1158/1538-7445.AM2017-DDT02-04

We discovered a novel somatic gene fusion, CD74–NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74–NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2–ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74–NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K–AKT pathway, and led to increased colony formation in soft agar. Thus, CD74–NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. Significance: CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease. Cancer Discov; 4(4); 415–22. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 377

Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold. ispartof: Bioscience Reports vol:35 issue:6 ispartof: location:England status: published

The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies. ispartof: PLOS ONE vol:11 issue:1 ispartof: location:United States status: published

Maternal embryonic leucine zipper kinase (MELK, hMP38, pEG3), a Ser/Thr protein kinase, is highly overexpressed in stem cells and cancer cells. The oncogenic role of MELK is attributed to disabling critical cell cycle check points as well as enhancing replication. Most functional studies have relied on the use of siRNA/shRNA mediated gene silencing, but this often can be associated with off-target effects. Here we want to present a novel, potent and selective small molecule inhibitor JNJ-47117096 that has enabled us to validate the biological function of MELK kinase. Outcome: MELK inhibition in cancer cells with an intact p53 signaling pathway is linked to a replicative senescence phenotype. This phenotype correlates with a rapid ATM activation and phosphorylation of CHK2 without any effects on the alternative ATR/ CHK1 DNA damage pathways. Furthermore, JNJ-47117096 induces strong phosphorylation of p53 and prolonged up-regulation of p21 without the induction of apoptosis. Strikingly, MELK inhibition in several p53 disabled cancer cells showed induction of a mitotic catastrophe phenotype followed by prominent cell killing. Finally, JNJ-47117096 triggers a rapid degradation of cellular MELK protein, independent of the cell cycle phase and its regulation by the E2F pathway. This observation can clearly be linked to a direct binding effect of JNJ-47117096 to cellular MELK protein confirmed by chemical proteomics. Conclusion: Our data generated with JNJ-47117096, confirmed by selective siRNAs, indicate MELK as a key stimulator of proliferation and replication by its ability to increase the threshold for DNA damage tolerance. Targeting MELK function by selective small molecule inhibitors might sensitise tumors to DNA-damaging agents or radiation therapy. Citation Format: Lijs Beke, Joannes T.m. Linders, Cenk Kig, An Boeckx, Erika van Heerde, Dirk Wuyts, Marc Parade, Lieven Meerpoel, Chris Johnson, Monique Beullens, Mathieu Bollen, Dirk Brehmer. JNJ-47117096, a selective small molecule inhibitor of the MELK oncogene decreases DNA damage tolerance in highly proliferating cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2936. doi:10.1158/1538-7445.AM2014-2936