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Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a serious therapy-limiting side effect of commonly used anticancer drugs. Previous studies suggest that lipids may play a role in CIPN. Therefore, the present study aimed to identify the particular types of lipids that are regulated as a consequence of paclitaxel administration and may be associated with the occurrence of post-therapeutic neuropathy., Methods: High-resolution mass spectrometry lipidomics was applied to quantify d=255 different lipid mediators in the blood of n=31 patients drawn before and after paclitaxel therapy for breast cancer treatment. A variety of supervised statistical and machine-learning methods was applied to identify lipids that were regulated during paclitaxel therapy or differed among patients with and without post-therapeutic neuropathy., Results: Twenty-seven lipids were identified that carried relevant information to train machine learning algorithms to identify, in new cases, whether a blood sample was drawn before or after paclitaxel therapy with a median balanced accuracy of up to 90%. One of the top hits, sphinganine-1-phosphate (SA1P), was found to induce calcium transients in sensory neurons via the transient receptor potential vanilloid 1 (TRPV1) channel and sphingosine-1-phosphate receptors.SA1P also showed different blood concentrations between patients with and without neuropathy., Conclusions: Present findings suggest a role for sphinganine-1-phosphate in paclitaxel-induced biological changes associated with neuropathic side effects. The identified SA1P, through its receptors, may provide a potential drug target for co-therapy with paclitaxel to reduce one of its major and therapy-limiting side effects., Funding: This work was supported by the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG, Grants SFB1039 A09 and Z01) and by the Fraunhofer Foundation Project: Neuropathic Pain as well as the Fraunhofer Cluster of Excellence for Immune-Mediated Diseases (CIMD). This work was also supported by the Leistungszentrum Innovative Therapeutics (TheraNova) funded by the Fraunhofer Society and the Hessian Ministry of Science and Arts. Jörn Lötsch was supported by the Deutsche Forschungsgemeinschaft (DFG LO 612/16-1)., Competing Interests: JL, KG, SM, LH, CA, YS, ST, SW, DT, NF, CB, BS, CS, GG, MS No competing interests declared, (© 2024, Lötsch, Gasimli et al.)

The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, disease maintenance and progression in acute myeloid leukemia (AML). Mitophagy is orchestrated by a variety of mitophagy receptors whose interplay is not well understood. Here, we established a pairwise multiplexed CRISPR screen targeting mitophagy receptors to elucidate redundancies and gain a deeper understanding of the functional interactome governing mitophagy in AML. We identified OPTN (optineurin) as sole non-redundant mitophagy receptor and characterized its unique role in AML. Knockdown and overexpression experiments demonstrated that OPTN expression is rate-limiting for AML cell proliferation. In a MN1-driven murine transplantation model, loss of OPTN prolonged overall median survival by 7 days (+21%). Mechanistically, we found broadly impaired mitochondrial respiration and function with increased mitochondrial ROS, that most likely caused the proliferation defect. Our results decipher the intertwined network of mitophagy receptors in AML for both ubiquitin-dependent and receptor-mediated mitophagy, identify OPTN as a non-redundant tool to study mitophagy in the context of leukemia and suggest OPTN inhibition as an attractive therapeutic strategy. Abbreviations: AML: acute myeloid leukemia; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; CTRL: control; DFP: deferiprone; GI: genetic interaction; KD: knockdown; KO: knockout; ldMBM, lineage-depleted murine bone marrow; LFC: log2 fold change; LIR: LC3-interacting region; LSC: leukemic stem cell; MAGeCK: Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout; MDIVI-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MOM: mitochondrial outer membrane; NAC: N-acetyl-L-cysteine; OA: oligomycin-antimycin A; OCR: oxygen consumption rate; OE: overexpression; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SEM: standard error of the mean; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; UBD: ubiquitin-binding domain; WT: wild type.

In colorectal cancers, the tumor microenvironment plays a key role in prognosis and therapy efficacy. Patient-derived tumor organoids (PDTO) show enormous potential for preclinical testing; however, cultured tumor cells lose important characteristics, including the consensus molecular subtypes (CMS). To better reflect the cellular heterogeneity, we established the colorectal cancer organoid-stroma biobank of matched PDTOs and cancer-associated fibroblasts (CAF) from 30 patients. Context-specific phenotyping showed that xenotransplantation or coculture with CAFs improves the transcriptomic fidelity and instructs subtype-specific stromal gene expression. Furthermore, functional profiling in coculture exposed CMS4-specific therapeutic resistance to gefitinib and SN-38 and prognostic expression signatures. Chemogenomic library screening identified patient- and therapy-dependent mechanisms of stromal resistance including MET as a common target. Our results demonstrate that colorectal cancer phenotypes are encrypted in the cancer epithelium in a plastic fashion that strongly depends on the context. Consequently, CAFs are essential for a faithful representation of molecular subtypes and therapy responses ex vivo., Significance: Systematic characterization of the organoid-stroma biobank provides a resource for context dependency in colorectal cancer. We demonstrate a colorectal cancer subtype memory of PDTOs that is independent of specific driver mutations. Our data underscore the importance of functional profiling in cocultures for improved preclinical testing and identification of stromal resistance mechanisms. This article is featured in Selected Articles from This Issue, p. 2109., (©2023 The Authors; Published by the American Association for Cancer Research.)

We performed a retrospective single-center analysis to investigate the diagnostic yield of bone marrow puncture in patients with liver cirrhosis and cytopenia. Liver cirrhosis patients receiving bone marrow aspiration or biopsy for the diagnostic work-up of otherwise unexplained peripheral blood cytopenia at our institution between 2004 and 2020 were enrolled in this study. We evaluated findings from cytologic, histologic and immunologic assessment and final diagnostic outcomes. A total of 118 patients with a median age of 55 years and a median Child-Pugh score of B (8 points) were enrolled. The main etiologies of liver cirrhosis were viral hepatitis (B and C) or chronic alcohol consumption. The majority of patients (60%) exhibited concurrent anemia, leukocytopenia and thrombocytopenia. Bone marrow assessment revealed normal, unspecific or reactive alterations in 117 out of 118 patients (99%). One patient was diagnosed with myelodysplastic syndrome. Our findings suggest that peripheral blood cytopenia in patients with liver cirrhosis is rarely associated with a primary bone marrow pathology.

Cranial imaging (CI) is a widely used diagnostic procedure, especially in acute myeloid leukemia (AML) patients with suspected bleeding or infection. However, common clinical decision rules to guide CI do not apply to AML patients and the diagnostic yield and outcomes of CI for AML patients are largely unknown. We retrospectively evaluated all CI from newly diagnosed non-promyelocytic AML patients receiving intensive induction or consolidation chemotherapy between 2007 and 2019 for imaging indications, diagnostic yield, and consequences. A total of 110 of 462 patients (24%) received CI for 152 imagings in distinct clinical situations. Forty-four patients (40%) had at least one new and acute pathological finding. Main indication was focal neurologic deficit, craniocerebral trauma, and suspected cerebral hypertension. The most common new finding was intracranial hemorrhage (13% of all imagings), followed by sinusitis (9%). CI led to therapy change in 21 patients. There were no clear associations between indications, laboratory values, and a positive imaging. Positive imaging was associated with adverse overall survival. Our study suggests that the overall rate of ordered CI was appropriate and that CI should generally be performed at a low threshold. A systematized approach to CI may further increase diagnostic yield but is complicated by variable clinical presentation., (© 2023. The Author(s).)

Objectives and Methods: Venous thromboembolic (VTE) events are emerging as frequent complications in acute myeloid leukemia (AML); however, there is insufficient data regarding epidemiology, risk factors, and impact on outcomes. The optimal approach to balance risks of thrombosis and hemorrhage remains unclear. This retrospective single-center study in AML patients undergoing induction chemotherapy between 2007 and 2018 assessed incidence, risk factors, features, and outcomes of early-onset VTE., Results: 423 patients (median age 59 years) were enrolled. VTE was diagnosed in 31 patients (7.3%) within 3 months of admission. The median time to VTE was 3 days. Non-central venous catheter (CVC)-related VTE occurred in 19 patients (61%). Main risk factor for VTE was leukocytosis at admission, independent of platelet counts/INR. Four patients (13%) exhibited VTE recurrence. No deaths directly related to VTE or major bleeding events associated with platelet-adjusted anticoagulation in patients with VTE were recorded. There was no clear impact of VTE on 1-year overall survival; however, non-CVC-related VTE may be associated with adverse outcomes., Conclusions: Early-onset VTE is a common complication in newly diagnosed AML patients admitted for induction chemotherapy. Leukocytosis is an independent VTE risk factor. The potentially adverse impact of non-CVC-related VTE merits further study., (© 2022 The Authors. European Journal of Haematology published by John Wiley & Sons Ltd.)

Sample loss and contamination are critical preanalytical pitfalls in microscale proteomic applications of nonadhering cells. Common assays and workflows are not easily adoptable to microscale sample sizes of suspension cells due to inadvertent sample loss. This impedes preanalytical experimental manipulation of limited suspension cell samples for microscale proteomics applications, such as encountered for primary human materials. Here, we describe and test a simple manual batch technique for single-step 100-fold concentration of scarce numbers of diluted suspension cells (down to 5000 cells) by volume reduction, facilitating microscale experiments with suspension cells. Pipette tips with heat-sealed orifices (SpinTips) are manufactured within 1 min and serve as versatile microcentrifugation vessels from which supernatant can be aspirated with minimal cell loss. A residual volume of approximately 3 μL can be achieved without visualization of the cell pellet. The results show that SpinTips enable the concentration, medium exchange, washing, and culture of highly limited amounts of suspension cells for functional manipulation and microscale proteomics and are readily incorporated into standard workflows. The application is illustrated by profiling ex vivo responses of primary acute myeloid leukemia (AML) cells from one AML patient to daunorubicin (DNR) to a depth of 3462 quantified proteins with excellent repeatability.

Background: Germline variant evaluation in precision oncology opens new paths toward the identification of patients with genetic tumor risk syndromes and the exploration of therapeutic relevance. Here, we present the results of germline variant analysis and their clinical implications in a precision oncology study for patients with predominantly rare cancers., Patients and Methods: Matched tumor and control genome/exome and RNA sequencing was carried out for 1485 patients with rare cancers (79%) and/or young adults (77% younger than 51 years) in the National Center for Tumor Diseases/German Cancer Consortium (NCT/DKTK) Molecularly Aided Stratification for Tumor Eradication Research (MASTER) trial, a German multicenter, prospective, observational precision oncology study. Clinical and therapeutic relevance of prospective pathogenic germline variant (PGV) evaluation was analyzed and compared to other precision oncology studies., Results: Ten percent of patients (n = 157) harbored PGVs in 35 genes associated with autosomal dominant cancer predisposition, whereof up to 75% were unknown before study participation. Another 5% of patients (n = 75) were heterozygous carriers for recessive genetic tumor risk syndromes. Particularly, high PGV yields were found in patients with gastrointestinal stromal tumors (GISTs) (28%, n = 11/40), and more specifically in wild-type GISTs (50%, n = 10/20), leiomyosarcomas (21%, n = 19/89), and hepatopancreaticobiliary cancers (16%, n = 16/97). Forty-five percent of PGVs (n = 100/221) supported treatment recommendations, and its implementation led to a clinical benefit in 40% of patients (n = 10/25). A comparison of different precision oncology studies revealed variable PGV yields and considerable differences in germline variant analysis workflows. We therefore propose a detailed workflow for germline variant evaluation., Conclusions: Genetic germline testing in patients with rare cancers can identify the very first patient in a hereditary cancer family and can lead to clinical benefit in a broad range of entities. Its routine implementation in precision oncology accompanied by the harmonization of germline variant evaluation workflows will increase clinical benefit and boost research., Competing Interests: Disclosure AJ: Honoraria: AstraZeneca. CH: Honoraria: Roche, Novartis; research funding: Boehringer Ingelheim; consulting or advisory board: Boehringer Ingelheim. CVG: Employed by Illumina since August 2021. LM reports non-financial support from Celgene outside the submitted work (travel expenses). MAu: Consulting or advisory board membership: Bristol-Myers Squibb, Ipsen, Merck KGaA, MSD Sharp & Dohme, Novartis, Pfizer, Roche, PharmaMar; research funding: AstraZeneca, Bristol-Myers Squibb, Eli Lilly, Exelixis, Ipsen, Merck KGaA, Novartis, Pfizer, PharmaMar, Roche; honoraria: Blueprint Medicines, Bristol-Myers Squibb, Ipsen, Janssen-Cilag, Merck KGaA, Pfizer, PharmaMar. PHoh: Advisory board membership: Roche, Deciphera, PharmaMar, Pfizer, BluMedicine, GSK; honoraria: Bristol-Meyers-Squibb, Astrazeneca; research funding: Novartis, Siemens. MK: Honoraria: Bayer, MSD, Eisai, Ipsen, Eli Lilly; research funding: Eli Lilly, Ipsen, Loxo Oncology. RFS: Consulting or advisory board membership: Astellas, Bristol-Myers Squibb, Celgene, Pfizer; research funding: AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, Pfizer, PharmaMar, Roche; honoraria: Daiichi Sankyo, Pfizer. GF: Honoraria: Amgen, Armo Biosciences, Bayer, Bristol-Myers Squibb, Eli Lilly, HRA Pharma, Merck KGaA, MSD Sharp & Dohme, Pierre Fabre, Roche, Sanofi, Servier, Shire; research funding: Merck KGaA. SB: Consulting or advisory board membership: ADC Therapeutics, Blueprint Medicines, Daichii-Sankyo, Deciphera, Eli Lilly, Exelixis, Janssen-Cilag, Mundipharma, Novartis, Plexxikon; honoraria: Bayer, Eli Lilly, GlaxoSmithKline, Novartis, Pfizer, PharmaMar; research funding: Blueprint Medicines, Incyte, Novartis. JTS: Consulting or advisory board membership: AstraZeneca, Bayer, Bristol Myers Squibb, Celgene, Immunocore, Novartis, Roche, Shire; honoraria: AstraZeneca, Aurikamed, Baxalta, Bristol Myers Squibb, Celgene, Falk Foundation, iomedico, Immunocore, Novartis, Roche, Shire; research funding: Bristol-Myers Squibb, Celgene, Roche; minor equity in iTheranostics and Pharma15; member of the Board of Directors for Pharma15, all outside the submitted work. ALI: Honoraria: Bristol-Myers Squibb, Janssen-Cilag, Takeda, Roche. NvB: Consulting or advisory board membership: Novartis; honoraria: Novartis, Takeda. PJJ: Consulting or advisory board membership, honoraria, research funding, travel or accommodation expenses: Abbvie, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Novartis, Pfizer, Roche, Servier. KHM: Consulting: Celgene/BMS, Novartis, Jazz Pharmaceuticals, Pfizer; honoraria: Celgene/BMS, Daiichi Sankyo, Astellas, AbbVie, Novartis; research funding: Celgene. AS: Consulting or advisory board membership, honoraria: AGCT, AstraZeneca, Bayer, Bristol-Myers Squibb, Chugai, Eli Lilly, Illumina, Janssen, MSD Sharp & Dohme, Novartis, Pfizer, Roche, Seattle Genetics, Takeda, Thermo Fisher; research funding: Bayer, Bristol-Myers Squibb, Chugai. WW: Consulting or advisory board membership, honoraria: Agilent, Amgen, Astellas, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, Illumina, Merck, MSD Sharp & Dohme, Pfizer, NewOncology, Novartis, Roche, Takeda; research funding: AstraZeneca, Bristol-Myers Squibb, MSD Sharp & Dohme, Roche. SF: Consulting or advisory board membership: Bayer, Illumina, Roche; honoraria: Amgen, Eli Lilly, PharmaMar, Roche; research funding: AstraZeneca, Pfizer, PharmaMar, Roche; travel or accommodation expenses: Amgen, Eli Lilly, Illumina, PharmaMar, Roche. ES: Honoraria: AstraZeneca, Illumina. All other authors have declared no conflicts of interest. Data sharing All evaluated germline variants can be found in Supplementary Table S5, available at https://doi.org/10.1016/j.annonc.2022.07.008. Sequencing data have been deposited in the European Genome-phenome Archive (https://www.ebi.ac.uk/ega/datasets) under accession EGAS00001005537., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction., (© 2022. The Author(s).)

Background: The multi-receptor tyrosine kinase inhibitor pazopanib is approved for the treatment of advanced soft-tissue sarcoma and has also shown activity in other sarcoma subtypes. However, its clinical efficacy is highly variable, and no reliable predictors exist to select patients who are likely to benefit from this drug., Patients and Methods: We analysed the molecular profiles and clinical outcomes of patients with pazopanib-treated sarcoma enrolled in a prospective observational study by the German Cancer Consortium, DKTK MASTER, that employs whole-genome/exome sequencing and transcriptome sequencing to inform the care of young adults with advanced cancer across histology and patients with rare cancers., Results: Among 109 patients with available whole-genome/exome sequencing data, there was no correlation between clinical parameters, specific genetic alterations or mutational signatures and clinical outcome. In contrast, the analysis of a subcohort of 62 patients who underwent molecular analysis before pazopanib treatment and had transcriptome sequencing data available showed that mRNA levels of NTRK3 (hazard ratio [HR] = 0.53, p = 0.021), IGF1R (HR = 1.82, p = 0.027) and KDR (HR = 0.50, p = 0.011) were independently associated with progression-free survival (PFS). Based on the expression of these receptor tyrosine kinase genes, i.e. the features NTRK3-high, IGF1R-low and KDR-high, we developed a pazopanib efficacy predictor that stratified patients into three groups with significantly different PFS (p < 0.0001). Application of the pazopanib efficacy predictor to an independent cohort of patients with pazopanib-treated sarcoma from DKTK MASTER (n = 43) confirmed its potential to separate patient groups with significantly different PFS (p = 0.02), whereas no such association was observed in patients with sarcoma from DKTK MASTER (n = 97) or The Cancer Genome Atlas sarcoma cohort (n = 256) who were not treated with pazopanib., Conclusion: A score based on the combined expression of NTRK3, IGF1R and KDR allows the identification of patients with sarcoma and with good, intermediate and poor outcome following pazopanib therapy and warrants prospective investigation as a predictive tool to optimise the use of this drug in the clinic., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S. Bauer: Consulting or advisory board membership: Adcendio, Bayer, Blueprint Medicines, Boehringer Ingelheim, Daiichi Sankyo, Deciphera, Eli Lilly, Exelixis, GlaxoSmithKline, Nanobiotix, Novartis, Roche; honoraria: Eli Lilly, Novartis PharmaMar; research funding: Blueprint Medicines, Incyte, Novartis; travel or accommodation expenses: PharmaMar. J.T. Siveke: Consulting or advisory board membership: AstraZeneca, Bayer, Bristol Myers Squibb, Celgene, Immunocore, Novartis, Roche, Shire; honoraria: AstraZeneca, Aurikamed, Baxalta, Bayer, Bristol Myers Squibb, Celgene, Falk Foundation, iomedico, Immunocore, Novartis, Roche, Shire; research funding: Bristol Myers Squibb, Celgene, Roche; board of directors membership: Pharma15; minor equity: iTheranostics, Pharma15. N. von Bubnoff: Honoraria: Novartis, Takeda. C.B. Westphalen: Consulting or advisory board membership, honoraria, travel or accommodation expenses: Bayer, Bristol Myers Squibb, Celgene, GlaxoSmithKline, Ipsen, Janssen, MedScape, MerckSerono, MSD Sharp & Dohme, Rafael Pharmaceuticals, RedHill, Roche, Servier, Shire/Baxalta, SirTex, Taiho; research funding: Roche. W. Weichert: Consulting or advisory board membership, honoraria: ADC, Agilent, Amgen, Astellas, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, Eisai, Eli Lilly, GlaxoSmithKline, Illumina, MerckSerono, Molecular Health, MSD Sharp & Dohme, Novartis, Pfizer, Roche, Siemens, Takeda; research funding: AstraZeneca, Bristol Myers Squibb, MSD Sharp & Dohme, Roche. U. Keilholz: Consulting or advisory board membership, honoraria, research support: Amgen, AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Eli Lilly, Glycotope, Innate, Medimmune, MerckSerono, MSD Sharp & Dohme, Novartis, Pfizer, Roche/Genentech, Sirtex. C. Heining: Consulting or advisory board membership: Boehringer Ingelheim; honoraria: Novartis, Roche; research funding: Boehringer Ingelheim. A. Stenzinger: Consulting or advisory board membership, honoraria: Aignostics, Amgen, AstraZeneca, AGCT, Bayer, Bristol Myers Squibb, Eli Lilly, Illumina, Incyte, Janssen, MSD Sharp & Dohme, Novartis, Pfizer, Roche, Seattle Genetics, Takeda, Thermo Fisher. Research Funding: Bayer, Bristol Myers Squibb, Chugai, Incyte. R.F. Schlenk: Consulting or advisory board membership: Daiichi Sankyo, Pfizer; honoraria: Daiichi Sankyo, Novartis, Pfizer; research funding: Abbvie, AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, Pfizer, PharmaMar, Roche; steering committee membership: Daiichi Sankyo; data monitoring committee membership: BerGenBio, Novartis. S. Fröhling: Consulting or advisory board membership: Bayer, Illumina, Roche; honoraria: Amgen, Eli Lilly, PharmaMar, Roche; research funding: AstraZeneca, Pfizer, PharmaMar, Roche; travel or accommodation expenses: Amgen, Eli Lilly, Illumina, PharmaMar, Roche. The other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The benefit of molecularly-informed therapies in cancer of unknown primary (CUP) is unclear. Here, we use comprehensive molecular characterization by whole genome/exome, transcriptome and methylome analysis in 70 CUP patients to reveal substantial mutational heterogeneity with TP53, MUC16, KRAS, LRP1B and CSMD3 being the most frequently mutated known cancer-related genes. The most common fusion partner is FGFR2, the most common focal homozygous deletion affects CDKN2A. 56/70 (80%) patients receive genomics-based treatment recommendations which are applied in 20/56 (36%) cases. Transcriptome and methylome data provide evidence for the underlying entity in 62/70 (89%) cases. Germline analysis reveals five (likely) pathogenic mutations in five patients. Recommended off-label therapies translate into a mean PFS ratio of 3.6 with a median PFS1 of 2.9 months (17 patients) and a median PFS2 of 7.8 months (20 patients). Our data emphasize the clinical value of molecular analysis and underline the need for innovative, mechanism-based clinical trials., (© 2022. The Author(s).)

The optimal follow-up care for relapse detection in acute myeloid leukemia (AML) patients in first remission after consolidation therapy with intensive chemotherapy is not established. In this retrospective study, we evaluate the diagnostic value of an intensive relapse surveillance strategy by regular bone marrow aspirations (BMA) in these patients. We identified 86 patients with newly diagnosed non-promyelocytic AML who had reached complete remission (CR) after intensive induction and consolidation chemotherapy between 2007 and 2019. Annual relapse rates were 40%, 17%, and 2% in years 1-3, respectively. Patients in CR were surveilled by BMA scheduled every 3 months for 2 years, followed by BMA every 6 months. This surveillance regimen detected 29 of 55 relapses (53%), 11 of which were molecular relapses (20%). The remaining 26 of 55 relapses (47%) were diagnosed by non-surveillance BMA prompted by specific suspicion of relapse. Most patients showed concurrent morphological abnormalities in peripheral blood (PB) at time of relapse. Seven percent of all morphological relapses occurred without simultaneous PB abnormalities and would have been delayed without surveillance BMA. Intensified monthly PB assessment paired with BMA every 3 months during the first 2 years may be a highly sensitive relapse surveillance strategy., (© 2022. The Author(s).)

Objectives and Methods: Intracranial hemorrhage (ICH) in acute myeloid leukemia (AML) patients is a major concern due to the increased risk of mortality. Few studies have examined ICH specifically in newly diagnosed AML patients receiving intensive induction chemotherapy (IC) and prophylactic platelet transfusions during thrombocytopenia <10/nL. This retrospective cohort study included 423 newly diagnosed AML patients without acute promyelocytic leukemia who underwent IC between 2007 and 2019. We assessed risk factors, clinical features, and outcomes of ICH., Results: 17 of 423 patients (4%) suffered ICH during hospital stay, and 4 patients (24%) died directly because of ICH despite routine prophylactic platelet transfusions. Patients with ICH had a negatively impacted overall survival (median OS, 20.1 vs. 104.8 months) and were more likely not to continue with curative treatment. Main risk factors were female gender, severe thrombocytopenia, and decreased fibrinogen. Patients with subsequent ICH also had laboratory signs of liver dysfunction., Conclusions: Intracranial hemorrhage remains a potentially deadly complication with notable incidence despite prophylactic platelet substitution, suggesting that additional prophylactic interventions may be required to further reduce the frequency of ICH in high-risk patients. Unrecognized genetic factors may simultaneously predispose to AML and platelet dysfunction with ICH., (© 2021 The Authors. European Journal of Haematology published by John Wiley & Sons Ltd.)

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies., (© 2022 by The American Society of Hematology.)

Autophagy is an important survival mechanism that allows recycling of nutrients and removal of damaged organelles and has been shown to contribute to the proliferation of acute myeloid leukemia (AML) cells. However, little is known about the mechanism by which autophagy- dependent AML cells can overcome dysfunctional autophagy. In our study we identified autophagy related protein 3 (ATG3) as a crucial autophagy gene for AML cell proliferation by conducting a CRISPR/Cas9 dropout screen with a library targeting around 200 autophagy-related genes. shRNA-mediated loss of ATG3 impaired autophagy function in AML cells and increased their mitochondrial activity and energy metabolism, as shown by elevated mitochondrial ROS generation and mitochondrial respiration. Using tracer-based NMR metabolomics analysis we further demonstrate that the loss of ATG3 resulted in an upregulation of glycolysis, lactate production, and oxidative phosphorylation. Additionally, loss of ATG3 strongly sensitized AML cells to the inhibition of mitochondrial metabolism. These findings highlight the metabolic vulnerabilities that AML cells acquire from autophagy inhibition and support further exploration of combination therapies targeting autophagy and mitochondrial metabolism in AML.

The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patients (31.8%) and resulted in significantly improved overall response and disease control rates (23.9% and 55.3%) compared with previous therapies, translating into a progression-free survival ratio >1.3 in 35.7% of patients. These data demonstrate the benefit of molecular stratification in rare cancers and represent a resource that may promote clinical trial access and drug approvals in this underserved patient population. SIGNIFICANCE: Rare cancers are difficult to treat; in particular, molecular pathogenesis-oriented medical therapies are often lacking. This study shows that whole-genome/exome and RNA sequencing enables molecularly informed treatments that lead to clinical benefit in a substantial proportion of patients with advanced rare cancers and paves the way for future clinical trials. See related commentary by Eggermont et al., p. 2677 . This article is highlighted in the In This Issue feature, p. 2659 ., (©2021 American Association for Cancer Research.)

Treatment-related complications contribute substantially to morbidity and mortality in acute myeloid leukemia (AML) patients undergoing induction chemotherapy. Although AML patients are susceptible to fluid overload (FO) (e.g., in the context of chemotherapy protocols, during sepsis treatment or to prevent tumor lysis syndrome), little attention has been paid to its role in AML patients undergoing induction chemotherapy. AML patients receiving induction chemotherapy between 2014 and 2019 were included in this study. FO was defined as ≥5% weight gain on day 7 of induction chemotherapy compared to baseline weight determined on the day of admission. We found FO in 23 (12%) of 187 AML patients undergoing induction chemotherapy. Application of >100 ml crystalloid fluids/kg body weight until day 7 of induction chemotherapy was identified as an independent risk factor for FO. AML patients with FO suffered from a significantly increased 90-day mortality rate and FO was demonstrated as an independent risk factor for 90-day mortality. Our data suggests an individualized, weight-adjusted calculation of crystalloid fluids in order to prevent FO-related morbidity and mortality in AML patients during induction chemotherapy. Prospective trials are required to determine the adequate fluid management in this patient population., (© 2021. The Author(s).)

Early results of the randomized placebo-controlled SORAML trial showed that, in patients with newly diagnosed acute myeloid leukaemia (AML), sorafenib led to a significant improvement in event-free (EFS) and relapse-free survival (RFS). In order to describe second-line treatments and their implications on overall survival (OS), we performed a study after a median follow-up time of 78 months. Newly diagnosed fit AML patients aged ≤60 years received sorafenib (n = 134) or placebo (n = 133) in addition to standard chemotherapy and as maintenance treatment. The 5-year EFS was 41 versus 27% (HR 0.68; p = 0.011) and 5-year RFS was 53 versus 36% (HR 0.64; p = 0.035). Allogeneic stem cell transplantation (allo SCT) was performed in 88% of the relapsed patients. Four years after salvage allo SCT, the cumulative incidence of relapse was 54 versus 35%, and OS was 32 versus 50%. The 5-year OS from randomization in all study patients was 61 versus 53% (HR 0.82; p = 0.282). In conclusion, the addition of sorafenib to chemotherapy led to a significant prolongation of EFS and RFS. Although the OS benefit did not reach statistical significance, these results confirm the antileukaemic activity of sorafenib., (© 2021. The Author(s).)

Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Acute kidney injury (AKI) complicates the clinical course of hospitalized patients by increasing need for intensive care treatment and mortality. There is only little data about its impact on AML patients undergoing intensive induction chemotherapy. In this study, we analyzed the incidence as well as risk factors for AKI development and its impact on the clinical course of AML patients undergoing induction chemotherapy. We retrospectively analyzed data from 401 AML patients undergoing induction chemotherapy between 2007 and 2019. AKI was defined and stratified according to KIDGO criteria by referring to a defined baseline serum creatinine measured on day 1 of induction chemotherapy. Seventy-two of 401 (18%) AML patients suffered from AKI during induction chemotherapy. AML patients with AKI had more days with fever (7 vs. 5, p = 0.028) and were more often treated on intensive care unit (45.8% vs. 10.6%, p < 0.001). AML patients with AKI had a significantly lower complete remission rate after induction chemotherapy and, with 402 days, a significantly shorter median overall survival (OS) (median OS for AML patients without AKI not reached). In this study, we demonstrate that the KIDGO classification allows mortality risk stratification for AML patients undergoing induction chemotherapy. Relatively mild AKI episodes have impact on the clinical course of these patients and can lead to chronic impairment of kidney function. Therefore, we recommend incorporating risk factors for AKI in decision-making considering nutrition, fluid management, as well as the choice of potentially nephrotoxic medication in order to decrease the incidence of AKI.

Patients with acute myeloid leukemia (AML) are often exposed to broad-spectrum antibiotics and thus at high risk of Clostridioides difficile infections (CDI). As bacterial infections are a common cause for treatment-related mortality in these patients, we conducted a retrospective study to analyze the incidence of CDI and to evaluate risk factors for CDI in a large uniformly treated AML cohort. A total of 415 AML patients undergoing intensive induction chemotherapy between 2007 and 2019 were included in this retrospective analysis. Patients presenting with diarrhea and positive stool testing for toxin-producing Clostridioides difficile were defined to have CDI. CDI was diagnosed in 37 (8.9%) of 415 AML patients with decreasing CDI rates between 2013 and 2019 versus 2007 to 2012. Days with fever, exposition to carbapenems, and glycopeptides were significantly associated with CDI in AML patients. Clinical endpoints such as length of hospital stay, admission to ICU, response rates, and survival were not adversely affected. We identified febrile episodes and exposition to carbapenems and glycopeptides as risk factors for CDI in AML patients undergoing induction chemotherapy, thereby highlighting the importance of interdisciplinary antibiotic stewardship programs guiding treatment strategies in AML patients with infectious complications to carefully balance risks and benefits of anti-infective agents.

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (n = 496) and a validation set (DKTK MASTER cohort, n = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately reanalyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background and Objectives: Red blood cell (RBC) transfusions are needed by almost every acute myeloid leukaemia (AML) patient undergoing induction chemotherapy and constitute a cornerstone in supportive measures for cancer patients in general. Randomized controlled trials have shown non-inferiority or even superiority of restrictive transfusion guidelines over liberal transfusion guidelines in specific clinical situations outside of medical oncology. In this study, we analysed whether more restrictive RBC transfusion reduces blood use without affecting hard outcomes., Materials and Methods: A total of 352 AML patients diagnosed between 2007 and 2018 and undergoing intensive induction chemotherapy were included in this retrospective analysis. In the less restrictive transfusion group, patients received RBC transfusion for haemoglobin levels below 8 g/dl (2007-2014). In the restrictive transfusion group, patients received RBC transfusion for haemoglobin levels below 7 g/dl (2016-2018). Liberal transfusion triggers were never endorsed., Results: A total of 268 (76·1%) and 84 (23·9%) AML patients fell into the less restrictive and restrictive transfusion groups, respectively. The less restrictive transfusion group had 1 g/dl higher mean haemoglobin levels, received their first RBC transfusions earlier and needed 1·5 more units of RBC during the hospital stay of induction chemotherapy. Febrile episodes, C-reactive protein levels, admission to the intensive care unit, length of hospital stay as well as response and survival rates did not differ between the two cohorts., Conclusion: From our retrospective analysis, we conclude that a more restrictive transfusion trigger does not affect important outcomes of AML patients. The opportunity to test possible effects of the more severe anaemia in the restrictive transfusion group on quality of life was missed., (© 2020 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

BACKGROUNDNeurofibroma/schwannoma hybrid nerve sheath tumors (N/S HNSTs) are neoplasms associated with larger nerves that occur sporadically and in the context of schwannomatosis or neurofibromatosis type 2 or 1. Clinical management of N/S HNSTs is challenging, especially for large tumors, and established systemic treatments are lacking.METHODSWe used next-generation sequencing and array-based DNA methylation profiling to determine the clinically actionable genomic and epigenomic landscapes of N/S HNSTs.RESULTSWhole-exome sequencing within a precision oncology program identified an activating mutation (p.Asp769Tyr) in the catalytic domain of the ERBB2 receptor tyrosine kinase in a patient with schwannomatosis-associated N/S HNST, and targeted treatment with the small-molecule ERBB inhibitor lapatinib led to prolonged clinical benefit and a lasting radiographic and metabolic response. Analysis of a multicenter validation cohort revealed recurrent ERBB2 mutations (p.Leu755Ser, p.Asp769Tyr, p.Val777Leu) in N/S HNSTs occurring in patients who met diagnostic criteria for sporadic schwannomatosis (3 of 7 patients), but not in N/S HNSTs arising in the context of neurofibromatosis (6 patients) or outside a tumor syndrome (1 patient), and showed that ERBB2-mutant N/S HNSTs cluster in a distinct subgroup of peripheral nerve sheath tumors based on genome-wide DNA methylation patterns.CONCLUSIONThese findings uncover a key biological feature of N/S HNSTs that may have important diagnostic and therapeutic implications.FUNDINGThis work was supported by grant H021 from DKFZ-HIPO, the University Cancer Center Frankfurt, and the Frankfurt Research Funding Clinician Scientist Program.

The hierarchical organization of the hematopoietic system requires hematopoietic stem cells (HSCs) capable of self-renewal and multilineage differentiation to produce all cellular lineages of the blood. Novel techniques of purification of hematopoietic subpopulations and their functional characterization have defined individual steps of their lineage commitment. A detailed molecular map of the selective autophagy landscape governing self-renewal, maintenance, and differentiation of HSCs and their progeny during early and terminal differentiation has not yet been drawn. However, the importance of selective autophagy pathways is increasingly being recognized, as this evolutionary conserved degradation pathway is instrumental in orchestrating intracellular turnover of macromolecular complexes and organelles to meet the specific needs of various hematopoietic cells. For instance, mitophagy has clearly been demonstrated to regulate mitochondrial homeostasis in HSCs, thereby defining their metabolic fate decisions. This review focuses on the emerging evidence supporting an important role of selective autophagy in regulating normal hematopoiesis. As this has uncovered important features in malignant hematopoiesis, the review also covers aspects of selective autophagy pathways affected in the development and progression of leukemia and lymphoma. A molecular understanding of the unique requirements of selective autophagy may open new avenues for specific therapeutic intervention., (Copyright © 2019. Published by Elsevier Ltd.)

Clonal hematopoiesis of indeterminate potential (CHIP) is caused by recurrent somatic mutations leading to clonal blood cell expansion. However, direct evidence of the fitness of CHIP-mutated human hematopoietic stem cells (HSCs) in blood reconstitution is lacking. Because myeloablative treatment and transplantation enforce stress on HSCs, we followed 81 patients with solid tumors or lymphoid diseases undergoing autologous stem cell transplantation (ASCT) for the development of CHIP. We found a high incidence of CHIP (22%) after ASCT with a high mean variant allele frequency (VAF) of 10.7%. Most mutations were already present in the graft, albeit at lower VAFs, demonstrating a selective reconstitution advantage of mutated HSCs after ASCT. However, patients with CHIP mutations in DNA-damage response genes showed delayed neutrophil reconstitution. Thus, CHIP-mutated stem and progenitor cells largely gain on clone size upon ASCT-related blood reconstitution, leading to an increased future risk of CHIP-associated complications., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Autophagy is essential for cellular homeostasis and when deregulated this survival mechanism has been associated with disease development. Inhibition of autophagy initiation by inhibiting the kinase ULK1 (Unc-51-like autophagy activating kinase 1) has been proposed as a potential cancer therapy. While inhibitors and crystal structures of ULK1 have been reported, little is known about the other closely related kinase ULK2 (Unc-51-like autophagy activating kinase 2). Here, we present the crystal structure of ULK2 in complex with ATP competitive inhibitors. Surprisingly, the ULK2 structure revealed a dimeric assembly reminiscent of dimeric arrangements of auto-activating kinases suggesting a role for this association in ULK activation. Screening of a kinase focused library of pre-clinical and clinical compounds revealed several potent ULK1/2 inhibitors and good correlation of inhibitor-binding behavior with both ULK kinases. Aurora A was identified as a major off-target of currently used ULK1 inhibitors. Autophagic flux assays demonstrated that this off-target activity by strongly inducing autophagy in different cellular systems conferred an additional layer of complexity in the interpretation of cellular data. The data presented here provide structural models and chemical starting points for the development of ULK1/2 dual inhibitors with improved selectivity for future exploitation of autophagy inhibition., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In many countries, a majority of cancer patients are not treated at comprehensive cancer centers. Even for those that are, parts of the treatment or follow-up may be carried out in local community hospitals or in private practices. How to assure quality in cancer care and create innovation? How to integrate decentralized versus centralized patient care, education, and cancer research? Outlined here is a 360° view of outreach to include all stakeholders-most importantly patients and their families, patient advocacy groups, healthcare providers, health insurers, and policymakers., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity., (© 2019 by The American Society of Hematology.)

Purpose The initial results of the APL0406 trial showed that the combination of all- trans-retinoic acid (ATRA) and arsenic trioxide (ATO) is at least not inferior to standard ATRA and chemotherapy (CHT) in first-line therapy of low- or intermediate-risk acute promyelocytic leukemia (APL). We herein report the final analysis on the complete series of patients enrolled onto this trial. Patients and Methods The APL0406 study was a prospective, randomized, multicenter, open-label, phase III noninferiority trial. Eligible patients were adults between 18 and 71 years of age with newly diagnosed, low- or intermediate-risk APL (WBC at diagnosis ≤ 10 × 10 9 /L). Overall, 276 patients were randomly assigned to receive ATRA-ATO or ATRA-CHT between October 2007 and January 2013. Results Of 263 patients evaluable for response to induction, 127 (100%) of 127 patients and 132 (97%) of 136 patients achieved complete remission (CR) in the ATRA-ATO and ATRA-CHT arms, respectively ( P = .12). After a median follow-up of 40.6 months, the event-free survival, cumulative incidence of relapse, and overall survival at 50 months for patients in the ATRA-ATO versus ATRA-CHT arms were 97.3% v 80%, 1.9% v 13.9%, and 99.2% v 92.6%, respectively ( P < .001, P = .0013, and P = .0073, respectively). Postinduction events included two relapses and one death in CR in the ATRA-ATO arm and two instances of molecular resistance after third consolidation, 15 relapses, and five deaths in CR in the ATRA-CHT arm. Two patients in the ATRA-CHT arm developed a therapy-related myeloid neoplasm. Conclusion These results show that the advantages of ATRA-ATO over ATRA-CHT increase over time and that there is significantly greater and more sustained antileukemic efficacy of ATO-ATRA compared with ATRA-CHT in low- and intermediate-risk APL.

Background: Preclinical data and results from non-randomised trials suggest that the multikinase inhibitor sorafenib might be an effective drug for the treatment of acute myeloid leukaemia. We investigated the efficacy and tolerability of sorafenib versus placebo in addition to standard chemotherapy in patients with acute myeloid leukaemia aged 60 years or younger., Methods: This randomised, double-blind, placebo-controlled, phase 2 trial was done at 25 sites in Germany. We enrolled patients aged 18-60 years with newly diagnosed, previously untreated acute myeloid leukaemia who had a WHO clinical performance score 0-2, adequate renal and liver function, no cardiac comorbidities, and no recent trauma or operation. Patients were randomly assigned (1:1) to receive two cycles of induction therapy with daunorubicin (60 mg/m(2) on days 3-5) plus cytarabine (100 mg/m(2) on days 1-7), followed by three cycles of high-dose cytarabine consolidation therapy (3 g/m(2) twice daily on days 1, 3, and 5) plus either sorafenib (400 mg twice daily) or placebo on days 10-19 of induction cycles 1 and 2, from day 8 of each consolidation, and as maintenance for 12 months. Allogeneic stem-cell transplantation was scheduled for all intermediate-risk patients with a sibling donor and for all high-risk patients with a matched donor in first remission. Computer-generated randomisation was done in blocks. The primary endpoint was event-free survival, with an event defined as either primary treatment failure or relapse or death, assessed in all randomised patients who received at least one dose of study treatment. We report the final analysis. This trial is registered with ClinicalTrials.gov, number NCT00893373, and the EU Clinical Trials Register (2008-004968-40)., Findings: Between March 27, 2009, and Nov 28, 2011, 276 patients were enrolled and randomised, of whom nine did not receive study medication. 267 patients were included in the primary analysis (placebo, n=133; sorafenib, n=134). With a median follow-up of 36 months (IQR 35·5-38·1), median event-free survival was 9 months (95% CI 4-15) in the placebo group versus 21 months (9-32) in the sorafenib group, corresponding to a 3-year event-free survival of 22% (95% CI 13-32) in the placebo group versus 40% (29-51) in the sorafenib group (hazard ratio [HR] 0·64, 95% CI; 0·45-0·91; p=0·013). The most common grade 3-4 adverse events in both groups were fever (71 [53%] in the placebo group vs 73 [54%] in the sorafenib group), infections (55 [41%] vs 46 [34%]), pneumonia (21 [16%] vs 20 [14%]), and pain (13 [10%] vs 15 [11%]). Grade 3 or worse adverse events that were significantly more common in the sorafenib group than the placebo group were fever (relative risk [RR] 1·54, 95% CI 1·04-2·28), diarrhoea (RR 7·89, 2·94-25·2), bleeding (RR 3·75, 1·5-10·0), cardiac events (RR 3·46, 1·15-11·8), hand-foot-skin reaction (only in sorafenib group), and rash (RR 4·06, 1·25-15·7)., Interpretation: In patients with acute myeloid leukaemia aged 60 years or younger, the addition of sorafenib to standard chemotherapy has antileukaemic efficacy but also increased toxicity. Our findings suggest that kinase inhibitors could be a useful addition to curative treatment for acute myeloid leukaemia. Overall survival after long-term follow-up and strategies to reduce toxicity are needed to determine the future role of sorafenib in treatment of this disease., Funding: Bayer HealthCare., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

FLT3 and c-KIT are crucial regulators of hematopoietic stem and progenitor cells. We investigated the role of STS1 and STS2 on FLT3 and c-KIT phosphorylation, activity, and function in normal and stress-induced hematopoiesis. STS1/STS2-deficient mice show a profound expansion of multipotent progenitor and lymphoid primed multipotent progenitor cells with elevated colony-forming capacity. Although long-term hematopoietic stem cells are not increased in numbers, lack of STS1 and STS2 significantly promotes long-term repopulation activity, demonstrating a pivotal role of STS1/STS2 in regulating hematopoietic stem and progenitor cell fitness. Biochemical analysis identified STS1/STS2 as direct phosphatases of FLT3 and c-KIT. Loss of STS1/STS2 induces hyperphosphorylation of FLT3, enhances AKT signaling, and confers a strong proliferative advantage. Therefore, our study reveals that STS1 and STS2 may serve as novel pharmaceutical targets to improve hematopoietic recovery after bone marrow transplantation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: All-trans retinoic acid (ATRA) with chemotherapy is the standard of care for acute promyelocytic leukemia (APL), resulting in cure rates exceeding 80%. Pilot studies of treatment with arsenic trioxide with or without ATRA have shown high efficacy and reduced hematologic toxicity., Methods: We conducted a phase 3, multicenter trial comparing ATRA plus chemotherapy with ATRA plus arsenic trioxide in patients with APL classified as low-to-intermediate risk (white-cell count, ≤10×10(9) per liter). Patients were randomly assigned to receive either ATRA plus arsenic trioxide for induction and consolidation therapy or standard ATRA-idarubicin induction therapy followed by three cycles of consolidation therapy with ATRA plus chemotherapy and maintenance therapy with low-dose chemotherapy and ATRA. The study was designed as a noninferiority trial to show that the difference between the rates of event-free survival at 2 years in the two groups was not greater than 5%., Results: Complete remission was achieved in all 77 patients in the ATRA-arsenic trioxide group who could be evaluated (100%) and in 75 of 79 patients in the ATRA-chemotherapy group (95%) (P=0.12). The median follow-up was 34.4 months. Two-year event-free survival rates were 97% in the ATRA-arsenic trioxide group and 86% in the ATRA-chemotherapy group (95% confidence interval for the difference, 2 to 22 percentage points; P<0.001 for noninferiority and P=0.02 for superiority of ATRA-arsenic trioxide). Overall survival was also better with ATRA-arsenic trioxide (P=0.02). As compared with ATRA-chemotherapy, ATRA-arsenic trioxide was associated with less hematologic toxicity and fewer infections but with more hepatic toxicity., Conclusions: ATRA plus arsenic trioxide is at least not inferior and may be superior to ATRA plus chemotherapy in the treatment of patients with low-to-intermediate-risk APL. (Funded by Associazione Italiana contro le Leucemie and others; ClinicalTrials.gov number, NCT00482833.).

Balancing the processes of hematopoietic stem cell (HSC) differentiation and self-renewal is critical for maintaining a lifelong supply of blood cells. The bone marrow (BM) produces a stable output of newly generated cells, but immunologic stress conditions inducing leukopenia increase the demand for peripheral blood cell supply. Here we demonstrate that the proinflammatory cytokine interferon-γ (IFN-γ) impairs maintenance of HSCs by directly reducing their proliferative capacity and that IFN-γ impairs restoration of HSC numbers upon viral infection. We show that IFN-γ reduces thrombopoietin (TPO)-mediated phosphorylation of signal transducer and activator of transcription (STAT) 5, an important positive regulator of HSC self-renewal. IFN-γ also induced expression of suppressor of cytokine signaling (SOCS) 1 in HSCs, and we demonstrate that SOCS1 expression is sufficient to inhibit TPO-induced STAT5 phosphorylation. Furthermore, IFN-γ deregulates expression of STAT5-mediated cell-cycle genes cyclin D1 and p57. These findings suggest that IFN-γ is a negative modulator of HSC self-renewal by modifying cytokine responses and expression of genes involved in HSC proliferation. We postulate that the occurrence of BM failure in chronic inflammatory conditions, such as aplastic anemia, HIV, and graft-versus-host disease, is related to a sustained impairment of HSC self-renewal caused by chronic IFN-γ signaling in these disorders.

Ubiquitination has long been recognised as a key determinator of protein fate by tagging proteins for proteasomal degradation. Most recently, the ability of conjugated ubiquitin chains to confer selectivity to autophagy was demonstrated. Although autophagy was first believed to be a bulk, non-selective 'self-eating' degradative process, the molecular mechanisms of selectivity are now starting to emerge. With the discovery of autophagy receptors - which bind both ubiquitinated substrates and autophagy specific light chain 3 (LC3) modifier on the inner sheath of autophagosomes - a new pathway of selective autophagy is being unravelled. In this review, we focus on the special role of ubiquitin signals and selective autophagy receptors in sorting a variety of autophagic cargos.

Activating mutations in the receptor tyrosine kinase FLT3 are frequently found in acute myelogenous leukemia patients and confer poor clinical prognosis. It is unclear how leukemic blasts escape cytokine control that regulates normal hematopoiesis. We have recently demonstrated that FLT3-internal tandem duplication (ITD), when localized to the biosynthetic compartment, aberrantly activates STAT5. Here, we show that one of the target genes induced by STAT5 is suppressor of cytokine signaling (SOCS)1-a surprising finding for a known tumor suppressor. Although SOCS1 expression in murine bone marrow severely impaired cytokine-induced colony growth, it failed to inhibit FLT3-ITD-supported colony growth, indicating resistance of FLT3-ITD to SOCS1. In addition, SOCS1 coexpression did not affect FLT3-ITD-mediated signaling or proliferation. Importantly, SOCS1 coexpression inhibited interferon-α and interferon-γ signaling and protected FLT3-ITD hematopoietic cells from interferon-mediated growth inhibitory effects. In a murine bone marrow transplantation model, the coexpression of SOCS1 and FLT3-ITD significantly shortened the latency of a myeloproliferative disease compared with FLT3-ITD alone (P < .01). Mechanistically, SOCS proteins shield FLT3-ITD from external cytokine control, thereby promoting leukemogenesis. The data demonstrate that SOCS1 acts as a conditional oncogene, providing novel molecular insights into cytokine resistance in oncogenic transformation. Restoring cytokine control may provide a new way of therapeutic intervention.

Introduction: Older patients with acute myeloid leukemia (AML) experience short survival despite intensive chemotherapy. Azacitidine has promising activity in patients with low proliferating AML. The aim of this dose-finding part of this trial was to evaluate feasibility and safety of azacitidine combined with a cytarabine- and daunorubicin-based chemotherapy in older patients with AML., Trial Design: Prospective, randomised, open, phase II trial with parallel group design and fixed sample size., Patients and Methods: Patients aged 61 years or older, with untreated acute myeloid leukemia with a leukocyte count of <20,000/µl at the time of study entry and adequate organ function were eligible. Patients were randomised to receive azacitidine either 37.5 (dose level 1) or 75 mg/sqm (dose level 2) for five days before each cycle of induction (7+3 cytarabine plus daunorubicine) and consolidation (intermediate-dose cytarabine) therapy. Dose-limiting toxicity was the primary endpoint., Results: Six patients each were randomised into each dose level and evaluable for analysis. No dose-limiting toxicity occurred in either dose level. Nine serious adverse events occurred in five patients (three in the 37.5 mg, two in the 75 mg arm) with two fatal outcomes. Two patients at the 37.5 mg/sqm dose level and four patients at the 75 mg/sqm level achieved a complete remission after induction therapy. Median overall survival was 266 days and median event-free survival 215 days after a median follow up of 616 days., Conclusions: The combination of azacitidine 75 mg/sqm with standard induction therapy is feasible in older patients with AML and was selected as an investigational arm in the randomised controlled part of this phase-II study, which is currently halted due to an increased cardiac toxicity observed in the experimental arm., Trial Registration: This trial is registered at clinical trials.gov (identifier: NCT00915252).

Objective: Platelet-derived growth factor receptors α and β (PDGFRA, PDGFRB) are frequently expressed on hematopoietic cells and regulate cellular responses such as proliferation, differentiation, survival, and transformation. Stimulation by autocrine loops or activation by chromosomal translocation makes them important factors in development of hematopoietic disorders. Interaction with the ligand PDGF results in activation of the tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for molecules containing Src homology 2 domains. We hypothesized that one such protein may be Lnk, a negative regulator of cytokine receptors, including Mpl, EpoR, c-Kit, and c-Fms., Materials and Methods: Interaction of Lnk with PDGFRA, PDGFRB, or leukemogenic FIP1L1-PDGFRA or TEL-PDGFRB was studied in cotransfected 293T cells. Effects of Lnk on PDGFR signaling were shown in 293T and NIH3T3 cells, whereas its influence on either PDGF-dependent or factor-independent growth was investigated using Ba/F3 or 32D cells expressing wild-type PDGFR, FIP1L1-PDGFRA, or TEL-PDGFRB., Results: We show that Lnk binds to PDGFR after exposure of cells to PDGF. Furthermore, Lnk can bind the FIP1L1-PDGFRA fusion protein. Mutation or deletion of the Lnk Src homology 2 domain completely abolished binding of Lnk to FIP1L1-PDGFRA, but just partly prevented binding to PDGFRA or PDGFRB. Expression of Lnk inhibited proliferation of PDGF-dependent Ba/F3 cells and diminished phosphorylation of Erk in PDGF-treated NIH3T3. 32D cells transformed by either FIP1L1-PDGFRA or TEL-PDGFRB stopped growing when Lnk was expressed., Conclusions: Lnk is a negative regulator of PDGFR signaling. Development of Lnk mimetic drugs might provide a novel therapeutic strategy for myeloproliferative disorders., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The M-CSFR (c-Fms) participates in proliferation, differentiation, and survival of macrophages and is involved in the regulation of distinct macrophage functions. Interaction with the ligand M-CSF results in phosphorylation of tyrosine residues on c-Fms, thereby creating binding sites for molecules containing SH2 domains. Lnk is a SH2 domain adaptor protein that negatively regulates hematopoietic cytokine receptors. Here, we show that Lnk binds to c-Fms. Biological and functional effects of this interaction were examined in macrophages from Lnk-deficient (KO) and WT mice. Clonogenic assays demonstrated an elevated number of M-CFUs in the bone marrow of Lnk KO mice. Furthermore, the M-CSF-induced phosphorylation of Akt in Lnk KO macrophages was increased and prolonged, whereas phosphorylation of Erk was diminished. Zymosan-stimulated production of ROS was increased dramatically in a M-CSF-dependent manner in Lnk KO macrophages. Lastly, Lnk inhibited M-CSF-induced migration of macrophages. In summary, we show that Lnk binds to c-Fms and can blunt M-CSF stimulation. Modulation of levels of Lnk in macrophages may provide a unique therapeutic approach to increase innate host defenses.

Acute myeloid leukemia (AML) is a malignant disease of the bone marrow. Despite intensive treatment only one third of AML patients are cured. Numerous genetic events have been identified in the last years that have shed light into the mechanisms dictating increased self-renewal, proliferation, survival and block in differentiation. It is increasingly recognized that AML represents a hierarchical disease, originating from a leukemia stem cell population. Sophisticated animal models have helped to elucidate rate-limiting steps in initiation, development and maintenance of AML. This review discusses the fundamental genetic events identified to date that determine the pathogenesis of AML, with particular emphasis on the oncogenic signaling events that result from translocation of myeloid transcription factors and mutations in receptor tyrosine kinases. Our current understanding of the biology of AML has fueled the development of promising anti-leukemic agents, which may improve the treatment of the disease in the future.