Abstract Book for the 27th Congress of the European Hematology Association : HemaSphere

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Abstract Book for the 27th Congress of the European Hematology Association

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doi: 10.1097/01.HS9.0000852292.38263.b8
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Presidential Symposium


H. Erba1,*, P. Montesinos2, R. Vrhovac3, E. Patkowska4, H.-J. Kim5, P. Zak6, P.-N. Wang7, T. Mitov8, J. Hanyok9, L. Liu9, A. Benzohra9, A. Lesegretain9, J. Cortes10, A. Perl11, M. Sekeres12, H. Dombret13, S. Amadori14, J. Wang15, M. Levis16, R. Schlenk17

1Duke Cancer Institute, Durham, NC, United States of America; 2La Fe University and Polytechnic Hospital, Valencia, Spain; 3University Hospital Centre Zagreb, Zagreb, Croatia; 4Institute of Hematology and Blood Transfusion, Warsaw, Poland; 5The Catholic University of Korea, Seoul St. Mary’s Hospital, Seoul, South Korea; 6University Hospital Hradec Kralove, Hradec Kralove, Czechia; 7Chang Gung Medical Foundation, Linkou, Taiwan; 8Daiichi Sankyo UK Ltd, Uxbridge, United Kingdom; 9Daiichi Sankyo, Inc, Basking Ridge, NJ; 10Augusta University Medical Center, Augusta, GA; 11University of Pennsylvania, Philadelphia, PA; 12University of Miami Health System, Miami, FL, United States of America; 13Saint Louis Hospital, University of Paris, Paris, France; 14Tor Vergata Polyclinic Hospital Rome, Rome, Italy; 15Institute of Hematology and Blood Diseases Hospital, Tianjin, China; 16Johns Hopkins University, Baltimore, MD, United States of America; 17Heidelberg University Hospital and German Cancer Research Center, Heidelberg, Germany

Background: Quizartinib (Quiz) is an oral, highly potent, and selective type II FLT3 inhibitor with single-agent activity in relapsed/refractory FLT3–internal tandem duplication positive (FLT3-ITD+) acute myeloid leukemia (AML). This is the first report of the global, randomized, double-blind, placebo (PBO)-controlled phase 3 QuANTUM-First trial (NCT02668653).

Aims: QuANTUM-First aimed to determine if the addition of Quiz to standard induction (IND) and post remission (including allogeneic hematopoietic cell transplant [allo-HCT]) in first complete remission [CR1]) consolidation followed by single-agent continuation therapy for up to 3 years improved survival compared with chemotherapy alone in patients (pts) with newly diagnosed FLT3-ITD+ AML.

Methods: Pts aged 18-75 y with newly diagnosed AML were centrally screened for FLT3-ITD prior to initiation of IND with cytarabine 100 mg/m2/day (200 mg/m2/day if institutional standard) for 7 days and anthracycline (daunorubicin 60 mg/m2/day or idarubicin 12 mg/m2/day) for 3 days. Pts at 193 sites in 26 countries who were FLT3-ITD+ provided informed consent and were randomized to Quiz (40 mg/day days 8-21) or PBO and were stratified by region (North America, Europe, and Asia/Other regions), age (<60 y, ≥60 y), and white blood cell count (<40×109/L, ≥40×109/L) at diagnosis. A second IND was allowed if residual AML was noted at the post-IND marrow exam. Pts who achieved CR or CR with incomplete hematologic recovery (CRi) received up to 4 cycles of high-dose cytarabine plus Quiz (40 mg/day) or PBO and/or allo-HCT followed by up to 3 y of continuation therapy with Quiz (30-60 mg/day) or PBO. The primary endpoint was overall survival (OS).

Results: Between September 2016 and August 2019, 3468 pts were screened, and 539 pts with FLT3-ITD+ AML were randomized to Quiz (n=268) or PBO (n=271). The median age was 56 y (range, 20-75 y). Baseline pt and disease characteristics, including FLT3-ITD variant allele frequency, were balanced between the 2 arms. At data cutoff (August 2021), the median follow-up was 39.2 months and 58 pts remained on continuation therapy. OS was significantly longer in the Quiz arm than the PBO arm (hazard ratio [HR], 0.776; 95% CI, 0.615-0.979; 2-sided P=.0324). Median OS was 31.9 mo with Quiz vs 15.1 mo with PBO (Figure). CR/CRi rates were 71.6% and 64.9%, respectively. Allo-HCT in CR1 was performed in 157 pts (Quiz, 31%; PBO, 27%). When censored for allo-HCT, OS trended longer with Quiz vs PBO (HR, 0.752; 95% CI, 0.562-1.008; 2-sided P=0.055). Relapse-free survival was longer with Quiz than PBO (HR, 0.733; 95% CI, 0.554-0.969). Although rates of grade ≥3 adverse events (AEs) were similar across arms, grade ≥3 neutropenia was more frequent in the Quiz arm (18.1% vs 8.6%). Discontinuations due to AEs occurred in 20.4% of Quiz and 8.6% of PBO pts. A total of 56 treatment-emergent AEs were associated with a fatal outcome (Quiz, 11.3%; PBO, 9.7%), mostly due to infections. Grade 3/4 electrocardiogram QT prolonged occurred in 3.0% of Quiz vs 1.1% of PBO pts.



Summary/Conclusion: These pivotal findings show that the addition of Quiz to standard chemotherapy and up to 3 years of continuation therapy yielded statistically significant and clinically meaningful improvements to OS in adults with newly diagnosed FLT3-ITD+ AML up to age 75 y. The manageable safety profile further supports use of Quiz in combination with standard therapy, including allo-HCT, in FLT3-ITD+ AML.


D. Noerenberg1,*, F. Briest1, C. Hennch1, K. Yoshida2,3, J. Nimo1, R. Hablesreiter1, Y. Takeuchi2, D. Sasca4, H. Ueno2, L. Mansouri5, Y. Inoue2, L. Wiegand1, A. M. Staiger6,7, B. Casadei8,9, M. Ziepert10, F. Asmar11, P. Korkolopoulou12, M. Kirchner13, P. Mertins13, J. Weiner14, E. Toth15, T. Weber16, A. Warth17, T. Schneider18, R.-M. Amini19, W. Klapper20, M. Hummel21,22, V. Poeschel23, G. Kanellis24, A. Rosenwald25, G. Held23,26, E. Campo27,28,29, K. Stamatopoulos5,30, I. Anagnostopoulos21,25, L. Bullinger1,22, N. Goldschmidt31, P. L. Zinzani9,32, C. Bödor33, R. Rosenquist5,34, T. P. Vassilakopoulos35, G. Ott6, S. Ogawa2,36,37, F. Damm1,22

1Department of Hematology, Oncology and Cancer Immunology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany; 2Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 3Wellcome Trust Sanger Institute, Hinxton, United Kingdom; 4Department of Hematology, Oncology, and Pulmonary Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany; 5Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 6Department of Clinical Pathology, Robert-Bosch-Krankenhaus; 7Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology Stuttgart, and University of Tuebingen, Stuttgart, Germany; 8Istituto di Ematologia “Seràgnoli”, IRCCS Azienda Ospedaliero-Universitaria di Bologna; 9Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy; 10Institute of Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany; 11Department of Hematology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 12First Department of Pathology, National and Kapodistrian University of Athens, Laikon General Hospital, Athens, Greece; 13Core Unit Proteomics, Berlin Institute of Health, Charité - Universitätsmedizin Berlin and Max-Delbrück-Center for Molecular Medicine; 14Core Unit Bioinformatics, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany; 15National Institute of Oncology, Budapest, Hungary; 16Department of Internal Medicine IV, Haematology and Oncology, University Hospital Halle (Saale), Martin-Luther-University Halle-Wittenberg, Halle; 17Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; 18National Institute of Oncology, Budapest, Hungary; 19Department of Immunology, Genetics and Pathology, Uppsala University and University Hospital, Uppsala, Sweden; 20Department of Pathology, Hematopathology Section and Lymph Node Registry, Universitätsklinikum Schleswig-Holstein, Kiel; 21Department of Pathology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin; 22German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg; 23Department of Internal Medicine 1 (Oncology, Hematology, Clinical Immunology, and Rheumatology), Saarland University Medical School, Homburg/Saar, Germany; 24Department of Hematopathology, Evangelismos General Hospital, Athens, Greece; 25Institute of Pathology, University of Würzburg and Comprehensive Cancer Center (CCC) Mainfranken, Würzburg; 26Department Internal Medicine I, Westpfalzklinikum Kaiserslautern, Kaiserslautern, Germany; 27Centro de Investigacion Biomedica en Red en Oncologia (CIBERONC), Madrid; 28Hospital Clinic of Barcelona, University of Barcelona; 29Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 30Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece; 31Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 32Istituto di Ematologia “Seràgnoli”, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 33HCEMM-SE Momentum Molecular Oncohematology Research Group, 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary; 34Clinical Genetics, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden; 35Department of Hematology and Bone Marrow Transplantation, National and Kapodistrian University of Athens, Laikon General Hospital, Athens, Greece; 36Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan; 37Department of Medicine, Centre for Haematology and Regenerative Medicine, Karolinska Institutet, Stockholm, Sweden

Background: Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive lymphoma affecting predominantly young female patients. Previous studies in this rare entity have focused on single genes or were limited in cohort size.

Aims: To unravel the underlying genetic pathogenesis and its impact on outcome, we embarked on a comprehensive large-scale genetic investigation.

Methods: Specimens of 486 previously untreated PMBCL patients were analyzed by paired tumor/normal whole-genome (WGS, n=14), whole-exome (WES, n=78) and targeted sequencing (TS, n=486). To understand the consequences of highly recurrent mutations in the chromatin-modifying gene ZNF217, we conducted functional and multi-omics analyses in CRISPR/Cas9 engineered cell lines.

Results: WGS/WES revealed a complex genomic landscape in PMBCL with a median of 85 structural variants, a mutational burden of 5 mutations/Mb, 12 mutated coding candidate driver genes (CDG) and 4 focal somatic copy-number aberrations per sample (Fig.1a). Besides known targets, significant breakpoints were identified in genes previously not implicated in B-lymphomagenesis such as TOX and TP73 (36% and 21%). In addition, non-coding mutations clustered within the PAX5 enhancer region. With the identification of 50 recurrently mutated CDGs, we significantly expand the repertoire of known PMBCL drivers. The 10 most frequently mutated CDGs were SOCS1 (86%), B2M (67%), ITPKB (64%), ACTB (58%), STAT6 (58%), IGLL5 (56%), TNFAIP3 (53%), NFKBIE (49%), GNA13 (47%), and ZNF217 (36%), respectively. The operative mutational processes were attributed to aging, AID/APOBEC activity, defective MMR, and an unexpected infidelity of DNA-Polymerase ε. Next, we performed TS in 486 samples using a PMBCL-specific 106-gene panel. Recurrent lesions in 25 epigenetic modifiers were found in >90%, with ZNF217 being among the most frequently mutated genes (Fig.1b). After knockdown of ZNF217 in Karpas1106P and L428 cells, we demonstrated altered proliferation, migration, and apoptosis. Using mass spectrometry, we showed that ZNF217 is acting in a LSD1, CoREST and HDAC containing histone modifier complex. Accordingly, knockout of ZNF217 led to global changes in chromatin accessibility with an enrichment of differentially accessible motifs for crucial lymphoma-associated transcription factors, especially of the NF-κB, BATF/AP1, and IRF family, but also of CTCF, a major regulator of global 3D chromatin architecture. Resulting gene expression was characterized by changes in interferon-responsive genes and inflammation-associated transcription (Fig 1c). Clinical data were available for 329 cases, including 84 cases from clinical trials. Multivariate analysis using an IPI-corrected Cox regression model was performed. The estimated 5-year PFS and OS were 77% and 86%. Among the genetic lesions with the strongest association for poor outcome, we identified patients with mutatedCD58 having a significantly shorter survival (PFS: HR 2.96; p<.001; OS: HR 2.55; p=.006). In contrast, mutated DUSP2 indicated longer survival (PFS: HR 0.28; p=.002; OS: HR 0.15; p=.011) (Fig1d). Notably, DUSP2 mutated patients (25%) showed a similar outcome for CR rate, PFS and OS when comparing CHOP-like and intensified treatment regimens, suggesting no further benefit from treatment intensification in this very-low risk patient population.



Summary/Conclusion: Here, we present the genetic landscape of PMBCL highlighting a previously underappreciated role of chromatin modifying genes, identify novel treatment targets and provide a solid basis for guiding precision medicine approaches.


P. Pölönen1,*, A. Elsayed1,2, L. Montefiori1, S. Kimura1, J. Myers3, D. Hedges3, J. Xu4, Y. Hui3, Z. Cheng3, Y. Fan3, Y. Chang1, R. Shraim5, M. Devidas6, S. Winter7, K. Dunsmore8, J. J. Yang9, T. L. Vincent10, K. Tan10,11,12,13,14, C. Chen10, H. Newman15, M. Loh16, E. Raetz17, S. P. Hunger18, E. Rampersaud3, T.-C. Chang3, G. Wu3, S. Pounds2, C. G. Mullighan1,19, D. T. Teachey5,12,20

1Pathology; 2Biostatistics; 3Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis; 4Perelman School of Medicine at the University of Pennsylvania; 5Department of Pediatrics and the Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia; 6Global Pediatric Medicine, St. Jude Children’s Research Hospital, Memphis; 7Minnesota Research Institute and Cancer and Blood Disorders Program, Children’s Minnesota Research Institute, Minneapolis; 8University of Virginia Children’s Hospital, Charlottesville; 9Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis; 10Division of Oncology and Center for Childhood Cancer Research; 11Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia; 12Perelman School of Medicine; 13Institute for regenerative medicine; 14Penn Epigenetics Institute, University of Pennsylvania; 15Division of Oncology and Center for Childhood Cancer, Children’s Hospital of Philadelphia, Philadelphia; 16Department of Pediatrics, Benioff Children’s Hospital and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco; 17Department of Pediatrics and Perlmutter Cancer Center, NYU Langone Health, New York; 18Department of Pediatrics and the Center for Childhood Cancer Research, Children’s Hospital of Philadelphia and The Perelman School of Medicine at The University of Pennsylvania, Philadelphia; 19Hematological Malignancies Program, St. Jude Children’s Research Hospital, Memphis; 20Divisions of Hematology and Oncology, Children’s Hospital of Philadelphia, Philadelphia, United States of America

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy including leukemias of early T cell precursor acute lymphoblastic leukemia (ETP-ALL) and transformed thymocytes. Prior genomic studies of T-ALL had limited cohort size, excluded refractory disease and focused on alterations in coding parts of the genome.

Aims: Investigate the genomic basis of T-ALL by identifying both coding and non-coding alterations and defining T-ALL subtypes.

Methods: We performed whole-genome sequencing (WGS), whole-exome sequencing (WES), RNA-sequencing of 1,313 cases enrolled on the Children’s Oncology Group AALL0434 trial.

Results: Uniform Manifold Approximation and Projection (UMAP) and gene expression clustering analyses of RNA-seq data identified 16 subtypes, of which 4 have not been reported previously. Furthermore, we could divide existing subtypes into smaller subgroups with common subtype-defining alterations, such as structural variation (SV) and copy number variation (CNV). TLX1 activation was linked with TLX1-TCR SVs and deletions in the TLX1 chromosomal domain, whereas TLX3 deregulation was associated with TLX3-BCL11B enhancer hijacking, but also through TLX3-TCR, TLX3-CDK6 rearrangements. We discovered two separate NKX2-1 deregulated groups, one characterized by TCR rearrangements and RPL10 mutations and the other by NKX2-1 CNVs, chromosome 14 chromothripsis, or MYB-TCR rearrangements. We also observed a distinct group of 9 cases aged 1-2 years, with recurrent STAG2-LMO2 rearrangements and a patient with inactivating STAG2 mutation and CELF1 enhancer hijacking by LMO2. Moreover, we found a group of 22 cases that were highly enriched for ETP-ALL with the following hallmark lesions: BCL11B enhancer amplification, BCL11B locus SVs involving enhancer hijacking of ARID1B, CCDC26, and novel CD34+ enhancer hijacking of lincRNA locus in chromosome 6.

Gene expression-based clustering was unable to stratify patients based on TAL1, TAL2, LMO1, LMO2, LYL1 expression, and their respective activation mechanisms. However, WGS enabled further characterization of these patients, by identifying several types of activation mechanisms and co-occurring alterations for each oncogene. We identified canonical events, such as TAL1-STIL fusions, TCR rearrangements, and activation by TAL1/LMO1/LMO2 regulatory region indels, but also novel events, such as CD34 specific enhancer duplications downstream of TAL1 and BCL11B enhancer hijacking by LMO2. We also observed two smaller clusters with TAL1/LMO2 activation, where one with 39 cases was associated with TAL1/LMO2 activation and RPL10 mutations and the other with 8 cases had refractory disease (Day 29 MRD >5%). HOXA gene expression-associated subtypes were defined by fusions involving KMT2A or MLLT10 and PICALM/DDX3X or HOXA9-TCR SVs. Furthermore, our analysis revealed segregation of patients by HOXA13 or ZFP36L2 rearrangements and NUP98/NUP214 fusions. Interestingly, HOXA locus breakpoints involving HOXA13 and HOXA9 were in different chromatin compartments and were associated with mutually exclusive activation of either HOXA13 or other HOXA genes. Interestingly, HOXA13-deregulation was associated with ETP-ALL and frequent BCL11B enhancer hijacking, whereas HOXA9 breakpoints typically involved TCRg and were non-ETP.



Summary/Conclusion: Large-scale analysis of all children enrolled on the AALL0434 study has identified new subtype-defining lesions in T-ALL, including candidate novel enhancer hijacking events and enhancer duplications that are likely to result in oncogene deregulation in T-ALL.


C. Fernandez De Larrea1,*, V. González-Calle2, A. Oliver-Caldés1, V. Cabañas3, P. Rodríguez-Otero4, M. Español-Rego1, J. L. Reguera5, L. López-Corral2, B. Martin-Antonio1, B. Paiva4, S. Inogés4, L. Rosiñol1, A. López-Díaz de Cerio4, N. Tovar1, M. López-Parra2, L. G. Rodríguez-Lobato1, A. Sánchez-Salinas3, S. Varea1, V. Ortiz-Maldonado1, J. A. Pérez Simón5, F. Prósper4, M. Juan1, J. M. Moraleda3, M. V. Mateos2, M. Pascal1, A. Urbano-Ispizua1

1Hospital Clínic de Barcelona. IDIBAPS. University of Barcelona, Barcelona; 2Hospital Universitario de Salamanca, Instituto de Investigacion Biomedica de Salamanca (IBSAL), Centro de Investigación del Cancer (IBMCC-USAL, CSIC), Salamanca; 3Hospital Clínico Universitario Virgen de la Arrixaca. IMIB-Arrixaca. University of Murcia, Murcia; 4Clínica Universidad de Navarra, Centro de Investigacion Medica Aplicada (CIMA), IDISNA, CIBERONC Pamplona, Pamplona; 5Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS/CSIC/CIBERONC), University of Sevilla, Sevilla, Spain

Background: ARI0002h is a lentiviral autologous CAR T-cell product with a 4-1BB co-stimulatory domain and a humanized single chain variable fragment targeting BCMA. In pre-clinical studies, this academic CAR-T has demonstrated potent in vitro and in vivo activity.

Aims: We report the safety and efficacy results of the CARTBCMA-HCB-01 multicenter clinical trial for patients with relapsed/refractory multiple myeloma (RRMM) (NCT04309981) who received ARI0002h in 5 Spanish centers.

Methods: Patients (pts) aged 18-75 years old with RRMM were eligible for this study if they had measurable disease, received ≥2 prior regimens, including a proteasome inhibitor, an immunomodulatory drug and an anti-CD38 antibody, and were refractory to the last line of treatment. Bridging therapy was allowed after apheresis. Cyclophosphamide (300 mg/m2) and fludarabine (30 mg/m2) were used as lymphodepletion regimen. The targeted dose was 3x106/kg CAR+ cells and was administered in a fractionated manner (10%/30%/60%), with at least 24h between infusions. A second dose of 3x106 CAR+ cells/kg was planned at least 4 months after the first dose in pts who achieved any grade of response any response and had not or serious complications after the first administration. Primary objectives were overall response rate (ORR; at least partial response -PR-) within 3 months of the first infusion and rate of cytokine release syndrome (CRS) and/or neurological toxicity in the first 30 days. Response was assessed as per IMWG criteria and bone marrow minimal residual disease (MRD) was analyzed by next-generation flow (NGF).

Results: As of February 9th 2022, 35 pts (median age 61 years) with RRMM were included in the trial. Four pts could not receive ARI-0002h due to MM progression and one died of infection. Therefore, 30 pts received ARI0002h cells (modified intention-to-treat population), of which 47% received bridging therapy. Median CAR-T cell production time was 11 days (range 9-14) with a 100% manufacture success.

Median follow-up after ARI0002h administration for surviving pts was 16 months. The ORR of 30 evaluable pts was 100%, with a stringent complete remission (sCR) plus very good partial response (VGPR) rate of 90%. Median time to first response was one month. Of 26 MRD-evaluable pts at day +100, 92% were MRD-negative in bone marrow by NGF. 53% of patients were alive and without progression at 16 months. Median overall survival (OS) was not reached and the 16-month OS rate was 80% (Figure 1).

AEs reported in >70% of pts were CRS (87%; grade [gr] 3/4 0%; gr 1 73%), neutropenia (97%; gr 3/4 100%), anemia (85%; gr 3/4 43%), and thrombocytopenia (79%; gr 3/4 70%). Median duration of CRS was 4 days (range 1-12). No CAR-T cell-related neurotoxicity cases were reported. Tocilizumab and corticosteroids were administered in 76% (mainly for persistent grade 1 CRS) and 12% of pts, respectively.

ARI0002h cells demonstrated peak expansion on day 14 (range 7 days-6 months). 24 out of 28 eligible pts (86%) received the second dose (range 1.2-3x106 CAR+ cells/kg). Median time after first infusion was 4 months and 38% received a second lymphodepletion regimen. No relevant toxicities after second infusions were reported. 7 pts (29%) improved their response after reinfusion.



Summary/Conclusion: ARI0002h is the first European academic CART for RRMM that has demonstrated deep and durable responses and a favorable safety profile, including the absence of neurotoxicity and the feasibility of a second booster dose.


P. Aguilar-Garrido1,*, M. Velasco1, M. Hernández Sánchez2, M. Á. Navarro Aguadero1, P. Malaney3, M. JL Aitken3, X. Zhang3, K. H Young3, R. Duan3, P. Hu3, S. Kornblau3, A. Fernández1, A. Ortiz1, Á. Otero-Sobrino1, P. J. de Andrés2, D. Megías1, M. Pérez1, J. Gómez1, G. Mata1, J. Martínez López1, S. Post3, M. Gallardo1

1CNIO; 2UCM, Madrid, Spain; 3MD Anderson, Houston, United States of America

Background: Nucleoli and ribosome cross-talk regulates cell translation capacity. Its dysregulation and impairment drive ribosome and nucleolus stress (NS), related to the biological mechanism of cancer. Ribosomopathies are a group of diseases characterized by ribosome defects leading to complex syndromes that include bone marrow failure.

hnRNP K is an RNA binding protein (RBP) in charge of processing nascent RNAs (nucleoli) into mature mRNAs (ribosome). Our research found a novel ribosome gain-of-function ribosomopathy phenotype by hyper-nucleoli generation due RBP hnRNP K dysregulation.

Aims: We aim to elucidate how hnRNP K dysregulation impact on haematopoietic stem cell biology.

Methods:Hnrnpk overexpression was established in MEFs using CRISPR/SAM (Konermann S. et al, Nature). Global protein synthesis was tested using a Click-iT OPP and proteasome function was evaluated by Proteasome 20S activity NS hallmarks were analyzed by confocal microscopy evaluating Ncl, NS sensor marker. To evoke NS in our cells, we used Actinomycin D insult. Cell cycle FACS analysis (DAPI) and senescence assays (β-galactosidase staining) were performed. Molecular mechanism underlying was elucidated by qRT-PCR and WB (p21, p16, c-Myc, and mTor). To study the impact of hnRNP K overexpression in vivo, we developed an inducible tamoxifen mouse model activated 30-60 days after birth (HnrnpkTg-Ubc-creERT2). Survival was evaluated by Kaplan-Meier, and phenotype described by symptoms, signs, CBC and bone marrow IHC panel (CD34, Gr1, B220, MPO).

Results:Hnrnpk overexpressing cells led to an increment in protein and gene expression of Ncl, mTor and c-Myc (Figure 1A-B). Moreover, we found an increase in global protein synthesis (Figure 2C-D). Nevertheless, the elevation of hnRNP K inversely correlated with the proteasome function, which dropped significantly (Figure 1E). NS induction promoted higher hnRNP K expression. Additionally, hnRNP K overexpressing cells showed NS hallmarks associated with an increase of the number of nucleoli, and total area of the nucleoli and nucleus (Figure 1F-G). Cell cycle analysis confirmed an increment of arrested G2/M phase cells (Figure 1H-I), linked to an increment in p21 and p16 levels all leading towards a senescent cell phenotype (Figure 1J-L).

HnrnpkTg-Ubc-creERT2 mice had widespread Hnrnpk overexpression (Figure 1M) and a reduction in lifespan (Figure 1N), mainly due to dysplastic and bone marrow failure phenotype, with dramatic reduction of CD34 and b-cells, leukopenia, anaemia and thrombocytopenia. (Figure 1O-Q).



Summary/Conclusion: The overexpression of hnRNP K drives to an increase in nucleoli activity, leading to ribosome biogenesis and higher global translation by the regulation of molecules involved in both systems: Ncl, c-Myc or mTor. Our work found that hnRNP K overexpression in vivo drives a bone marrow failure phenotype, promoting the exhaustion of haematopoietic stem cells by ribosome dysregulation that triggered cell senescence.

Of note, this is the first time reported that a nucleoli/ribosome-gain-of-function induce bone marrow failure ribosomopathies phenotype.

This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295), ISCIII Miguel Servet (CP19/00140), Cancer Research UK [C355/A26819], FC AECC and AIRC under the Accelerator Award Program and National Cancer Institutes of Health Award (R01CA207204, SMP) Leukemia and Lymphoma Society (6577-19, SMP).

Oral Sessions


A.-K. Wirth1,*, L. Wange2, S. Vosberg3, A. K. Jayavelu4, W Enard2, T Herold5, I Jeremias1

1Apoptosis in Hematopoietic stem cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), Munich; 2Anthropology and Human Genomics, Faculty of Biology, Ludwig Maximilian University (LMU), Martinsried, Germany; 3Clinical Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; 4Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried; 5Department of Medicine III, and Laboratory for Leukemia Diagnostics, Ludwig Maximilian University (LMU), Munich, Germany

Background: Acquired resistance to conventional polychemotherapy regimens leads to relapse and poor prognosis, and remains a major unmet clinical need.

Aims: To identify therapeutic options to overcome acquired chemo-resistance.

Methods: We studied acute lymphoblastic leukemia (ALL) as model disease and combined long-term in vivo treatment in orthotopic patient-derived xenograft (PDX) models with multi-omics profiling and functional genomic CRISPR/Cas9 screens.

Results: We adapted conventional chemotherapeutic protocols to allow treatment of mice for up to 18 consecutive weeks, using a combination of the widely used drugs cyclophosphamide and vincristine. Three luciferase-transgenic PDX models were monitored by repetitive in vivo imaging. Polychemotherapy strongly reduced PDX ALL cells within the first weeks, proving initial sensitivity of the sample. Under continuous treatment, tumor load persisted in mice at the level of minimal residual disease for several weeks, until tumors resumed growth despite treatment, indicating acquired resistance.

In an exemplary PDX model, eight resistant derivatives were generated in replicate mice and characterized individually. Genomic profiling revealed profound genomic heterogeneity between distinct derivatives; individual resistant derivatives acquired different copy number alterations in regions associated with resistance and distinct point mutations, e.g. in TP53. In contrast to genomic heterogeneity, transcriptome and proteome profiling identified a group of genes differentially expressed between sensitive and resistant cells, but similar across all derivatives.

To gain insights into underlying mechanisms and to identify therapeutic targets to overcome acquired resistance, a customized CRISPR/Cas9 in vivo dropout screen was performed in individual resistant PDX derivatives, to test the relevance of around 200 candidate genes under treatment. Among others, sgRNAs targeting BCL2, BRIP1 or COPS2 dropped out in the in vivo screen specifically during treatment; single knockout experiments confirmed that knockout of either BCL2, BRIP1 or COPS2 re-sensitized PDX ALL cells towards chemotherapy. Of direct translational relevance, treatment of mice with the BCL2 inhibitor ABT-199 sensitized resistant PDX cells towards polychemotherapy in vivo. Interestingly, BCL2 inhibition restored treatment response in resistant derivatives independently from the highly diverse underlying genetic alterations, e.g., in clones with and without mutation in TP53.

Summary/Conclusion: Taken together, we established a highly clinically relevant PDX in vivo model of acquired resistance to conventional chemotherapy. Using this model, we demonstrate that heterogeneous genomic alterations evolved in parallel in replicate mice, which could be overcome by a single therapeutic approach to re-sensitize tumors towards conventional chemotherapy.


L. Bastian1,*, A. Hartmann1, T. Beder1, S. Hänzelmann1, J. Kässens1, M. Bultmann1, M. P. Höppner2, S. Franzenburg2, M. Wittig2, A. Franke2, I. Nagel3, M. Spielmann3, N. Reimer4, H. Busch4, S. Schwartz5, B. Steffen6, A. Viardot7, K. Döhner7, M. Kondakci8, G. Wulf9, K. Wendelin10, A. Renzelmann11, A. Kiani12, H. Trautmann1, M. Neumann1, N. Gökbuget6, M. Brüggemann1, C. Baldus1

1Department of Medicine II, Hematology and Oncology, University Medical Center Schleswig-Holstein, Kiel; 2Institute for Clinical Molecular Biology, Kiel University, Kiel; 3Institute of Human Genetics, University Medical Center Schleswig-Holstein, Kiel and Lübeck, Kiel and Lübeck; 4Medical Systems Biology Group and Institute for Cardiogenetics, University of Lübeck, Lübeck; 5Department of Hematology, Oncology and Tumor Immunology (Campus Benjamin Franklin), Charité - Universitätsmedizin Berlin, Berlin; 6Department of Medicine II, Hematology/Oncology, Goethe University Hospital, Frankfurt / Main; 7Department of Internal Medicine III, University Hospital Ulm, Ulm; 8Department of Hematology, Oncology and Clinical Immunology, University Hospital Düsseldorf, Düsseldorf; 9Department of Hematology and Oncology, University Hospital Göttingen, Göttingen; 10Medical Department V, Hospital Nürnberg, Paracelsus Medizinische Privatuniversität, Nürnberg; 11Medical Department Oncology and Hematology, University Medical Center Oldenburg, Oldenburg; 12Department of Medicine IV, Hematology/Oncology, Klinikum Bayreuth, Bayreuth, Germany

Background: Response to induction chemotherapy assessed by quantification of minimal residual disease (MRD) is the strongest independent prognosticator in B precursor acute lymphoblastic leukemia (BCP-ALL). Molecular underpinnings of MRD poor response are insufficiently understood.

Aims: We aimed to identify novel high-risk subtypes in adult BCP-ALL as cell-intrinsic determinants of MRD poor response.

Methods: Adult BCP-ALL patients (n=565) were treated according to pediatric inspired protocols of the German Acute Lymphoblastic Leukemia Study group (GMALL) and profiled for integrative analyses by RNA-Seq (n=565), SNP-arrays (n=115), whole exome sequencing (WES; n=84) and whole genome sequencing (WGS; n=3).

Results: Concordance between transcriptomic and genomic profiles was used to allocate samples to one of 15 established molecular driver subgroups (Figure A). Unsupervised clustering of gene expression from the remaining samples revealed a distinct cluster (n=12/565, 2.1%) with an in-frame gene fusion between upstream binding transcription factor (UBTF) and ataxin-7-like protein 3 (ATXN7L3) as exclusive event in these patients. Both fusion partners are in direct neighborship at 17q21.31. WGS revealed a 10.08 kb genomic deletion which truncated UBTF at exon 17/21 and comprised most of the intergenic region between both genes (Figure B). UBTF-ATXN7L3 rearranged cases frequently harbored 1q gains (n=5/7). Further genomic profiling showed a remarkable paucity of additional cooperating events compared to other molecular subtypes, supporting a prominent driver function of the newly identified fusion. UBTF and ATXN7L3 are global epigenetic regulators involved in transcriptional control. Both genes were highly expressed across the entire cohort. The gene fusion was associated with a marked increase of Caudal Homeobox 2 (CDX2) expression. Analysis of functional modules related CDX2 to upregulated HOXA9 and MEIS1, described essential co-regulators of KMT2A-driven leukemogenesis. NTRK3 expression was also strongly upregulated, suggesting a possible rationale for specific inhibitors.

UBTF-AXTN7L3 rearranged patients were older, more frequently female and presented with normal leukocyte counts, low bone marrow infiltration and pro-B immunophenotypes or common ALL with reduced CD10 expression. Response to induction chemotherapy in evaluable patients (n=11) was poor with only 3 patients achieving MRD negativity after consolidation I compared to n=271/402 (67%; p=0.019) in the remaining cohort. Four patients suffered either cytologic (n=2) or molecular (n=2) relapse. Immunotherapeutic treatment intensification using blinatumomab (n=5) or inotuzumab ozogamizin (n=1) and / or allogenic stem cell transplantation (n=7) in MRD poor responders or relapsed cases resulted in an overall survival probability of 80% (+/- 12%) vs. 73% (+/- 2%; p=0.07) in the remaining cohort. Heterogeneous MRD responses were observed for other molecular subtypes (poor: ZNF384 - 48.2% MRD neg., p=0.056; Ph-like - 54.0% MRD neg., p=0.003; KMT2A - 55.8% MRD neg., p=0.127 / good: High Hyperdiploid - 90.9% MRD neg., p=0.01; TCF3-PBX1 - 94.1% MRD neg., p=0.016) indicating how molecular drivers affect chemo-sensitivity in adult BCP-ALL.



Summary/Conclusion: Molecular driver alterations determine sensitivity to induction chemotherapy in adult BCP-ALL. UBTF-ATXN7L3 ALL represents a novel subtype with poor induction chemotherapy response which could be successfully salvaged by MRD-based treatment intensification using immunotherapeutic strategies.


N. K. Ibrahim1,*, S. Schreek1, B. Cinar1, L. Loxha1, B. Fehlhaber1, J.-P. Bourquin2, B. Bornhauser2, C. Eckert3, G. Cario4, M. Forster5, M. Stanulla1, A. Gutierrez6, L. Hinze1

1Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany; 2Department of Pediatric Hematology and Oncology, University Children’s Hospital Zurich, Zurich, Switzerland; 3Department of Pediatric Hematology and Oncology, Charité Universitätsmedizin, Berlin; 4Department of Pediatrics I, Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein; 5Institute of Clinical Molecular Biology, Kiel, Germany; 6Division of Pediatric Hematology and Oncology, Boston Children’s Hospital, Boston, United States of America

Background: The ability of cells to tolerate amino acid starvation is fundamental for survival under cellular stress conditions. Some cancer cells are vulnerable to asparagine depletion, which is exploited therapeutically using asparaginase. However, the mechanisms of adaptation to amino acid starvation in leukemia cells remain incompletely understood.

Aims: We recently performed a genome-wide CRISPR/Cas9 loss-of-function screen in the resistant T-ALL cell line CCRF-CEM to identify molecular pathways that promote asparaginase resistance. We found that Wnt-dependent stabilization of proteins (Wnt/STOP) induces a profound therapeutic vulnerability to asparaginase in acute leukemias and colorectal cancers (Hinze et. al., 2019; Hinze et al., 2020). Another unrelated gene on the top of the screen included SOD2, a mitochondrial superoxide dismutase. Intriguingly, to date, SOD2 activity has not been linked to a cellular amino acid starvation response, whose biologic basis we thus sought to further investigate.

Methods: To evaluate the significance of SOD2 in mediating an asparaginase response, we employed genetic epistasis experiments as well as phenotypic assays including short hairpin RNA (shRNA) mediated knockdowns, quantitative PCRs, Western blots, amino acid starvation, and viability assays.

Results: Knockdown of SOD2 (shSOD2) resulted in a profound sensitivity to asparaginase in several T-ALL and B-ALL cell lines (p<0.0001), and an increase in apoptosis, as assessed by caspase 3/7 activity (p<0.001). The sensitization was rescued by either overexpressing SOD2 cDNA (p<0.0001), or by adding the functional SOD2 mimetic MnTBAP (p<0.01). Of note, shSOD2 mediated sensitization was selective to asparaginase, as it could not be observed for other commonly used chemotherapeutic agents including vincristine, doxorubicin, dexamethasone, and 6-mercaptopurine (p=ns). Due to the selectivity to asparagine depletion, we then investigated whether SOD2 inhibition mediates a broader amino acid starvation response. Indeed, culturing SOD2-inhibited T-ALL cells in the absence of essential amino acids (EAA) or non-EAA, induced a significant decrease in cell viability (p<0.05). Sensitization appeared to be specific to the SOD2 isoform, and distinct from known SOD2-associated pathways including reactive oxygen species, cell cycle changes, alterations of mTOR signaling, or glutamine anaplerosis. To better understand the molecular underpinnings of SOD2 in regulating an amino acid starvation response, we leveraged the Bioplex Interactome database (Huttlin et al., 2020), and identified UBR2, an E3 ubiquitin ligase in the N-degron pathway, as a unique binding partner of SOD2. Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation (Yang et al., 2010). Indeed, SOD2 and UBR2 were co-immunoprecipitated, suggesting the formation of a complex that can drive proteasome-dependent protein catabolism. In line, inhibition of SOD2 significantly decreased ubiquitin levels, suggesting that SOD2 positively regulates catabolic protein degradation through the N-degron pathway to promote cancer cell fitness in amino acid starved conditions.

Summary/Conclusion: The interaction of SOD2 and the N-degron pathway represents a previously unknown molecular adaptation of cancer cells in response to amino acid starvation. These results serve as a strong proponent for an in-depth characterization of the N-degron pathway in mediating leukemia cell fitness upon amino acid starvation and thus provide a basis for therapeutic intervention in refractory leukemias.


P. Richter-Pechanska1 2,*, J. Kunz1 2, T. Rausch2 3, B. Erarslan-Uysal1 2, B. Bornhauser4, V. Frismantas4, Y. Assenov5, M. Zimmermann6, M. Happich1, C. von Knebel-Doeberitz1, N. von Neuhoff7, R. Koehler8, M. Stanulla6, M. Schrappe9, G. Cario9, G. Escherich10, R. Kischner-Schwabe11, C. Eckert11, S. Avigad12, S. Pfister5 13, M. Muckenthaler1 2, J.-P. Bourquin4, J. Korbel2 3, A. Kulozik1 2 5

1University Hospital Heidelberg; 2MMPU; 3EMBL, Heidelberg, Germany; 4University Children’s Hospital, Zürich, Switzerland; 5DKFZ, Heidelberg; 6Hannover Medical School, Hannover; 7University Hospital, University of Duisburg-Essen, Essen; 8University Heidelberg, Heidelberg; 9University Hospital Schleswig-Holstein, Kiel; 10University Medical Center Hamburg-Eppendorf, Hamburg; 11Charite, Berlin, Germany; 12Schneider Children’s Medical Center of Israel, Petah Tikva, Israel; 13KiTZ, Heidelberg, Germany

Background: Relapse is the main cause of death from pediatric acute precursor T-cell leukemia (T-ALL), but the underlying mechanisms of disease evolution from initial disease to relapse remain incompletely understood and show remarkable interpatient heterogeneity.

Aims: As cross-sectional studies failed to identify unifying determinants of relapse, we adopted a longitudinal strategy and performed multi-omic analyses in 13 matched pairs of initial diagnosis and relapse samples and their matched PDXs. We extended this set by WES and methylome analyses in an additional cohort of 25 matched DNA samples from patient cells collected at initial diagnosis, remission, and relapse.

Methods: Thirty-eight patients were recruited from the ALL‐BFM 2000/2009, CoALL97/03/09, and ALL‐REZ BFM 2002 trials or from Schneider Children’s Medical Center of Israel at time points of initial diagnosis, remission, and relapse. Material from 13 matched pairs of PDXs (RNA, cells) was used for multi-omic analyses, including DNA-Seq (WES), RNA-Seq, ATAC-Seq and methylation analysis with EPIC arrays.

Results: Based on the profile of SNVs and InDels we distinguished 18 (47%) type-1 (derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). We observed stronger remodeling on the way to type 2 than to type 1 relapses reflected by more evident changes in methylation, chromatin accessibility and gene expression. At the time of relapse, 3/20 type 2 patients exhibited a hypermutator phenotype, probably caused by gains of mutations in TP53, BLM and BUB1B combined with PMS2. Moreover, type 2 T-ALLs were predominantly TAL1-driven (4/8) in contrary to type 1 (0/5). T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. The fraction of subclonal mutations of those T-ALLs that later developed into a type-2 relapse was significantly higher already at the time of initial diagnosis than in those T-ALLs that later developed into a type-1 relapse (p=0.0387; Fisher’s exact), a difference that became even more pronounced at the time of relapse (p<0.0001). On the other hand, relapse type 1 T-ALLs exhibited overexpression of IL7R, its ligand HGF, and repressors of cytokine signaling (SOCS1, SOCS2, SOCS3) which regulates the IL7R pathway via negative feedback loop. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. Moreover, an analysis of remission samples revealed a significant enrichment of mutations in constitutional cancer predisposition genes (CPG) in type 2 patients, thus indicating fundamental differences between these two groups of patients. In both types of relapse, we observed known and novel drivers of drug resistance including MDR1 and MVP and NT5C2.



Summary/Conclusion: In sum, our comprehensive analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia. Leukemias of patients with type-1 relapses were often characterized by upregulation of the IL7R pathway, whereas type-2 relapses were characterized by (i) an enrichment of TAL-1 fusion, (ii) and of constitutional mutations in CPG, (iii) divergent genetic and epigenetic remodeling, and (iv) an enrichment of somatic hypermutator phenotypes.


P. Ntziachristos1,*, Q. Jin2, B. Gutierrez3

1Ghent University, Ghent, Belgium; 2MD Anderson, Houston; 3Northwestern University, Chicago, United States of America

Background: Dysregulation of kinase signaling pathways via mutations favors tumor cell survival and resistance to therapy and it is common in cancer. Our data unveil how dysregulated deubiquitination controls signaling pathways, leading to cancer cell survival and drug non-response, and suggest novel therapeutic combinations towards targeting T-cell acute lymphoblastic leukemia (T-ALL). Here, we reveal a novel mechanism of post-translational regulation of kinase signaling and nuclear receptor activity via deubiquitination in acute leukemia.

Aims: This study aims at 1) characterizing the function of an oncogenic complex composed by two deubiquitinating enzymes in in vitro and in vivo leukemia systems and 2) testing the association of deubiquitinase activity with resistance to therapy in acute lymphoblastic leukemia.

Methods: We use genetic mouse and human:mouse xenograft models of T-cell leukemia, biochemical studies (quantitative global proteomics, phosphoproteomics and ubiquitination analysis) and high-throughput molecular biology (chromatin conformation capture (HiC), chromatin accessibility (ATAC-Seq) and gene expression (RNA-Seq)) analyses.

Results: We observed that the ubiquitin specific protease 11 (USP11) is highly expressed in lymphoblastic leukemia and associates with poor prognosis in this disease. USP11 ablation inhibits leukemia growth in vitro and in vivo, sparing normal hematopoiesis and thymus development, suggesting that USP11 could be a therapeutic target in leukemia. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK). Deubiquitination of LCK controls its activity, thereby altering T cell receptor signaling. Impairment of LCK activity leads to increased expression of the glucocorticoid receptor transcript, culminating into transcriptional activation of pro-apoptotic target genes, and sensitizes cells to glucocorticoids in T cell leukemia patient samples. The transcriptional activation of pro-apoptotic target genes, such as BCL2L11, is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Pharmacological inhibition of USP7 or genetic knockout of USP7 in combination treatment of glucocorticoid displayed improved anti-T-ALL efficacy in vivo.



Summary/Conclusion: Our data unveil how dysregulated deubiquitination controls signaling pathways, leading to cancer cell survival and drug non-response, and suggest novel therapeutic combinations towards targeting T-cell leukemia.


J. Yang1, J. Li1, X. Zhang1, L. Qiu1, P. Lu1,*

1Hebei Yanda Lu Daopei Hospital, Langfang, China

Background: T cell malignancies represent a group of hematologic cancers with high relapse and mortality rates. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T due to unintended CAR-T fratricide. Here, we develop a fratricide-resistant anti-CD7 CAR-T modified by CD7 ablation through CRISPR/CAS9 gene editing (KO7CAR).

Aims: In a phase I clinical trial, we explored the efficacy and safety of KO7CAR T-cells for relapsed or refractory (R/R) T-cell malignancies (NCT04916860 & NCT04938115).

Methods: Peripheral blood mononuclear cells were collected from patients (n=13) or the transplant donor (n=2) by leukapheresis. CD7-ablated CAR T cells (KO7CAR) were derived by electroporation of bulk T cells with CD7-targeting Cas9-gRNA RNP 24 hours before 7CAR transduction. This KO7CAR is a second-generation CAR-T with the co-stimulatory domain of 4-1BB and CD3ζ targeting CD7. Intravenous fludarabine (30mg/m2/d) and cyclophosphamide (300mg/m2/d) were given to all patients on day -5 to day -3 prior to KO7CAR-T cells infusion.

Results: From Oct. 2020 to Oct. 2021, 15 patients with T-cell acute lymphoblastic leukemia (n=10), T-cell lymphoblastic lymphoma (n=3), and mixed phenotype acute leukemia (n=2) were enrolled and received KO7 CAR-T cells (Table 1). The median age was 28 (8-46) years old. Four patients had prior hematopoietic stem cell transplantation (HSCT). At enrollment, 10 patients had bone marrow (BM) blasts >5% by morphology, and 10 patients had the extramedullary disease (EMD, diffuse involvement, n=8, and bulky mediastinal masses, n=2). Both patient- and donor-derived KO7CAR-T cells were successfully generated with a transduction efficiency of 59.0% (22.5%-97.4%). A single dose of KO7CAR-T cells was infused to patients at low dose (1.5~5x105 cells/kg, n=8), medium dose (1x106 cells/kg, n=6) or high dose (2x106 cells/kg, n=1).

On day 28, 15/15 (100%) patients achieved minimal residual disease (MRD) negative complete remission (CR). Among the 10 patients with EMD, 7 achieved EMD CR on day 30, 2 achieved partial response (PR), and 1 who relapsed post 2nd transplant had no response on day 35 then withdrew. Up to data cutoff Feb.10, 2022, the median follow-up time was of 309 days (35~407 days). About 2 months post KO7CAR, 12 patients bridged into allogeneic HSCT, and all remained progression-free after a median time of 253 (30~388) days after HSCT except for 1 who relapsed on day 147 then died from intracerebral hemorrhage on day 249. The other 2 patients without subsequent HSCT (all had a prior transplant) died from infection on day 78 and GVHD on day 103, respectively, post KO7CAR.

Mild cytokine release syndrome (CRS, ≤grade II) occurred in 10/15 (66.7%) patients, and 5/15(33.3%) patients had grade III CRS. One patient had grade I neurotoxicity, and 2 had grade III/IV neurotoxicity. All was controlled after the administration of corticosteroids and/or tocilizumab.

Following infusion, the median peak of circulating KO7CAR-T cells was 1.77×105 (0.279~14.3×105) copies/μg genomic DNA which occurred around day 20 (day10~ day25) and 63.47% (23.1%~94.18%) occurring on day15 (day 9~day25) by q-PCR and flow cytometry respectively.



Summary/Conclusion: This study demonstrated KO7CAR-T therapy had a high efficacy for CD7+ T-cell malignancies even for those who relapsed post-transplant. Safety was manageable, however, more data on additional patients and longer observation time are needed to evaluate the efficacy of KO7 CAR-T products further.


G. F. Torelli1,*, S. Chiaretti1, N. Peragine1, W. Barberi1, L. Santodonato2, G. D’Agostino2, E. Abruzzese3, M. I. Del Principe4, A. Mancino5, M. Matarazzo1, M. S. Bafti1, M Mancini1, M. Messina5, L. Castiello2, A. Guarini1, R. Foà1

1Hematology, Department of Translational and Precision Medicine, Sapienza University; 2FaBioCell Cell Factory, Istituto Superiore di Sanità; 3Hematology, Sant’Eugenio Hospital, ASL Roma2, Tor Vergata University; 4Hematology, Department of Biomedicine and Prevention, Tor Vergata University; 5Fondazione GIMEMA Onlus, Rome, Italy

Background: Due to age and co-morbidities, many Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients are ineligible to undergo high-dose chemotherapy or allogeneic transplant as consolidation treatment. Our group reported the promising results of the chemo-free scheme D-ALBA based on dasatinib/blinatumomab in induction/consolidation, underlying the potential role of immunotherapy in this setting (Foà et al, NEJM 2020;383:1616-23).

Considerable interest has been raised by natural killer (NK) cells. We developed a GMP protocol for NK cell ex vivo expansion in the presence of IL-2 and IL-15, and report the results of a phase 1 protocol of adoptive immunotherapy with activated and expanded autologous NK cells for Ph+ ALL patients in complete hematologic remission (CHR) but with persistent/recurrent minimal residual disease (MRD) ≥60 years or ineligible for other post-CHR treatment modalities.

Aims: The primary endpoint was to determine the maximum tolerated dose of NK cells and the recommended dose for subsequent studies. Secondary endpoints were the assessment of safety and tolerability of the treatment, the immunologic modifications induced by the procedure and the clinical response to treatment.

Methods: The planned 6 patients were enrolled: 5 in 1st CHR and 1 in 2nd CHR. Patients underwent repeated infusions (maximum 5) of escalating doses of NK cells, ranging from 1x106 to 5x107/kg of body weight (BW). No conditioning therapies were administered before the infusion; patients were allowed to continue tyrosine kinase inhibitors. Patients underwent a comprehensive MRD monitoring by Q-RT-PCR with a one-year follow-up. Immunophenotypic analysis on the NK cell product was performed before and after the expansion. Intracellular cytokine production and PBMC cytotoxic activity against K562 cells, allogeneic and autologous blasts were evaluated after expansion and at time 0 and 7 days from each NK cell infusion.

Results: NK cells presented a 12.3-fold ex vivo expansion. Expanded cells showed an increased expression of activating receptors and measurable cytotoxicity against primary allogeneic and autologous blasts. One patient received a maximum NK cell dose of 5x106 cells/kg, 2 patients 1x107 cells/kg and 3 5x107 cells/kg/BW. No patient experienced infusion-related toxicities. Two adverse events were recorded (grade 1 and 2), both judged not treatment-related, that resolved after TKI suspension. The higher cell dose infusion resulted in a significantly increased expression of natural cytotoxicity receptors, a greater cytokine production by NK, T and NKT cells, and in an increased capacity of PBMC to lyse K562 cells. These modifications appear persistent over time.

At a 1-year follow-up from the last infusion, 5/6 patients are alive in CHR (Table 1). The MRD levels reduced over time and 4/6 patients reached a complete molecular response (CMR) or a positive-not-quantifiable (PNQ) status during the study period. At a median follow-up of 30.8 months from the last infusion, the 5 patients who received the NK treatment in 1st CHR are still in CMR or PNQ, though 1 patient required additional treatment. The patient in 2nd CHR at the time of the infusions showed a rise in MRD and died of disease progression.



Summary/Conclusion: This phase 1 study demonstrates that autologous NK cells can be efficiently expanded ex vivo from MRD-positive Ph+ ALL patients in CHR. The infusion of these expanded cells is safe and induces a marked in vivo host immune response, suggesting that this approach represents a tolerable and feasible model worthy of being investigated in larger clinical studies.


S. Rives1,*, S. L. Maude2, H. Hiramatsu3, A. Baruchel4, P. Bader5, H. Bittencourt6, J. Buechner7, T. Laetsch2, B. De Moerloose8, M. Qayed9, H. E. Stefanski10, K. L. Davis11, P. L. Martin12, E. Nemecek13, C. Peters14, G. Yanik15, A. Balduzzi16, N. Boissel17, S. L. Khaw18, J. Krueger19, J. Levine20, S. Davies21, G. D. Myers22, A. Yeo23, D. O’Donovan24, R. Ramos23, M. Pulsipher25, S. Grupp2

1Department of Pediatric Hematology – Oncology, Hospital Sant Joan de Déu Barcelona, and Institut de Recerca Sant Joan de Déu, Barcelona, Spain; 2Division of Oncology, Center for Childhood Cancer Research and Cancer Immunotherapy Program, Children’s Hospital of Philadelphia and Department of Pediatrics, Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 3Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 4University Hospital Robert Debré (APHP) and Université de Paris, Paris, France; 5Division of Stem Cell Transplantation and Immunology, Hospital for Children and Adolescents, University Hospital Frankfurt, Frankfurt, Germany; 6Department of Pediatrics, Faculty of Medicine, University of Montreal, and the Hematology Oncology Division and Charles-Bruneau Cancer Center, Centre Hospitalier Universitaire Sainte-Justine Research Centre, Montreal, QC, Canada; 7Department of Pediatric Hematology and Oncology, Oslo University Hospital, Oslo, Norway; 8Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium; 9Aflac Cancer and Blood Disorders Center, Emory University, Atlanta, GA; 10National Bone Marrow Donor Program, Be the Match, Division of Pediatric Blood and Marrow Transplant, University of Minnesota, Minneapolis, MN; 11Division of Hematology, Oncology and Stem Cell Transplant, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA; 12Pediatric Transplant and Cellular Therapy, Duke University Medical Center, Durham, NC; 13Oregon Health and Science University, Portland, OR, United States of America; 14Stem Cell Transplantation Unit, St. Anna Children’s Hospital, Vienna, Austria; 15Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, MI, United States of America; 16Clinica Pediatrica Università degli Studi di Milano Bicocca, Fondazione MBBM, Ospedale San Gerardo, Monza, Italy; 17Saint-Louis Hospital (APHP) and Université de Paris, Paris, France; 18Children’s Cancer Centre, Royal Children’s Hospital and Murdoch Children’s Research Institute, Parkville, VIC, Australia; 19Division of Haematology/Oncology/Bone Marrow Transplantation, Department of Paediatrics, The Hospital for Sick Children, Toronto, ON, Canada; 20Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY; 21Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 22Children’s Mercy Hospital and Clinics, Kansas City, MO; 23Novartis Pharmaceuticals Corporation, East Hanover, NJ, United States of America; 24Novartis Pharmaceuticals Corporation, Dublin, Ireland; 25Division of Pediatric Hematology and Oncology, Intermountain Primary Children’s Hospital, Huntsman Cancer Institute at the University of Utah, Salt Lake City, UT, United States of America

Background: Pediatric and young adult pts with R/R B-ALL experience a treatment journey characterized by diminishing likelihood of cure and increasing morbidity. Tisagenlecleucel is an autologous CD19-directed chimeric antigen receptor (CAR) T-cell therapy approved for use in pediatric and young adults with B-ALL and adults with B-cell lymphomas. Tisagenlecleucel provided high rates of remission (>80%) in children and young adults with R/R B-ALL in ELIANA, with 62% of responders remaining relapse-free at 24 mo (Grupp et al, Blood, 2018).

Aims: Here, we report the final efficacy and safety analyses in pts followed up to 5.9 years post-tisagenlecleucel infusion.

Methods: ELIANA (NCT02435849) was a pivotal, Phase II, open-label, multicenter, global study of tisagenlecleucel in pediatric and young adult pts with R/R B-ALL. Pts received a single infusion of tisagenlecleucel at 0.2-5.0×106 CAR+ viable T cells/kg body weight for pts ≤50 kg and 0.1-2.5×108 CAR+ viable T cells for pts >50 kg. Endpoints included overall remission rate (ORR) within 3 mo, relapse-free survival (RFS), duration of remission (DOR), overall survival (OS), persistence of B-cell aplasia, and short- and long-term safety events.

Results:Results: As of September 24, 2021, 97 pts were enrolled and 79 pts (81%) received tisagenlecleucel. Median time from infusion to data cutoff was 5.5 y; 64 pts had ≥5 y of follow-up. At study entry, the median age was 11 y (range, 3-24). Pts were heavily pretreated with a median of 3 prior lines of therapy (range, 1-8) and 61% had a history of prior stem cell transplant (SCT). ORR (complete remission [CR] or CR with incomplete hematologic recovery within 3 mo after infusion) was 82% (95% CI, 72-90). Among pts in remission (CR/CRi), the 5y RFS rate was 49% (95% CI, 34-62), and the median RFS was not reached (Figure, 46.8 mo when censoring for SCT; n=15). The median time to B-cell recovery was 38.6 mo (95% CI, 23-not reached) and the probability of B-cell aplasia at 6 mo and 12 mo was 83% (95% CI, 71-91) and 71% (95% CI, 57-82), respectively. Pts with B-cell recovery (<6 mo, n=10; 6-12 mo, n=4; >12 mo, n=7) experienced a 2y cumulative incidence of relapse of 25.2% (with SCT treated as a competing risk). Among all pts, the 5y EFS and OS rates were 42% (95%CI, 29-54) and 55% (95% CI, 43-66), respectively. There were no significant differences in any efficacy endpoint between pediatric (<18 y; n=65) and young adult (≥18 y; n=14) pts. No new or unexpected AEs were reported during long-term follow-up. Among pts in remission, the most commonly reported grade ≥3 AEs occurring >1 y post-infusion were infection (20%) and cytopenias (6%). Ten (14%) pts in remission experienced long-term cytopenias persisting for >1 y; however, none of these pts experienced cytopenias persisting for >5 y (median 2 y; range, 1.1-5y). Eighty-two percent of pts received IVIG any time post-infusion.



Summary/Conclusion: This >5 y follow-up study demonstrates continued durable efficacy of tisagenlecleucel without late adverse effects in heavily pretreated pediatric and young adult pts with R/R B-ALL. Tisagenlecleucel continues to be a potentially curative treatment option for pediatric and young adult patients with R/R B-ALL.


R. Bassan1,*, S. Chiaretti2, I. Della Starza3, O. Spinelli4, A. Santoro5, L. Elia3, M. S. De Propris3, A. M. Scattolin1, F. Paoloni6, M. Messina7, E. Audisio8, L. Marbello9, E. Borlenghi10, P. Zappasodi11, C. Vetro12, G. Martinelli13, D. Mattei14, N. Fracchiolla15, M. Bocchia16, P. De Fabritiis17, M. Bonifacio18, A. Candoni19, V. Cassibba20, P. Di Bartolomeo21, G. Latte22, S. Trappolini23, A. Guarini24, A. Vitale3, P. Fazi6, M. Vignetti6, A. Rambaldi4, R. Foà3

1Hematology, Ospedale dell’Angelo, Venice; 2Translational and Precision Medicine, Sapienza Univesrity of Rome; 3Translational and Precision Medicine, Sapienza University of Rome, Rome; 4UOC Ematologia, ASST-Papa Giovanni XXIII, Bergamo; 5Divisione di Ematologia con UTMO, Ospedali Riuniti Villa Sofia-Cervello, Palermo; 6GIMEMA Data Center; 7GIIMEMA Data Center, Fondazione GIMEMA – Franco Mandelli Onlus, Rome; 8Ematologia, Città della Salute, Torino; 9Hematology, Niguarda Ca’ Granda Hospital, Milan; 10Hematology, Spedali Civili, Brescia, Italy, Spedali Civili, Brescia; 11Hematology, Foundation IRCCS Policlinico San Matteo, Pavia; 12General Surgery and Medical-Surgical Specialties, University of Catania, Catania; 13Institute of Hematology, L. and A. Seràgnoli, Bologna; 14Hematology, Ospedale S. Croce, Cuneo, Italy, Cuneo; 15UOC Oncoematologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milan; 16Hematology Unit, Azienda Ospedaliera Universitaria Senese, Siena; 17Hematology Division, S. Eugenio Hospital, Rome; 18Ospedale Policlinico “G.B. Rossi”, University of Verona, Verona; 19Clinica Ematologica, Azienda Sanitaria Universitaria Integrata di Udine, Udine; 20Divisione di Ematologia, Ospedale Civile, Bolzano; 21Oncology Hematology, Ospedale Civile, Pescara; 22Hematology, S. Franceso Hospital, Nuoro; 23Clinica di Ematologia, Azienda Ospedaliero - Universitaria Ospedali Riuniti Umberto I,, Ancona; 24Molecular Medicine, Sapienza University of Rome, Rome, Italy

Background: Pediatric-inspired chemotherapy is standard of care for younger adults with Ph− ALL/LL. An essential component of these regimens is pegaspargase, here incorporated into a national treatment program for patients 18-65 years.

Aims: To assess in the GIMEMA Phase 2 LAL 1913 study the feasibility and efficacy of a pegaspargase-containing induction and consolidation regimen sustaining a risk-oriented strategy for adult Ph− ALL/LL (ClinicalTrials.gov ID NCT02067143).

Methods: Our prior, reference 8-block chemotherapy protocol (Blood Cancer J 2020;10:119) was modified to include pegaspargase 2000 IU/m2 at courses 1 (d10), 2 (d8), 5 (d3, with HD-MTX) and 6 (d8), with dose reductions in patients >55 years (pegaspargase 1000 IU/m2). Serum drug activity was not assessed in this study. Responders were risk-stratified for allogeneic stem cell transplantation (SCT) or maintenance according to a mixed risk model based on WBC count, immunophenotype, genetics and post-remission molecular minimal residual disease (MRD): patients with high-risk (HR) features or MRD ≥ 10-4 at weeks 10-16 or positive at week 22 were eligible to SCT; standard-risk (SR) patients were eligible to maintenance.

Results: Two hundred and three patients entered the study (median age 39.8 years; 139 B- and 64 T-phenotype). The complete remission (CR) rate was 91% (100% in T-ALL/LL), with a 3-year cumulative relapse incidence and non-relapse mortality of 24.2% and 12.6%, respectively; 60 patients underwent a SCT. Overall (OS), event-free (EFS) and disease-free (DFS) survival were 66.7% (95% CI, 60.1-74.1%), 57.7% (95% CI, 51.0-65.3%) and 63.3% (95% CI, 56.3-71.1%) at 3 years. HR class (n=95) and LL diagnosis (n=20) did not affect prognosis. T-cell phenotype (CR 100%, P=0.001; EFS 67.1%, P=0.038), age 18-40 years (EFS 72.6%, P<0.0001) and MRD <10-4 after courses 1 (55%: DFS 77.9%, P=0.023) and 3 (79%: DFS 75.2%, P=0.048) were prognostically favorable. One hundred and eighty-seven patients had pegaspargase at course 1 (92.1%, 11 delayed, 3 reduced), 154 at course 2 (84.6%; 11 delayed, 12 reduced), 110 at course 5 (83.9%; 2 delayed, 11 reduced) and 73 at course 6 (68.8%; 3 delayed, 7 reduced). Dose reductions and delays were related to high-risk profile (liver dysfunction/steatosis, obesity etc.) or treatment toxicity. Toxicity of grade 2 or more was mainly observed at course 1 (hepatic 12.8%, coagulation/thrombosis 3.2% [enoxaparin prophylaxis recommended with platelets >30-50], pancreatic 1.6%), contributing to an induction death in 3 patients (1.4%), but was rare afterwards.



Summary/Conclusion: This pegaspargase-based ALL regimen was safely applicable to the majority of study patients, resulting in 3-year OS, EFS and DFS rates >50% in a patient population aged 18-65. The results were more favorable in patients up to the age of 55, especially in those aged 18-40 years, and in those who achieved maximum MRD response regardless of age (Figure). Subsequently, a pegaspargase dosing algorithm based on patient age, body mass index, hepatosteatosis and selected toxicities at first or prior drug exposure was developed to minimize toxicity, and was used in a successor GIMEMA trial of sequential chemotherapy-blinatumomab for CD19+ adult B-ALL (EHA Congress 2021, abstract S114).


N. Short1,*, H. Kantarjian1, M. Konopleva1, N. Jain1, F. Ravandi1, X. Huang2, W. Macaron1, W. Wierda1, G. Borthakur1, T. Kadia1, K. Sasaki1, G. Issa1, G. Montalban-Bravo1, Y. Alvarado1, G. Garcia-Manero1, C. Dinardo1, J. Thankachan1, R. Delumpa1, E. Mayor1, W. Deen1, A. Milton1, J. Rivera1, L. Waller1, C. Loiselle1, R. Garris1, E. Jabbour1

1Department of Leukemia; 2Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Ponatinib and blinatumomab are both highly effective therapies for Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). The combination of these two agents may offer an effective chemotherapy-free strategy in these patients (pts).

Aims: We evaluated the efficacy and safety of ponatinib and blinatumomab in pts with newly diagnosed (ND), relapsed/refractory (R/R) Ph+ ALL or CML in lymphoid blast phase (CML-LBP). For pts with ND Ph+ ALL, the primary endpoint was the complete molecular response (CMR) rate. For pts with R/R Ph+ ALL, the primary endpoint was the CR/CRi rate. Secondary endpoints included safety, event-free survival (EFS) and overall survival (OS).

Methods: In this phase II study, adults with ND Ph+ ALL, R/R Ph+ ALL, or CML-LBP were eligible. Pts were required to have a performance status of ≤2, total bilirubin ≤2x the upper limit of normal (ULN), and ALT and AST ≤3x the ULN. Pts with uncontrolled cardiovascular disease or clinically significant central nervous system (CNS) comorbidities (except for CNS leukemia) were excluded. Pts received up to 5 cycles of blinatumomab as a continuous infusion at standard doses. Ponatinib 30mg daily was given during cycle 1 and was decreased to 15mg daily once CMR was achieved. After 5 cycles of blinatumomab, ponatinib was continued for at least 5 years. Twelve doses of prophylactic IT chemotherapy with alternating cytarabine and methotrexate were administered.

Results: Between 2/2018 to 1/2022, 55 pts were treated (35 with ND Ph+ ALL, 14 with R/R Ph+ ALL and 6 with CML-LBP). Baseline characteristics are shown in Table 1.

Among the 35 pts with ND Ph+ ALL, 12 were in CR at enrollment (including 2 pts in CMR). 22 of the 23 evaluable pts (96%) achieved CR/CRi. One pt died on day 18 from intracranial hemorrhage in the setting of chemotherapy administered prior to enrollment. After one cycle, 21/33 pts (64%) achieved CMR, and 28/33 pts (85%) achieved CMR at any time. 11 of 15 tested pts (73%) also became MRD-negative by an NGS assay with sensitivity of 1x10-6.

CR/CRi was achieved in 12/13 (92%) evaluable pts with R/R Ph+ ALL. CMR was achieved in 10 pts (71%) after cycle 1 and in 11 pts (79%) overall. 5 of 6 pts with CML-LBP achieved CR/CRi, and 1 pt achieved PR as best response. 2 pts (40%) achieved CMR.

In the ND Ph+ ALL cohort, 1 of 34 pts who received at least 1 full cycle died in CR; the other 33 are in ongoing hematologic remission. Only one pt underwent stem cell transplant (SCT) in first remission for persistently detectable BCR/ABL1 transcripts. Among 13 responding pts in the R/R Ph+ ALL cohort, 6 proceeded to SCT, 4 did not undergo SCT and subsequently relapsed, 1 died in CR, and 2 are in ongoing remission without SCT. In the CML-LBP cohort, 3 of the 5 responding pts subsequently relapsed.

The median follow-up is 11 months (range, 1-46+). For ND Ph+ ALL, the 2-year EFS and OS are both 93% (Figure 2). There were no relapses or leukemia-related deaths in this cohort. In the R/R Ph+ ALL cohort, the 2-year EFS rate was 42% and the 2-year OS rate was 61%. In the CML-LBP cohort, the 2-year EFS was 33% and the 2-year OS was 60%.

The treatment was well-tolerated, and most toxicities were grade 1-2 and consistent with the known toxicities of the two agents. Two pts discontinued ponatinib due to toxicity (1 due to stroke and 1 due to DVT). One pt discontinued blinatumomab due to persistent grade 2 tremor.



Summary/Conclusion: The chemotherapy-free regimen of simultaneous ponatinib and blinatumomab is safe and effective in pts with Ph+ ALL. For pts with ND Ph+ ALL, SCT does not appear to be needed in first remission.


H. Uckelmann1,*, S. Armstrong1, E. Haarer1, E. Wong1, C. Hatton1, F. Perner1, C. Marinaccio1, C.-W. Chen2

1Pediatric Oncology, Dana-Farber Cancer Institute, Boston; 2Department of Systems Biology, Beckman Research Institute, City of Hope, United States of America

Background: The dysregulation of stem cell and self-renewal associated genes is a common phenomenon during leukemia development. In acute myeloid leukemia (AML) around 50 % of cases express high levels of HOXA cluster genes and MEIS1. Most of these AML cases harbor an NPM1 mutation (NPM1c), which encodes for an oncogene that is mislocalized from the nucleolus to the cytoplasm. Hence, most studies of NPM1c have focused on a potential cytoplasmic role. However, it remains unclear how NPM1c expression in hematopoietic cells leads to its characteristic gene expression pattern. Furthermore, NPM1c AMLs are highly sensitive to the disruption of the MLL1 histone methyltransferase complex. Small molecule inhibitors that block the interaction between MLL1 and its adaptor protein Menin have been shown to impair binding of MLL1 to a subset of its target genes and to inhibit leukemia cell proliferation and self renewal. Several MLL1-Menin inhibitors are currently in Phase I/II clinical trials and show promising activity in patients with NPM1c AML. The effectiveness of these molecules in NPM1c AML prompts the question whether NPM1c and the wildtype MLL complex cooperate directly on chromatin to drive leukemic self-renewal.

Aims: In this study we investigated the potential role of NPM1c in regulating oncogenic transcription on chromatin and the interplay between NPM1c and the histone methyltransferase complex KMT2A (MLL1).

Methods: We used an endogenously degrader tagged NPM1c leukemia cell line that allows rapid small molecule induced degradation to show that NPM1c occupies specific chromatin targets in AML. To characterize the effects of NPM1c degradation on the chromatin landscape and transcriptional output at genomic loci that are bound by NPM1c we used ChIPseq, PROseq and nascent RNAseq methods.

Results: Our results show that endogenous NPM1c directly binds to chromatin at specific target genes, such as HOXA9 and MEIS1, which are highly expressed in NPM1c patient samples. The loss of NPM1c from its targets leads to specific alterations in active chromatin marks and RNA Polymerase II (Pol II) chromatin occupancy which are accompanied by rapid changes in gene expression as well as Pol II transcriptional activity. The recruitment of NPM1c to chromatin is dependent on the nuclear exporter CRM1 as well as one of the acidic domains of NPM1c. We further show that NPM1c is lost from specific loci after treatment with small molecules that disrupt the MLL1-Menin complex interaction thus functionally linking targeted epigenetic therapy and NPM1c function.

Summary/Conclusion: Overall, we demonstrate that NPM1c directly regulates a network of leukemia self-renewal associated genes through direct chromatin interaction. We further found that NPM1c acts in collaboration with the MLL1 complex and define the mechanism by which MLL1-Menin small molecule inhibitors produce clinical responses in patients with NPM1-mutated AML.


Hugues-Etienne Châtel-Soulet1, Sabine Juge1, Ana Luisa Pereira2, Frederik Otzen Bagger3, Alexandar Tzankov4, Mineo Kurokawa5, Athimed El Taher6, Jonathan Seguin1, César Nombela Arrieta2, Juerg Schwaller1

1Biomedicine, University Children’s Hospital, Basel, Switzerland; 2Medical Oncology & Hematology, University Hospital, Zürich, Switzerland; 3Center for Genomic Medicine, University Hospital, Kopenhagen, Denmark; 4Institute for Pathology, University Hospital, Basel, Switzerland; 5Hematology & Oncology,The University of Tokyo, Tokyo, Japan; 6Biomedicine, University of Basel, Basel, Switzerland

Background: Expression of a doxycycline (DOX)-inducible acute myeloid leukaemia (AML)-associated iMLL-AF9 fusion transgene in long-term haematopoietic stem cells (LT-HSC) can lead to an invasive and chemo-resistant disease expressing the transcription factor EVI1. High EVI1 expression has been suggested as marker of poor outcome in AML patients even without rearrangements of the EVI1 locus at 3q26.

Aims: We addressed the association between EVI1 expression, the cellular origin and poor disease outcome in AML driven by the iMLL-AF9 fusion gene.

Methods: The role of EVI1 expression was studied in iMLL-AF9 transgenic mice carrying an Evi1-IRES GFP reporter in vitro using flow cytometry, colony formation and RT-qPCR assays, ex vivo with high-resolution bone marrow (BM) imaging, and in vivo, by transplantation of enriched naïve Evi1+ iMLL-AF9 hematopoietic stem and progenitor cells (HSPC) into irradiated recipients on DOX. Haematopoiesis of symptomatic mice was analysed by flow cytometry and histology. For mechanistic studies, single cell and bulk RNA sequencing was performed on enriched HSPC or BM samples from diseased mice.

Results: Analysis of BM cells from Evi1-IRES-GFP reporter mice revealed that not only the mostly quiescent LT-HSC but also fractions of the more proliferating multipotent progenitors (MPP1-3) express abundant Evi1 (“Evi1high”). Induction of the iMLL-AF9 fusion did not result in significant changes in numbers of Evi1+ cells nor levels of Evi1 mRNA expression in the LT-HSC and MPP1 compartments. However, in colony assays, Evi1high iMLL-AF9 cells retained a more immature phenotype and produced more colonies with an invasive morphology than Evi1low cells (n=11, p<0.05). While Evi1 expression did not influence disease induction upon transplantation of LT-HSC, recipients of Evi1+ MPP1 cells developed AML earlier than Evi1- MPP1 (n=11, 79 vs. 269d, p<0.05). Disease induced by Evi1+ cells presented with more extensive leukemic organ infiltration than Evi1- AML. Evi1 expression also correlated with in vitro Ara-C resistance. We also examined whether some exogenous factors may increase AML susceptibility by expanding the Evi1+ HSPC. Although a single injection of recombinant mouse thrombopoietin (TPO) only increased the number of LT-HSC and not of MPP1, the Evi1high cell fraction was enlarged in both compartments (LT-HSC: 23 vs. 50%; MPP1: 22 vs. 47%; n=29, p<0.0001) supported by high-resolution imaging. Interestingly, increased TPO induced HSPC cycling was confined to the Evi1high cell population (n=3, p<0.05). Transplantation of TPO-treated iMLL-AF9 LT-HSC or MPP1 resulted in a significantly faster induction of Evi1+ AML than controls (n=19, MPP1: 35 vs. 79d, p<0.001; LT-HSC: 41 vs. 90d, p<0.001). To better understand mechanisms of aggravated AML after TPO mediated expansion of Evi1+ HSCP we performed multiplexed single cell RNA sequencing of highly enriched HSPC cells in iMLL-AF9 and control mice. While we observed no changes of cellular cluster organisation 2 days after TPO injection, we found some differentially expressed genes in TPO-stimulated cycling iMLL-AF9 HSPC, including potential stemness regulators.

Summary/Conclusion: Our results suggest that expansion of Evi1-expressing HSPC by exogenous factors can result in a more aggressive MLL-AF9-driven AML. Ongoing data exploration and validation may characterize aberrantly expressed genes in TPO-stimulated Evi1+ iMLL-AF9-expressing HSPC as potential therapeutic targets to impair stemness of AML cells.


J. Schmoellerl1,*, I. Barbosa1, M. Minnich1, F. Andersch1, L. Smeenk2, M. Havermans2, T. Eder3, T. Neumann1, J. Jude1, M. Fellner1, A. Ebert1, M. Steininger1, R. Delwel2, F. Grebien3, J. Zuber1

1Research Institute of Molecular Pathology (IMP), Vienna, Austria; 2Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, Netherlands; 3Institute for Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria

Background: Chromosomal rearrangements leading to overexpression of EVI1 (MECOM) on chromosome 3q26 define a distinct subtype of acute myeloid leukemia (AML) that is associated with chemotherapy resistance and a 2-year survival of <10%. While genetic events driving aberrant expression of EVI1 are increasingly understood, the molecular functions of EVI1 that drive leukemogenesis are unclear, which has so far precluded the development of targeted therapeutics.

Aims: We aimed to elucidate transcriptional programs that are maintained by aberrant EVI1 expression and to systematically identify vulnerabilities of EVI1-driven AML.

Methods: We developed a panel of mouse models that recapitulate phenotypic and transcriptional hallmarks of patients suffering from EVI1-driven AML, allow tetracycline-controllable EVI1 expression and the functional interrogation of genetic targets using CRISPR/Cas9. We mapped transcriptional programs upon acute EVI1 repression in vivo and in vitro, profiled global EVI1 chromatin occupancy in human AML cell lines and primary patient-derived AML cells and performed comparative genome-wide CRISPR/Cas9-based loss-of-function screens in murine and human EVI1-driven AML.

Results: Integration of these datasets revealed a conserved core of genes that is transcriptionally regulated by EVI1 in murine and human AML, among which we identified the ETS transcription factor ERG as the only dependency that is highly selective for EVI1-driven AML. Suppression of ERG specifically triggered cellular differentiation and apoptosis of EVI1-driven leukemia cells while other AML cell lines were unaffected. Strikingly, ectopic expression of ERG was sufficient to functionally rescue loss of EVI1 in EVI1-driven AML cells, suggesting that the major oncogenic function of EVI1 in AML is the aberrant activation of ERG.

Summary/Conclusion: Interfering with the EVI1/ERG regulatory axis may provide entry points for the development of rational targeted therapies that are urgently needed for this group of AML patients.


S. Troester1,*, J. Schmoellerl2, T. Eder1, G. Manhart1, G. Winter3, J. Zuber2, F. Grebien1

1Institute for Medical Biochemistry, University for Veterinary Medicine Vienna; 2Research Institute of Molecular Pathology (IMP); 3Research Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), Vienna, Austria

Background: Oncogenic fusion proteins involving the Nucleoporin 98 (NUP98) gene are recurrently found in acute myeloid leukemia (AML) with a particular prevalence in pediatric patients. A chromosomal rearrangement resulting in the fusion of NUP98 to the gene encoding the lysine-specific demethylase 5A (KDM5A) is the most frequent NUP98-fusion in infant leukemia and is associated with particularly poor prognosis. The urgent need for the development of tailored treatments requires a better understanding of the effects of NUP98-KDM5A on the deregulation of gene expression programs. Although it has been shown that oncogenic NUP98-fusion proteins act as transcriptional regulators, it is unclear if and how NUP98-KDM5A directly regulates gene expression to drive leukemia.

Aims: In this study, we aimed to identify immediate critical effectors of the NUP98-KDM5A fusion protein and to characterize the transcriptional programs through which they regulate the development and maintenance of NUP98-KDM5A-driven AML.

Methods: We conducted a genome-scale CRISPR/Cas9 loss-of-function screen in a NUP98-KDM5A-driven murine AML cell line to unravel functional genetic dependencies that could be exploited to target leukemia cells. In parallel, we developed a new model for degradation tag (dTAG)-mediated ligand-induced degradation of the NUP98-KDM5A protein to gain a detailed understanding of direct transcriptional effects of NUP98-fusion-dependent gene regulation. We used this model to measure immediate changes in transcription upon acute NUP98-KDM5A degradation by nascent RNA-seq (SLAM-seq). An inducible shRNA system was used to assess the requirements of direct NUP98-KDM5A target genes for leukemia cell growth and to measure global gene expression changes by RNA-seq.

Results: Analysis of the CRISPR/Cas9 screen identified 4105 genes that are required for the proliferation and survival of NUP98-KDM5A-driven AML cells. Complete loss of the dTAG-NUP98-KDM5A fusion protein was achieved within one hour after ligand addition, resulting in cell cycle arrest, terminal differentiation and apoptosis of leukemia cells. Global analysis of nascent mRNA expression by SLAM-seq revealed 45 immediate NUP98-KDM5A target genes, as their transcription was significantly downregulated upon fusion protein degradation. Among these genes, 12 were classified as essential factors for NUP98-KDM5A cell growth from the CRISPR/Cas9 screen. This list included known target genes of NUP98-fusion proteins, such as members of the Hoxa gene cluster, but also other transcription factors, enzymes and RNA binding proteins. shRNA-mediated knockdown of the 12 candidate genes confirmed their essentiality for NUP98-KDM5A leukemia cell proliferation. Through RNA-seq studies, we found that the knockdown of a small subset among the 12 candidate genes was able to recapitulate global patterns of gene deregulation that are induced by NUP98-KDM5A knockdown.

Summary/Conclusion: Using a combination of CRISPR/Cas9 screening and ligand-induced degradation, we identified direct transcriptional target genes of NUP98-KDM5A that are functionally essential in AML. Multi-layered investigations of the interplay between the members of this small network of genes using a variety of models including primary patient samples will allow us to further dissect their role in the regulation of aberrant gene expression in NUP98-KDM5A-expressing leukemia cells and might identify novel therapeutic targets.


E. Heyes1,*, A. S. Wilhelmson2 3 4, A. Wenzel2 3 4, M. B. Schuster2 3 4, M. Ali3 4, T. D’Altri2 3 4, T. Eder1, G. Manhart1, E. Rzepa1, L. Schmidt1, M. Meggendorfer5, T. Haferlach5, G. Volpe6, C. Nerlov7, J. Frampton6, K. Jae Won3 4, F. Grebien1, B. Porse2 3 4

1Institute of Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria; 2The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences; 3Biotech Research and Innovation Center (BRIC); 4Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 5MLL Munich Leukemia Laboratory, München, Germany; 6Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham; 7MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

Background: The transcription factor CCAAT-enhancer-binding protein alpha (C/EBPα) is a master regulator of granulopoiesis and is mutated in 10-15 % of Acute Myeloid Leukemia (AML) patients. N-terminal frameshifts represent the predominant type of lesions and ablate the expression of the full-length protein p42, leading to overexpression of the shorter isoform p30. Mutations in the C-terminal basic-region leucine zipper (bZip) can disrupt the DNA-binding ability of C/EBPα. AML patients harbor mono- or biallelic CEBPA mutations (CEBPAmo or CEBPAbi). The most commonly co-occurring mutations are loss-of-function mutations in the methylcytosine dioxygenase TET2, resulting in adverse overall survival. We hypothesized that combinatorial effects of CEBPA mutations together with TET2 loss specifically rewire transcriptional and epigenetic circuitries in AML cells, thereby strongly influencing disease outcome.

Aims: We aimed to elucidate the molecular mechanisms behind cooperative effects of CEBPA and TET2 mutations through state-of-the-art transcriptomic and epigenomic analyses of relevant in vitro and in vivo models as well as data from AML patients.

Methods: We used the CRISPR/Cas9 technology to introduce Tet2 mutations in murine cell lines expressing only p30 (Cebpap30/p30) or mimicking biallelic CEBPA mutations (Cebpap30/C-mut.) to study the functional cooperation of these mutations in vitro. A Cebpa-/p30Tet2-/- mouse model was used to study effects of Tet2 loss in CEBPA mutated AML. We performed RNA-, C/EBPα-ChIP-, ATAC-, Bisulfite-, CUT&RUN and CRISPR/Cas9-mediated enhancer screens to generate a comprehensive dataset for in-depth comparative analysis and correlation with relevant patient data from the beatAML collection.

Results: Integration of transcriptomic and epigenomic data from in vitro and in vivo models of CEBPA-TET2 co-mutated AML, in combination with gene expression analyses in AML patients, identified the transcription factor GATA2 as a conserved target of the CEBPA-TET2 axis. p30 and TET2 were strongly bound to the -77 kb enhancer of the Gata2 gene, and CRISPR/Cas9-induced enhancer deletions diminished Gata2 expression. Furthermore, TET2 loss reduced chromatin accessibility and increased DNA methylation of the Gata2 promoter, resulting in decreased Gata2 mRNA levels. RNAi-mediated silencing revealed a dose-dependent effect of Gata2 expression on leukemia cell fitness in vivo. Reduction of Gata2 levels by 25-50 % provided a strong competitive advantage to Cebpap30/p30 cells while near-complete downregulation of Gata2 expression (>75 %) dramatically reduced cellular fitness. Finally, treatment with the demethylating agent 5-azacytidine restored Gata2 expression in an AML model with Cebpa and Tet2 mutations and caused a significant survival benefit.

Summary/Conclusion: The datasets generated from these models enable deeper insights into the epigenetic and transcriptomic changes that depend on CEBPA and TET2 mutations in a physiologically relevant mutational context. Our results reveal that mutational disruption of CEBPA and TET2 results in down-regulated GATA2 expression, causing aggressive AML. We propose a mechanism in which C/EBPα p30 mediates recruitment of TET2 to regulatory regions in the GATA2 gene to maintain its expression. Conversely, loss of TET2 leads to reduced GATA2 levels, which is restored by 5-azacytidine treatment. Thus, interference with the C/EBPα-TET2 axis may provide entry points for the development of rational targeted therapies in AML patients with these mutations.


A. Narr1 2 3,*, H. Alborzinia1 2, E. Donato1 2, F. Vogel4, T. Boch1, A.-M. Leppä1 2, A. Waclawiczek1 2, S. Renders1 2, A. Schulze4, A. Trumpp1 2

1Division of Stem Cells & Cancer, German Cancer Research Center (DKFZ); 2Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH); 3University Heidelberg; 4Division of Tumor Metabolismus und Microenvironment, German Cancer Research Center (DKFZ), Heidelberg, Germany

Background: Despite advances in the treatment of Acute Myeloid Leukemia (AML), the 5-year patient survival rate remains poor with 15-20%. The majority of patients develop recurrence partly due to resistance mechanisms to classical cell death programs. Exploiting new forms of cell death therefore may allow novel therapeutic options to limit survival or occurrence of resistant clones. Ferroptosis has been identified as non-apoptotic and iron-dependent form of cell death, characterized by an excessive ROS-induced peroxidation of cell membranes. Recently it has been implicated with a higher sensitivity in cancer stem cells and drug-resistant cancer cells, potentially making ferroptosis-inducing (FIN) therapies a promising option.

Aims: We aim to elucidate whether AML represents a ferroptosis-sensitive cancer entity and is susceptive for potential FIN-therapies.

Methods: IC50 values of ferroptosis inducers were determined and used in combination with the TCGA-LAML and a pan-cancer CRISPR screen dataset (CERES) to describe ferroptosis sensitivity of AML. A drug screen with known ROS-inducers was performed in 9 AML cell lines to identify ferroptosis induction. To mechanistically characterize ferroptotic cell death by the identified drug on the genetic and metabolic level, we performed SLAM-RNA-, RNA-Sequencing, Mass Spectrometry and Metabolite Tracing (Cystine/Ser/Gln) in HL-60 cells in vitro and in vivo. Genetic (CRISPR-Cas9-KO) and chemical inhibition were used to characterize the role of identified hits and pathways, aiming to design new synergistic combination therapies.

Results: We find that AML belongs to the most dependent cancer entities for various ferroptosis-associated genes and represents a ferroptosis-sensitive cancer entity. Furthermore, a 12-gene-ferroptosis-signature allows us to predict the Overall Survival of AML patients and shall help to identify a potential cohort for FIN-therapies. By screening known ROS-inducing drugs for ferroptosis induction, we identify one drug capable of inducing lipid peroxidation and subsequently ferroptosis in multiple AML and other tumor cell lines. Notably, in detail characterization of the drug’s mechanism on the genetic and metabolic level reveal the activity of the iron-metabolism gene HMOX-1 as essential for ferroptosis induction. Mechanistically, we further find that this drug dir