Introduction: Persisting cancer related fatigue (CRF) has impact on health related quality of life (HRQoL) and social re-integration of patients with Hodgkin lymphoma (HL). The GHSG HD18 trial established treatment PET-2 guided de-escalation of treatment for advanced-stage HL as new standard. Here, we investigate the impact of treatment de-escalation in HD18 on long-term HRQoL domains and time-to-recovery from fatigue (TTR-F). Methods: Mean fatigue scores (FA) of the EORTC QLQ-C30 questionnaire are reported descriptively for baseline, interim, end-of-treatment, and yearly follow-up. TTR-F was defined as time from end of chemotherapy until first occurrence of FA <30 or the time of last questionnaire (censored). TTR-F was analyzed and compared using time-to-event methods including cumulative incidence and cox proportional hazard models, as recommended by the SISAQoL consortium. Effect of disease, patient and treatment characteristics on 2y HRQoL domains was analyzed using multiple regression. Results: 2101 patients aged 18–60 years with advanced-stage HL were recruited in HD18, of whom 156 were found ineligible before or after randomization. PET-2 negative Patients were randomized between 8x eBEACOPP (arm C, n = 288) and 4x eBEACOPP (arm D; n = 285), and between 6x eBEACOPP (arm C6; n = 216) and 4x eBEACOPP (D4; n = 216). HRQoL questionnaires at baseline were available in 83.9% of all randomized patients. Overall, baseline FA and age were significantly associated with TTR-F, whereas sex was not. TTR-F differed between trial arms for PET-2 negative, but not for PET-2 positive patients. Particularly, treatment reduction from 8 to 4 cycles of eBEACOPP led to a significantly shorter TTR-F (HR 1.41, p = 0.008). Reducing the cycle number of eBEACOPP from 8 to 6 cycles (HR 1.21, p = 0.2) or 6 to 4 cycles (HR 1.22, p = 0.18) speeded TTR-F accordingly but was not statistically significant. For PET-positive patients a significantly slower TTR-F was observed with addition of Rituximab (HR 0.7, p = 0.0163). In PET-2-negative patients, median TTR-F was 19 months (CI95: 13–28) in arm C, 13 months (CI95: 10–20) in arm C6, 12 months (CI95: 8–15) in arm D and 10 months (CI95: 8–13) arm D4. HRQoL at baseline and age were the main determinants of 2y HRQoL domains. The research was funded by: Deutsche Krebshilfe, Swiss State Secretariat for Education and Research, and Roche Pharma AG. Keywords: Chemotherapy, Hodgkin lymphoma, Late Effects in Lymphoma Survivors No conflicts of interests pertinent to the abstract.

Measurable residual disease (MRD) is an important biomarker in acute myeloid leukemia (AML) that is used for prognostic, predictive, monitoring, and efficacy-response assessments. The European LeukemiaNet (ELN) MRD Working Party evaluated standardization and harmonization of MRD in an ongoing manner and has updated the 2018 ELN MRD recommendations based on significant developments in the field. New and revised recommendations were established during in-person and online meetings, and a 2-stage Delphi poll was conducted to optimize consensus. All recommendations are graded by levels of evidence and agreement. Major changes include technical specifications for next-generation sequencing-based MRD testing and integrative assessments of MRD irrespective of technology. Other topics include use of MRD as a prognostic and surrogate end point for drug testing; selection of the technique, material, and appropriate time points for MRD assessment; and clinical implications of MRD assessment. In addition to technical recommendations for flow- and molecular-MRD analysis, we provide MRD thresholds and define MRD response, and detail how MRD results should be reported and combined if several techniques are used. MRD assessment in AML is complex and clinically relevant, and standardized approaches to application, interpretation, technical conduct, and reporting are of critical importance.

Background The German Hodgkin Study Group's HD18 trial established the safety and efficacy of PET-guided eBEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone in escalated doses) for the treatment of advanced-stage Hodgkin lymphoma. However, because of a protocol amendment during the enrolment period (June 1, 2011) that changed standard treatment from eight to six cycles, the results of the HD18 trial have been partially immature. We report a prespecified 5-year follow-up analysis of the completed HD18 trial. Methods HD18 was an international, open-label, randomised, phase 3 trial done in 301 hospitals and private practices in five European countries. Patients aged 18–60 years with newly diagnosed, advanced-stage Hodgkin lymphoma and an Eastern Cooperative Oncology Group performance status of 0–2 were recruited. After receiving an initial two cycles of eBEACOPP (1250 mg/m2 intravenous cyclophosphamide [day 1], 35 mg/m2 intravenous doxorubicin [day 1], 200 mg/m2 intravenous etoposide [day 1–3], 100 mg/m2 oral procarbazine [day 1–7], 40 mg/m2 oral prednisone [day 1–14], 1·4 mg/m2 intravenous vincristine [day 8], and 10 mg/m2 intravenous bleomycin [day 8]), patients underwent a contrast-enhanced CT and PET scan (PET-2). Patients with positive PET-2 were randomly assigned to receive standard therapy (an additional six cycles of eBEACOPP; ie, eight cycles in total) or experimental therapy (an additional six cycles of eBEACOPP plus 375 mg/m2 intravenous rituximab; ie, eight cycles in total) until June 1, 2011. After June 1, 2011, all patients with positive PET-2 were assigned to the updated standard therapy with an additional four cycles of eBEACOPP (ie, six cycles in total). Patients with negative PET-2 were randomly assigned (1:1) to receive standard therapy (an additional six cycles of eBEACOPP [ie, eight cycles in total] until June 1, 2011; an additional four cycles of eBEACOPP [ie, six cycles in total] after June 1, 2011) or experimental therapy (an additional two cycles of eBEACOPP; ie, four cycles in total). Randomisation was done centrally with the minimisation method, including a random component, stratified by centre, age, stage, international prognostic score, and sex. The primary endpoint was progression-free survival. HD18 aimed to improve 5-year progression-free survival by 15% in the PET-2-positive intention-to-treat cohort and to exclude inferiority of 6% or more in 5-year progression-free survival in the PET-2-negative per-protocol population. This study is registered with ClinicalTrials.gov, NCT00515554, and is completed. Findings Between May 14, 2008, and July 18, 2014, 2101 patients were enrolled and 1945 were assigned to a treatment group according to their PET-2 result. In the PET-2-positive cohort, with a median follow-up of 73 months (IQR 59 to 94), 5-year progression-free survival was 89·9% (95% CI 85·7 to 94·1) in 217 patients assigned to eight cycles of eBEACOPP before the protocol amendment and 87·7% (83·1 to 92·4) in 217 patients assigned to eight cycles of rituximab plus eBEACOPP (p=0·40). Among 506 patients who received six cycles of eBEACOPP after the protocol amendment, 5-year progression-free survival was 90·1% (95% CI 87·2 to 92·9), with a median follow-up of 58 months (IQR 39 to 66). In the PET-2-negative cohort, with a median follow-up of 66 months (IQR 54 to 85) in the combined pre-amendment and post-amendment groups, 5-year progression-free survival was 91·2% (95% CI 88·4 to 93·9) in 446 patients who received eight or six cycles of eBEACOPP and 93·0% (90·6 to 95·4) in 474 patients who received four cycles of eBEACOPP (difference 1·9% [95% CI −1·8 to 5·5]). In the subgroup of PET-2-negative patients randomly assigned after protocol amendment, 5-year progression-free survival was 90·9% (95% CI 86·8 to 95·1) in 202 patients assigned to receive six cycles of eBEACOPP and 91·0% (86·6 to 95·5) in 200 patients assigned to receive four cycles of eBEACOPP (difference 0·1% [–5·9 to 6·2]). Interpretation Long-term follow-up confirms the efficacy and safety of PET-2-guided eBEACOPP in patients with advanced-stage Hodgkin lymphoma. The reduction from eight to four cycles of eBEACOPP represents a benchmark in the treatment of early-responding patients, who can now be potentially cured with a short and safe treatment approach. Funding Deutsche Krebshilfe, Swiss State Secretariat for Education, Research and Innovation SERI (Switzerland), and Roche Pharma. Translation For the German translation of the abstract see Supplementary Materials section.

Acute myeloid leukemia (AML) is characterized by relapse and treatment resistance in a major fraction of patients, underlining the need of innovative AML targeting therapies. Here we analysed the therapeutic potential of an innovative biohybrid consisting of the tumor-associated peptide somatostatin and the photosensitizer ruthenium in AML cell lines and primary AML patient samples. Selective toxicity was analyzed by using CD34 enriched cord blood cells as control. Treatment of OCI AML3, HL60 and THP1 resulted in a 92, and 99 and 97% decrease in clonogenic growth compared to the controls. Primary AML cells demonstrated a major response with a 74 to 99% reduction in clonogenicity in 5 of 6 patient samples. In contrast, treatment of CD34+ CB cells resulted in substantially less reduction in colony numbers. Subcellular localization assays of RU-SST in OCI-AML3 cells confirmed strong co-localization of RU-SST in the lysosomes compared to the other cellular organelles. Our data demonstrate that conjugation of a Ruthenium complex with somatostatin is efficiently eradicating LSC candidates of patients with AML. This indicates that receptor mediated lysosomal accumulation of photodynamic metal complexes is a highly attractive approach for targeting AML cells.

Background: CPX-351, a liposomal formulation of daunorubicin and cytarabine in the fixed molar ratio (1:5), is approved for the treatment of adult patients (pts) with newly diagnosed acute myeloid leukemia (AML) with myelodysplasia-related changes and therapy-related AML (t-AML). To explore the potential benefit of CPX-351 in a broader indication, we initiated a randomized phase III study of CPX-351 vs "3+7" in pts ≥18 years (yrs) of age with AML and intermediate or adverse genetics according to 2017 European LeukemiaNet (ELN) risk categorization (AMLSG 30-18, NCT03897127). In the younger pts (18-60 yrs) we sought to investigate a higher dose of CPX-351. We here report data from an interim safety analysis for this higher CPX-351 dose. Methods: Pts are randomized to receive first induction cycle (ind 1) with either CPX-351 or daunorubicin + cytarabine ("3+7": daunorubicin 60 mg/m2 on days 1, 2, 3 + cytarabine 200 mg/m2 on days 1-7); in pts aged 18-60 yrs (performance status 0-1) CPX-351 is given at a dose of 55 mg/m2 daunorubicin/125 mg/m2 cytarabine (125 U/m²; 1 U/m2=0.44 mg/m2 daunorubicin/1 mg/m2 cytarabine; days 1, 3, 5); pts >60 yrs receive the standard dose CPX-351 100 U/m² (days 1, 3, 5). There was no age-adapted dosing in the control arm. For induction cycle 2 (ind 2), pts on the CPX-351 arm receive the same dosage on day 1+2 only; pts on the control arm receive intermediate-dose cytarabine + daunorubicin (both in age-adapted dosing). Continuous assessment for safety is performed for two endpoints: 60-day mortality with a maximally tolerated rate (MTR) of 15%; and hematologic recovery times with i) neutropenia 4° and / or ii) thrombocytopenia 3° or 4° after each ind lasting longer than day 42 after start of treatment cycle (without evidence of persistent leukemia) with a MTR of 25%. Median hematologic recovery times were analyzed using Kaplan-Meier estimates, p-values are mentioned in a descriptive manner (log-rank test). Results: As of July 20, 2020, 36 patients have been randomized to the study (CPX-351, n=19; "3+7", n=17) with following characteristics: de novo AML, n=27, secondary or t-AML, n=9; median age 60.5 yrs (range 47-75; ≤60 yrs, n=18; >60 yrs, n=18); intermediate and adverse risk genetics were found in 7 and 10 pts, respectively (not available yet, n=19). On the CPX-351 arm, 9 of 19 pts were ≤60 yrs of age and received the higher CPX-351 dose. So far, 36 pts received ind 1, 25 pts ind 2. Overall, the median time to neutrophil recovery with absolute neutrophil count (ANC) >0.5 G/l was longer in the CPX-351 arm compared to the "3+7" arm: 39 vs 28 days (p=0.07) after ind 1, and 26.5 vs 19 days after ind 2 (p=0.06; table 1). Time to platelet recovery >50 G/l was significantly prolonged in the CPX-351 arm after ind 1 (40 vs 26 days; p<0.0001), currently not after ind 2 (33 and 18 days; p=0.35). When comparing the higher dose (125 U/m²; pts 18-60 yrs) with the standard CPX-351 dose (100 U/m², pts >60 yrs), the median time to neutrophil recovery after ind 1 was significantly longer with the higher dose (40 and 31 days, respectively; p=0.03); after ind 2 median times were 38 and 20.5 days (p=0.26); platelet recovery (>50 G/l) was also significantly delayed after ind 1 with the higher compared to the standard CPX-351 dose (median 43 vs 32 days; p=0.002); platelet recovery after ind 2 was after a median of 38.5 and 26.5 days, respectively (p=0.17). There was no treatment-related death (60-day mortality 0%) in both arms. So far, 6 of the 9 pts (67%) treated with the higher CPX-351 dose reached the safety endpoint of persisting neutropenia (n=4) or thrombocytopenia (n=5) during ind beyond day 42. The MTR was exceeded for thrombocytopenia (0.63; 95% confidence interval (CI) [0.31; 0.86]), but not for neutropenia (0.50; 95% CI [0.22; 0.78]). Overall, there were 18 serious adverse events (SAEs); among the most frequent SAEs were infections and fever in neutropenia (n=10). Conclusion: The higher dose of CPX-351 administered in pts 18-60 yrs of age led to significantly prolonged hematologic recovery times during ind 1 and 2 exceeding the MTR for thrombocytopenia without treatment-related death. Based on the prolonged hematologic recovery, the protocol will be amended, in that the CPX-351 dose for ind in pts 18-60 yrs of age is reduced to the current Package Insert for CPX-351 44 mg/m2 daunorubicin / 100 mg/m2 cytarabine (100 U/m²). Data on hematologic response as well as on measurable residual disease using multi-parameter flow cytometry will be presented. Disclosures Kapp-Schwoerer: Jazz Pharmaceuticals: Honoraria, Research Funding. Thol:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Heuser:Karyopharm: Research Funding; Abbvie: Consultancy; Astellas: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Roche: Research Funding; BerGenBio ASA: Research Funding; Janssen: Consultancy; Stemline Therapeutics: Consultancy; Bayer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Amgen: Research Funding; PriME Oncology: Honoraria. Faderl:Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Wagner:JAZZ Pharmaceuticals: Current Employment; JAZZ Pharmaceuticals: Current equity holder in publicly-traded company. Ganser:Celgene: Consultancy; Novartis: Consultancy. Döhner:Abbvie: Consultancy; Daiichi Sankyo: Honoraria; Celgene: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Amgen: Consultancy, Research Funding; Astellas Pharma: Consultancy; Janssen: Consultancy, Honoraria; Sunesis Pharmaceuticals: Research Funding; Agios: Consultancy; Arog: Research Funding. Paschka:BerGenBio ASA: Research Funding; Janssen Oncology: Other; Amgen: Other; Otsuka: Consultancy; Novartis: Consultancy, Speakers Bureau; Sunesis Pharmaceuticals: Consultancy; Pfizer: Consultancy, Speakers Bureau; Astellas Pharma: Consultancy, Speakers Bureau; Celgene: Consultancy, Other: Travel, accommodations or expenses; Jazz Pharmaceuticals: Consultancy, Speakers Bureau; Agios Pharmaceuticals: Consultancy, Speakers Bureau; Astex Pharmaceuticals: Consultancy; AbbVie: Other: Travel, accommodation or expenses, Speakers Bureau. Döhner:Abbvie: Consultancy, Honoraria; Sunesis: Research Funding; Roche: Consultancy, Honoraria; Pfizer: Research Funding; Oxford Biomedicals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Helsinn: Consultancy, Honoraria; Jazz: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; AROG: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Astex: Consultancy, Honoraria; GEMoaB: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. OffLabel Disclosure: CPX-351 is approved for the treatment of adult patients with newly diagnosed acute myeloid leukemia (AML) with myelodysplasia-related changes and therapy-related AML (t-AML). To explore the potential benefit of CPX-351 in a broader indication, a randomized phase III study of CPX-351 vs 3+7 in patients older than 18 years of age with AML and intermediate or adverse genetics according to 2017 European LeukemiaNet (ELN) risk categorization (AMLSG 30-18, NCT03897127) was initiated. In the younger patients (18-60 yrs) a higher dose of CPX-351 is evaluated.

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection. Here we present the validation experiments of the assay in several large AML research centres, both in Europe and the United States. Variability within instruments and sample processing showed high correlations between different instruments (Rpearson > 0·91, P < 0·001). Multi-centre testing introduced variation in reported LSC percentages but was found to be below the clinical relevant threshold. Clear gating protocols resulted in all laboratories being able to perform LSC assessment of the validation set. Participating centres were nearly unanimously able to distinguish LSChigh (>0·03% LSC) from LSClow (<0·03% LSC) despite inter-laboratory variation in reported LSC percentages. This study proves that the LSC assay is highly reproducible. These results together with the high prognostic impact of LSC load at diagnosis in AML patients render the one-tube LSC assessment a good marker for future risk classification.

characterized by skewed epigenetic patterns, raising the possibility of therapeutically targeting epigenetic factors in this disease. Here we report that among different cancer types, epigenetic 38 factor TET1 is highly expressed in T-ALL and is crucial for human T-ALL cell growth in vivo . 39 Tet1 knockout mice and knockdown in human T-cells did not perturb normal T-cell proliferation, 40 indicating that TET1 expression is dispensable for normal T-cell growth. The promotion of 41 leukemic growth by TET1 was depending on its catalytic property to maintain global 5- 42 hydroxymethylcytosine (5hmC) marks, thereby regulating cell cycle, DNA repair genes and T- 43 ALL associated oncogenes. Furthermore, overexpression of the Tet1 catalytic domain was 44 sufficient to augment global 5hmC levels and leukemic growth of T-ALL cells in vivo . We 45 demonstrate that PARP enzymes, which are highly expressed in T-ALL patients, participate in 46 establishing H3K4me3 marks at the TET1 promoter and that PARP1 interacts with the TET1 47 protein. Importantly, the growth related role of TET1 in T-ALL could be antagonized by the 48 clinically approved PARP inhibitor Olaparib, which abrogated TET1 expression, induced loss of 49 5hmC marks and antagonized leukemic growth of T-ALL cells, opening a therapeutic avenue for 50 this disease. targeting epigenetic factors for treating this malignancy. Recent studies in healthy human CD4 naïve T-cells indicate that TET1 negatively regulates Th1/Th2 differentiation by suppressing the expression of pro-differentiation genes such as GATA3 , CD69 and IFNG in vitro growth promoting. In this study, we now demonstrate that in human T-ALL cells, high TET1 expression maintains global hydroxymethylome which positively regulates gene expression, safeguards genome integrity, and thereby promotes leukemic growth. Furthermore, our data indicate that the growth promoting activity of TET1 can be pharmacologically targeted via inhibition of PARPs, 82 which act as TET1 upstream regulators, opening a potential treatment modality for T-ALL patients.

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

About 30% of all Hodgkin lymphoma (HL) patients are ≥60 years old. As lenalidomide has promising single agent activity in multiple relapsed HL, we replaced bleomycin in ABVD with lenalidomide in this phase-I trial. Patients aged ≥60 years with early-unfavourable- or advanced-stage HL (Eastern Cooperative Oncology Group performance status ≤2, Cumulative Illness Rating Scale for Geriatrics score 0–7) received 4–8 cycles of AVD (doxorubicin, vinblastine, dacarbazine) and lenalidomide in escalation with overdose control. Dose-limiting toxicities (DLTs) included thromboembolism ≥grade 2, severe haematological toxicity, neutropenic fever and prolonged therapy delay. Twenty-five patients with a median age of 68 years were included, 68% had advanced-stage HL. A pre-defined stopping criterion for dose escalation after DLT evaluation of 20/24 patients suggested a recommended phase II dose (RPTD) of 20 mg. DLTs occurred in 10/24 evaluable patients, all treated with ≥20 mg, however, median relative dose intensity was 97% (interquartile range 49–104%). Grade 3 or higher toxicities occurred in all 22 patients at ≥20 mg lenalidomide but no treatment-related deaths occurred. Overall response rate was 80% for all patients (20/25) and 86% (19/22) at ≥20 mg lenalidomide. Three-year estimates for progression-free survival and OS were 69·7% (95% CI: 50·3–89·1%) and 83·8% (95%-CI: 69·3–98·4%), respectively. In conclusion, AVD with lenalidomide 20 mg is feasible and highly effective in older HL patients.

Acute erythroid leukemia (AEL) is a rare and aggressive form of acute leukemia, the biology of which remains poorly understood. Here we demonstrate that the ParaHox gene CDX4 is expressed in patients with acute erythroid leukemia, and that aberrant expression of Cdx4 induced homogenously a transplantable acute erythroid leukemia in mice. Gene expression analyses demonstrated upregulation of genes involved in stemness and leukemogenesis, with parallel downregulation of target genes of Gata1 and Gata2 responsible for erythroid differentiation. Cdx4 induced a proteomic profile that overlapped with a cluster of proteins previously defined to represent the most primitive human erythroid progenitors. Whole-exome sequencing of diseased mice identified recurrent mutations significantly enriched for transcription factors involved in erythroid lineage specification, as well as TP53 target genes partly identical to the ones reported in patients with AEL. In summary, our data indicate that Cdx4 is able to induce stemness and inhibit terminal erythroid differentiation, leading to the development of AEL in association with co-occurring mutations.

This study explores biocompatible amino‐functionalized gold nanoparticles (Au‐NH 2 ) as nanotherapeutics for the selective eradication of leukemia cells, elucidates the mechanism of cytotoxicity, and it confirms in vivo efficacy of the engineered nanomaterial. Au‐NH 2 trigger apoptotic cell death of myeloid leukemia cell lines and primary acute myeloid leukemia (AML) cells by i) inhibition of mitochondrial respiration, ii) ATP depletion, iii) loss of mitochondrial membrane potential, and iv) mitochondrial release of cytochrome c. Au‐NH 2 act selectively on leukemia cells inasmuch as the viability of normal peripheral blood mononuclear cells and macrophages as well as the colony formation of hematopoietic stem cells remain basically unaffected. The selectivity of Au‐NH 2 for AML cells can be attributed to both the preferential accumulation of AuNH 2 in AML cells and the strong dependence of those cells on mitochondrial oxidative phosphorylation for ATP production. Importantly, Au‐NH 2 applied either as monotherapy or as a cytarabine combination regimen possess antileukemic efficacy in the absence of adverse events in mice xenografted with primary human AML in vivo. The engineered material may pave the way for a novel nanotherapeutic treatment of AML.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is propagated by leukemic stem cells (LSCs), often characterized by deregulated Wnt signaling. We previously showed that the central transcriptional mediator of Wnt signaling LEF1 is able to cause AML in mice and acts as an independent prognostic factor in normal karyotype AML. Here, we show that treatment naïve normal karyotype AML as well as samples AML LSCs predominantly express the long β-catenin-binding isoform of LEF1 in sharp contrast to normal human hematopoietic stem cells, which lack expression of the long isoform, but express the short N-terminally truncated isoform with loss of the β-catenin-binding site. Gene expression and ChiP-Seq analyses in mice linked the long isoform to Wnt-β-catenin signaling and oncogenic pathways, the N-terminally truncated isoform to stemness associated genes. Approaches impairing binding of LEF1 to β-catenin significantly impaired AML growth, but spared normal hematopoietic stem cells. This report now demonstrates a striking difference of LEF1 isoform expression between normal and AML cells, contributing to higher vulnerability of leukemic cells to approaches targeting β-catenin/LEF1 interaction.

Acute myeloid leukemia (AML) is characterized by high mortality, underlining the necessity for identifying tumor suppressors that counteract the leukemogenic potential of bona fide oncogenes such as the homeobox genes HOXA9 and CDX2. Homeobox genes are aberrantly expressed in the majority of patients with cytogenetically normal (CN)-AML and expression of CDX2 positively correlates with HOXA9 expression. Aberrant expression of Cdx2 in murine hematopoietic cells rapidly induced aggressive AML in mice. Recently, it was shown that a microRNA, miR-196, is encoded in the mammalian paralogous HOX gene cluster and that it has extensive evolutionarily conserved complementarity to sites in the 3' prime untranslated regions (3' UTR) of HOX genes (e.g. HOXA9, A7 and B8), directly regulating their expression in MLL-rearranged leukemic cells. To understand the role of miR-196b in AML more precisely, we first tried to identify transcripts generating mature miR-196b in human hematopoietic stem and progenitor cells (HSPC). We identified two novel non-coding transcripts encoding the miR-196b hairpin precursor sequence expressed from the HOXA9-10 locus in bone marrow HSPC (Online Supplementary Figure S1A, B) [NCBI accession number: MF139050, 486 basepairs (bp) and MF139051, 396 bp]. MF139051, a splice variant of the MF139050 transcript, showed 99.47% homology to MF139050. Both transcripts displayed high homology to the transcripts of other vertebrates and mammalian species. Retrovirally engineered expression of both transcripts resulted in significant overexpression of the mature miR-196b in HEK293T cells compared to the vector control (Figure 1A; Online Supplementary Figure S1C, D). Endogenous expression levels of both transcripts in the human CD34 bone marrow compartment were higher than those in mononuclear cells (Figure 1B). Second, we identified a highly conserved 802 bp long miR-196b promoter region, validated by luciferase reporter assay, located 201 bp upstream of the miR-196b stem loop precursor sequence on human chromosome 7 (Figure 1C; Online Supplementary Figure S1E, F). Transcription factor binding site prediction tools and published chromatin immunoprecipitation sequencing data showed, respectively, potential binding of transcription factors including SP1 and enrichment for proteins such as EZH2 on this promoter (Online Supplementary Figure S1G, H; Online Supplementary Table SW1). Previously, the miR-196b promoter region was described in the context of murine development, demonstrating in vivo enrichment for Cdx2 and Hoxd13. Next, we asked whether expression of miR-196b itself as well as the ratio between miR-196b expression and the expression of its direct target HOXA9 is perturbed in CN-AML patients: miRNA-sequencing analysis of healthy bone marrow-derived hematopoietic subpopulations revealed highest expression of miR-196b in the lymphoid-primed multi-potent progenitor subpopulation, following the same expression pattern as known for its target HOXA9, whose expression is also highest in immature cells (Figure 1D; Online Supplementary Table SE1). This resulted in a significant correlation between the expression levels of mature miR-196b or MF139051 and HOXA9 in HSPC (Online Supplementary Figure S2A; Online Supplementary Table SW2). In contrast, expression levels of miR-196b and of HOXA9 remained at similar levels across all three functionally validated leukemic bone marrow subpopulations from CN-AML patients (Figure 1D; Online Supplementary Table SE1). In contrast to the expression levels of the miR-196b transcript MF139051 and miR-196b, HOXA9 expression was dramatically increased in leukemic CD34 bone marrow compared to CD34 normal bone marrow cells. This difference could be confirmed in functionally validated leukemic stem cells compared to normal HSPC and in published data (Online Supplementary Figures S2B and S3; Online Supplementary Tables SW3-4). Based on this, the correlation between the expression levels of the mature miR-196b and its transcripts versus the expression of HOXA9 was lost in leukemic cells (Supplementary Figures S4). The difference in expression patterns resulted in a 111-fold higher ratio between the expression values of HOXA9 and mature miR-196b in CN-AML compared to normal CD34 bone marrow and a 57-fold higher ratio between NPM1c AML and normal CD34 bone marrow, which also held true for the short transcript MF139051 calculated for the same AML groups (Figure 1E; Online Supplementary Table SW4). This implies that compared to normal CD34 hematopoietic cells there are many fewer miR-196b transcripts and mature miRNA per HOXA9 transcript in CN-AML, including NPM1-mutated cases, previously shown to have particularly high HOX gene expression. Overexpression of miR-196b in normal murine HSPC did not affect clonogenicity and cell growth in vitro (Figure 2A; Online Supplementary Figure S5A, B). miR196b increased the proportion of circulating myeloid cells and three of 14 mice developed AML after a long latency, indicating that cooperating events may contribute to disease induction. (Figure 2B, F; Online Supplementary Table SW5). To test the impact of miR-196b expression in the context of Cdx2-induced AML, we retrovirally coexpressed Cdx2/miR-196b or Cdx2 alone in murine HSPC (Online Supplementary Figure S5C). Co-expression of miR-196b considerably reduced the Cdx2-induced proliferation, clonogenicity and spleen colony formation compared to those of Cdx2-transduced cells (Figure 2C, D; Online Supplementary Figure S5D-F). Cdx2-transplanted mice developed AML with a median latency of 193 days. In contrast, only 40% of the Cdx2/miR-196b-transplanted mice developed AML with a significantly longer latency (Figure 2E; Online Supplementary Table SW5). Moreover, miR-196b impaired growth and colony formation in the CDX2 and HOX gene-positive human AML cell lines OCI-AML3 and NB4 in vitro, in contrast to the CDX2 and HOX gene-negative human AML cell line Kasumi-1. Furthermore, miR-196b reduced engraftment of OCI-AML3 in NSG mice (Online Supplementary Figure S6A-K; Online Supplementary Table SW6). Overexpression of miR-196b did not induce apoptosis, cell cycle arrest or senescence in OCI-AML3 and NB4 cells (data not shown), suggesting that miR-196b preferentially targets selfrenewal by reducing HOX gene expression, thereby reducing clonogenicity and engraftment potential. Overexpression of miR-196b in HSPC alone resulted in 155 differentially expressed genes compared to the control. Among these 155 genes, 44% of the downregulated ones were significantly enriched for known miR-196b targets, whereas the upregulated ones did not show any miR-196b target enrichment (Online Supplementary Tables SE2-5; Online Supplementary Figure S7A-C). In Cdx2-transduced cells, overexpression of miR-196b induced 524 differentially expressed genes compared to Cdx2 alone (Online Supplementary Figure S7D; Online Supplementary Tables SE6 and SE7). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

During DNA replication and transcription, RNA:DNA hybrids are formed as part of three stranded nucleic acid structures known as R-loops. R-loops occur frequently in the genome at highly transcribed regions, ribosomal genes, mitochondria and intergenic regions, and are predominantly resolved by Ribonuclease (RNase) H family of enzymes. However, unscheduled and unresolved R-loops represent a potent source of DNA damage, especially in rapidly dividing cells such as cancer cells. It is imperative for cancer cells to prevent accumulation of unresolved R-loops in order to limit DNA damage. So far, the mechanism how leukemic cells prevent accumulation of R-loops is not well understood. In this study, we show that an RNase H-like protein, PIWIL4, is aberrantly and highly expressed in AML patients, prevents R-loop accumulation via its RNase H activity and thereby acts as important regulator of leukemic growth. In our initial analysis, we observed that the recombinant human PIWIL4 protein digested radiolabeled-RNA-containing R-loops in vitro, exhibiting an RNase H-like activity with increasing efficiency, in incremental concentrations and time durations. Moreover, immunoprecipitation of PIWIL4 followed by liquid chromatography mass spectrometry (LC/MS) in HEK cells showed that PIWIL4 was bound with multiple nuclear and nucleolar RNA processing factors that are associated with formation of R-loops. Published RNA-seq and microarray datasets revealed that, among all cancers, PIWIL4 was significantly highest expressed in myeloid leukemia. Quantitative real time PCR (qRT-PCR) of acute myeloid leukemia (AML) patients revealed that PIWIL4 showed an average of 21.6 ± 5.0-fold higher expression in AML patients (n=68; p<0.0001), compared to healthy CD34+ bone marrow (BM) and BM mononuclear cells (n=3). Western blot of AML patient samples and intracellular (IC) staining confirmed higher PIWIL4 protein expression levels in AML cells compared to cord blood CD34+ HSPCs. Piwil4 expression increased by 6-8 fold in murine BM healthy HPSCs within 48h after transduction with MLL-AF9, AML1-ETO9A and CDX2 oncogenes compared to empty vector (n=3, p<0.0001). Stable knockdown of PIWIL4 in AML cell lines and primary AML samples using shRNA, followed by IC staining and confocal microscopy using an antibody against R-loops (S9.6) revealed a marked increase in accumulation of R-loops within 72h post-transduction in PIWIL4 depleted cells, in contrast to healthy cord blood HSPCs which remained unaffected (n=3). PIWIL4 depleted AML cells exhibited an accumulation of DNA damage associated gH2AX foci, replication stress associated BrdU foci, higher levels of phosphorylated ATR (p-ATR), a marked increase in apoptosis and block in the G2M phase of the cell cycle. Depletion of PIWIL4 significantly impaired clonogenic potential of AML patient samples in vitro (avg. 4.9 ± 0.9-fold reduction, p<0.0001, n=3). In vivo, PIWIL4 depletion in cell lines delayed onset of leukemia (n=8, p<0.001) and in AML patient cells reduced leukemic engraftment in xenografts 12 weeks post-transplantation (avg. scr - 50.6±21% vs avg. shRNA-14.6±10, n=6). Of note, PIWIL4 depletion in cord blood CD34+ HSPCs had no impact on colony formation or differentiation in vitro. RNA-seq of PIWIL4 depleted THP-1 cell line followed by GSEA revealed a significant reduction in expression of ribosomal genes and increased expression of G2M checkpoint repair pathway (n=2, p<0.05, FDR<0.05). qRT-PCR of pre-rRNA (45S rRNA) showed a significant reduction in rRNA transcription in shRNA transduced cell lines (avg. 2.5 ± 0.3-fold reduction, n=3, p<0.01). Overexpression of PIWIL4 or RNase H1 in PIWIL4 depleted AML cell lines rescued R-loop and gH2AX signals, induced a decrease in p-ATR and gH2AX protein levels, and rescued the impact on apoptosis and growth phenotype in colony assays. RNA polymerase I inhibitor CX-5461, known to stabilize R-loop associated secondary structures, acted synergistically with PIWIL4 depletion and induced complete cell death of PIWIL4 depleted AML cells compared to scrambled control at IC50 concentrations. Thus, collectively, we could show for the first time that PIWIL4 is a functional RNase H like enzyme in AML cells, suppresses formation of R-loops, thereby preventing DNA damage and apoptosis of AML cells. Our data also suggest that impairing resolution of R-loops is a powerful therapeutic tool in AML.

Despite progress in the understanding of the biology of acute myeloid leukemia (AML) in recent years, mortality of the disease is still high. One reason for this is drug resistance of leukemic stem cells (LSCs), which are responsible for leukemic growth and relapse. Thus, there is great interest in understanding factors driving LSCs and in key differences between normal hematopoietic stem cells (HSC) and their leukemic counterparts. LEF1 acts via the canonical Wnt pathway by interacting with β-catenin (long isoform). Of note, a natural isoform of Lef1 is expressed in hematopoiesis, which lacks the β-catenin binding domain at the N-terminus and whose function is not well defined in normal and leukemic hematopoiesis (short isoform). Here we demonstrate in a cohort of 111 AML primary patient samples with normal karyotype, that exclusively the long isoform of LEF1 is expressed. These analyses were extended to functionally validated LSC populations, using the NSG xenograft model, and confirmed the highly predominant expression of the long isoform of LEF1 and the virtual lack of expression of the short isoform. This stood in clear contrast to normal HSCs: in highly purified CD34-/CD38-/CD93high HSCs, categorized as most immature human quiescent HSC population, as well as CD34+/CD49f+/CD90+ and CD34+/CD49f+/CD90- human HSCs, no expression of the long isoform could be detected with an exclusive expression of the short isoform of LEF1. In multipotent progenitors MPPs (CD34+/CD49f-/CD90- ) there was a shift towards expression of both isoforms. Thus, LSCs show an inverse expression pattern of LEF1 isoforms compared to their normal counterparts. To understand the function of the long versus short isoform, we cloned the murine long isoform and the Lef1 isoform lacking the ß-catenin binding domain at the N-terminus into retroviral expression vectors and transduced primary murine bone marrow cells. In line with our observation of the exclusive expression of the long isoform of LEF1 in human AML stem cells, we previously showed that expression of the long isoform induces AML in transplanted mice (Petropoulos et al., JEM 2008). In contrast, expression of the short isoform did not have any major effect at the level of repopulating stem cells as determined by quantifying CRU frequency in limit dilution transplantation assays. Furthermore, ChiP-Seq analyses showed predominant binding of the short isoform on genes associate re-entry of HSCs into quiescence such as like CD81 and Wnt5a, in line with our observation of exclusive expression of the short isoform in the human CD34-/CD38-/CD93high HSC population. Based on the observation that AML cells exclusively express the long isoform, which acts via β-catenin binding, we hypothesized that AML cells would be particularly vulnerable to drugs blocking the binding of LEF1-β-catenin binding. The small molecule inhibitors Cercosporin and Calphostin C (100nM), previously shown to block LEF1-β-catenin binding in CLL cells, reduced colony growth of the AML cell line THP1 by 100 % (p<0,0001) and impaired engraftment in NSG mice (26d control vs 32d Cercosporin 100nM, p=0,0013, and 53d Calphostin C, p= 0,0022). In contrast, normal CD34+ cord blood cells were much less vulnerable to these compounds (0,2% reduction of CFC growth with Cercosporin, 8% reduction in Calphostin C, not significant). Taken together, these data point to distinct LEF1 isoform expression in AML, thereby creating high vulnerability towards blockage of LEF1-β-catenin binding and forming a potentially targetable Achilles' heel at the level of leukemic stem cells.

AML1-ETO (AE) is the most commonly occurring fusion gene in AML and shows a distinct methylation pattern, the underlying mechanisms for which is poorly understood. TET1 dioxygenase regulates methylation patterns via conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby regulating a variety of biological processes. However, the role of TET1 in AE+ AML cases is yet unexplored. Using qRT- PCR we observed that TET1 was significantly higher expressed in the majority of AE+ patients compared to other AML subtypes and CD34+ normal BM cells. This observation was consistent with published cDNA microarray and transcriptome data. Knockdown (KD) of TET1 using two shRNAs in AE+ AML cell lines impaired their cell growth and clonogenicity in vitro. KD of Tet1 in AE9a+ murine leukemic cell line inhibited its clonogenicity in vitro and delayed onset of leukemia in vivo. Tet1-ko mouse derived HSPCs transduced with AE9a showed impaired serial replating capacity compared to AE9a transduced Tet1-wt HSPCs in vitro. hMeDIP and MeDIP-seq performed on TET1 depleted KASUMI1 cells revealed 8,562 5hmC-enriched promoters (-5kbTSS) in the scrambled arm and a 50% decrease in 5hmC-enriched promoters in KD arm. In RNA-seq the genes associated with AML, Pleuripotency and WNT signaling were downregulated upon TET1 KD and over 200 of these genes exhibited loss of 5hmC on their promoters. MeDIPseq and RNA-seq data revealed a global decrease of promoter methylation and increased expression of myeloid differentiation associated genes. IP analysis confirmed that TET1 physically interacted with PARP1 in AE+ cell line. Oncogenic TET1 expression was antagonized by PARP inhibitor Olaparib in AE+ human leukemic cell lines, which induced reduction of H3K4me3 marks on the TET1 promoter and 5hmC levels in AML cell lines. Furthermore, Olaparib treatment decreased cell growth and clonogenicity of human and murine AE+ cell lines. In conclusion, our data indicate that aberrant TET1 expression contributes to the growth of AE+ AML by maintain 5hmC marks and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic effect of TET in AML.

Organelle-targeted photosensitization represents a promising approach in photodynamic therapy where the design of the active photosensitizer (PS) is very crucial. In this work, we developed a macromolecular PS with multiple copies of mitochondria-targeting groups and ruthenium complexes that displays highest phototoxicity toward several cancerous cell lines. In particular, enhanced anticancer activity was demonstrated in acute myeloid leukemia cell lines, where significant impairment of proliferation and clonogenicity occurs. Finally, attractive two-photon absorbing properties further underlined the great significance of this PS for mitochondria targeted PDT applications in deep tissue cancer therapy.

Background Advanced stage Hodgkin's lymphoma represents a heterogeneous group of patients with different risk profiles. Data suggests that interim PET assessment during chemotherapy is superior to baseline international prognostic scoring in terms of predicting long-term treatment outcome in patients with Hodgkin's lymphoma. We therefore hypothesised that early interim PET-imaging after two courses of bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone (BEACOPP) might be suitable for guiding treatment in patients with advanced stage Hodgkin's lymphoma. We aimed to assess whether intensifying standard chemotherapy (BEACOPP(escalated)) by adding rituximab would improve progression-free survival in patients with positive PET after two courses of chemotherapy.Methods In this open-label, international, randomised, phase 3 study, we recruited patients aged 18-60 years with newly diagnosed, advanced stage Hodgkin's lymphoma from 160 hospitals and 77 private practices in Germany, Switzerland, Austria, the Netherlands, and the Czech Republic. Interim PET-imaging was done after two cycles of BEACOPP(escalated) and centrally assessed by an expert panel. Patients with a positive PET after 2 cycles of BEACOPP(escalated) chemotherapy (PET-2) were randomly assigned (1: 1) to receive six additional courses of either BEACOPP(escalated) (BEACOPP(escalated) group) or BEACOPP(escalated) plus rituximab (R-BEACOPP(escalated) group). PET-2 was assessed using a 5-point scale with.. FDG uptake higher than the mediastinal blood pool (corresponding to Deauville scale 3) defined as positive. BEACOPP(escalated) was given as previously described; rituximab was given intravenously at a dose of 375 mg/m(2) (maximum total dose 700 mg), the first administration starting 24 h before starting the fourth cycle of BEACOPP(escalated) (day 0 and day 3 in cycle 4, day 1 in cycles 5-8). Randomisation was done centrally and used the minimisation method including a random component, stratified according to centre, age, stage, international prognostic score, and sex. The primary efficacy endpoint was 5 year progression-free survival, analysed in the intention-to-treat population. We are reporting this second planned interim analysis as the final report of the trial. The trial is registered with ClinicalTrials. gov, number NCT00515554.Findings Between May 14, 2008, and May 31, 2011, we enrolled 1100 patients. 440 patients had a positive PET-2 and were randomly assigned to either the BEACOPP(escalated) group (n= 220) or the R-BEACOPP(escalated) group (n= 220). With a median follow-up of 33 months (IQR 25-42) for progression-free survival, estimated 3 year progression-free survival was 91.4% (95% CI 87.0-95.7) for patients in the BEACOPP(escalated) group and 93.0% (89.4-96.6) for those in the R-BEACOPP(escalated) group (difference 1.6%, 95% CI -4.0 to 7.3; log rank p= 0.99). Common grade 3-4 adverse events were leucopenia (207 [95%] of 218 patients in the BEACOPP(escalated) group vs 211 [96%] of 220 patients in the R-BEACOPP(escalated) group), and severe infections (51 [23%] vs 43 [20%] patients). Based on a futility analysis, the independent data monitoring committee recommended publication of this second planned interim analysis as the final result. Six (3%) of 219 patients in the BEACOPP(escalated) group and ten (5%) of 220 in the R-BEACOPP(escalated) group died; fatal treatment-related toxic effects occurred in one (< 1%) patient in the BEACOPP(escalated) group and three (1%) in the R-BEACOPP(escalated) group, all of them due to infection.Interpretation The addition of rituximab to BEACOPP(escalated) did not improve the progression-free survival of PET-2 positive patients with advanced stage Hodgkin's lymphoma. However, progression-free survival for PET-2 positive patients was much better than expected, exceeding even the outcome of PET-2-unselected patients in the previous HD15 trial. Thus, PET-2 cannot identify patients at high-risk for treatment failure in the context of the very effective German Hodgkin Study Group standard treatment for advanced stage Hodgkin's lymphoma.

The Ten-eleven translocation 1 (TET1) enzyme catalyzes the oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and plays a key role in active DNA demethylation and regulation of gene expression. TET1 has been shown play a tumor supper function in B-cell malignancies however, its roles in T-cell acute lymphoblastic leukemia (T-ALL) is yet unknown. qRTPCR revealed that TET1 was significantly higher expressed in all human T-ALL cell lines and 95% of primary T-ALL patients compared to B-ALL cell lines, other primary leukemia types and healthy T-cells. This was further confirmed by cDNA microarray analysis on larger cohort of primary leukemia patient samples. Knockdown (KD) of TET1 using shRNAs in T-ALL cell lines adversely affected their cell growth and clonogenicity in vitro, and significantly reduced their leukemic engraftment potential in xenografts. Global analysis of 5hmC and 5mC marks using hMeDIP-seq and MeDIP-seq performed on TET1 depleted JURKAT cells revealed lower global 5hmC levels and increased 5mC levels (n=2). Promoters (-5kbTSS) demonstrating loss of 5hmC or gain of 5mC marks belonged to gene families involved in DNA repair, Cell cycle, T-cell development, Cancer and Wnt signaling. Changes in promoter 5hmC/5mC marks were paralleled by changes in expression of DNA repair and T-cell associated genes as measured by RNA-Seq, and protein levels as measured by western blot. Furthermore, KD of TET1 in T-ALL cell lines induced DNA damage and cell cycle arrest in the G2M phase. Aberrant expression of TET1 could be reverted by Olaparib, an inhibitor of DNA repair enzyme PARP, which induced a reduction of euchromatic marks on the TET1 promoter accompanied by decrease in TET1 mRNA and protein levels and global reduction of 5-hmC levels in T-ALL cell lines. Olaparib treatment adversely affected cell growth, clonogenicity and leukemic engraftment potential of T-ALL cell lines. In conclusion, these data demonstrate for the first time that TET1 plays an important oncogenic role in T-ALL. We also identify PARPs as upstream regulators of TET1 in T-ALL, opening the way to antagonize TET1 pharmacologically in this poor prognosis leukemia.