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We performed a systematic analysis of gene expression features in early (10-21 days) development of human vs mouse embryonic cells (hESCs vs mESCs). Many development features were found to be conserved, and a majority of differentially regulated genes have similar expression change in both organisms. The similarity is especially evident, when gene expression profiles are clustered together and properties of clustered groups of genes are compared. First 10 days of mESC development match the features of hESC development within 21 days, in accordance with the differences in population doubling time in human and mouse ESCs. At the same time, several important differences are seen. There is a clear difference in initial expression change of transcription factors and stimulus responsive genes, which may be caused by the difference in experimental procedures. However, we also found that some biological processes develop differently; this can clearly be shown, for example, for neuron and sensory organ development. Some groups of genes show peaks of the expression levels during the development and these peaks cannot be claimed to happen at the same time points in the two organisms, as well as for the same groups of (orthologous) genes. We also detected a larger number of upregulated genes during development of mESCs as compared to hESCs. The differences were quantified by comparing promoters of related genes. Most of gene groups behave similarly and have similar transcription factor (TF) binding sites on their promoters. A few groups of genes have similar promoters, but are expressed differently in two species. Interestingly, there are groups of genes expressed similarly, although they have different promoters, which can be shown by comparing their TF binding sites. Namely, a large group of similarly expressed cell cycle-related genes is found to have discrepant TF binding properties in mouse vs human.

Enzyme kinetic parameters can differ between different species and isoenzymes for the same catalysed reaction. Computational approaches to calculate enzymatic kinetic parameters from the three-dimensional structures of proteins will be reviewed briefly here. Enzyme kinetic parameters may be derived by modelling and simulating the rate-determining process. An alternative, approximate, but more computationally efficient approach is the comparison of molecular interaction fields for experimentally characterized enzymes and those for which parameters should be determined. A correlation between differences in interaction fields and experimentally determined kinetic parameters can be used to determine parameters for orthologous enzymes from other species. The estimation of enzymatic kinetic parameters is an important step in setting up mathematical models of biochemical pathways in systems biology. [ABSTRACT FROM AUTHOR]

Abstract: Patients with acute myeloid leukemia (AML) who experience relapse following allogeneic hematopoietic cell transplantation (alloHCT) face unfavorable outcomes regardless of the chosen relapse treatment. Early detection of relapse at the molecular level by measurable residual disease (MRD) assessment enables timely intervention, which may prevent hematological recurrence of the disease. It remains unclear whether molecular MRD assessment can detect MRD before impending relapse and, if so, how long in advance. This study elucidates the molecular architecture and kinetics preceding AML relapse by using error-corrected next-generation sequencing (NGS) in 74 patients with AML relapsing after alloHCT, evaluating 140 samples from peripheral blood collected 0.6 to 14 months before relapse. At least 1 MRD marker became detectable in 10%, 38%, and 64% of patients at 6, 3, and 1 month before relapse, respectively. By translating these proportions into monitoring intervals, 38% of relapses would have been detected through MRD monitoring every 3 months, whereas 64% of relapses would have been detected with monthly intervals. The relapse kinetics after alloHCT are influenced by the functional class of mutations and their stability during molecular progression. Notably, mutations in epigenetic modifier genes exhibited a higher prevalence of MRD positivity and greater stability before relapse, whereas mutations in signaling genes demonstrated a shorter lead time to relapse. Both DTA (DNMT3A, TET2, and ASXL1) and non-DTA mutations displayed similar relapse kinetics during the follow-up period after alloHCT. Our study sets a framework for MRD monitoring after alloHCT by NGS, supporting monthly monitoring from peripheral blood using all variants that are known from diagnosis., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Treatment options for relapsed and refractory acute myeloid leukemia patients (R/R AML) are limited. This retrospective cohort study compares safety and efficacy of fludarabine, cytarabine, and idarubicin (FLA-IDA) without or with venetoclax (FLAVIDA) in patients with R/R AML. Thirty-seven and 81 patients received one course FLA-IDA with or without a 7-day course of venetoclax, respectively. The overall response rate (ORR) was significantly higher in FLAVIDA compared to FLAIDA- treated patients (78% vs. 47%; P=0.001), while measurable residual disease was negative at a similar proportion in responding patients (50% vs. 57%), respectively. Eighty-one percent and 79% of patients proceeded to allogeneic hematopoietic cell transplantation or donor lymphocyte infusion after FLAVIDA and FLA-IDA, respectively. Event-free and overall survival were similar in FLAVIDA- and FLA-IDA-treated patients. Refractory patients could be salvaged more successfully after FLA-IDA compared to FLAVIDA pretreatment. Neutrophil and platelet recovery times were similar in the venetoclax and the control group. In conclusion, short-term venetoclax in combination with FLA-IDA represents an effective treatment regimen in R/R AML identifying chemosensitive patients rapidly and inducing measurable residual disease-negative remission in a high proportion of R/R AML patients.

Recent evidence revealed important interactions between clonal hematopoiesis (CH) and cellular therapies established for the treatment of hematologic malignancies. The impact of CH on safety, efficacy, and outcome of chimeric antigen receptor (CAR) T-cell therapy is currently under investigation. We analyzed 110 patients with relapsed/refractory B-cell non-Hodgkin lymphoma (n = 105) or acute lymphoblastic leukemia (ALL) (n = 5), treated with Axicabtagene-Ciloleucel (39%), Tisagenlecleucel (51%), or Brexucabtagene autoleucel (10%). Using error-corrected targeted sequencing, a high CH prevalence of 56.4% (variant allele frequency [VAF] ≥1%) at the time of CAR T-cell infusion was detected. The most frequently mutated gene was PPM1D followed by DNMT3A , TET2 , ASXL1 , and TP53 . Variant allele frequencies were significantly lower in B and T cells compared with monocytes and granulocytes. CH did not increase the risk of CAR T-related toxicities. The incidences of cytokine release syndrome and immune effector-cell-associated neurotoxicity syndrome were similar between CH pos and CH neg patients, regardless of clone size, age, or CAR T product. Prolonged cytopenias were not associated with CH. Best overall response rates (ORRs) were numerically but not significantly higher in CH pos patients (ORR 76.7% versus 62.2%; P = 0.13). Furthermore, CH status did not predict progression-free survival or overall survival. Lastly, sequential analysis showed a modest VAF increase of 1.3% and acquisition of novel mutations within 100 days postinfusion. CH was frequent in large B-cell lymphoma/ALL patients receiving CAR T-cells but did not affect toxicity nor treatment response or outcome., Competing Interests: MH reports fees for advisory or consultancy services from Abbvie, Agios, BMS, Daiichi Sankyo, Eurocept, Glycostem, Janssen, Jazz Pharmaceuticals, Kura Oncology, Novartis, Pfizer, PinotBio, Roche, Takeda, and Tolremo. FD reports personal fees from Gilead, Incyte, Roche, Novartis, AbbVie, Astra Zeneca outside the submitted work. JFK reports personal fees from Gilead and Janssen outside the submitted work. GW reports honoraria from Gilead, Novartis, Clinigen, and Janssen outside the submitted work. OP has no conflicts of interest directly related to this work. OP has received honoraria or travel support from Gilead, Jazz, MSD, Novartis, Pfizer, and Therakos. He has received research support from Incyte and Priothera. He is member of advisory boards to Equillium Bio, Jazz, Gilead, Novartis, MSD, Omeros, Priothera, Sanofi, Shionogi, and SOBI. All the other authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Donor lymphocyte infusions (DLIs) can directly target leukemic cells through a graft-versus-leukemia effect and play a key role in the prevention and management of relapse after allogeneic hematopoietic cell transplantation (alloHCT). Predictors of response to DLIs are not well established. We evaluated measurable residual disease (MRD) before, 30 and 90 days after DLI treatment as biomarkers of response. MRD was assessed by next-generation sequencing in 76 DLI-treated acute myeloid leukemia patients. MRD status before DLI treatment was independently prognostic for event-free survival (EFS, p < 0.001) and overall survival (OS, p < 0.001). Within 90 days of DLI treatment, 73% of MRD + patients converted to MRD - and 32% of patients without remission achieved remission. MRD status 90 days after DLI treatment was independently prognostic for the cumulative incidence of relapse (CIR, p = 0.011) and relapse-free survival (RFS, p = 0.001), but not for OS. To evaluate the role of DLI treatment in MRD - patients, 23 MRD - patients who received DLIs were compared with a control cohort of 68 MRD - patients not receiving DLIs. RFS ( p = 0.23) and OS ( p = 0.48) were similar between the two cohorts. In conclusion, MRD is prognostic before (EFS, OS) and after (CIR, RFS) DLI treatment and may help in the selection of patients who benefit most from DLIs.

Relapse in patients with acute myeloid leukemia (AML) is common and is associated with a dismal prognosis. Treatment options are limited and the understanding of molecular response patterns is still challenging. We analyzed the clonal response patterns of 15 patients with relapsed/refractory AML treated with selinexor in a phase II trial (SAIL). DNA was analyzed at three time points and showed a decline of mutated alleles in FLT3, SF3B1, and TP53 under SAIL treatment. Overall survival (OS) was similar between patients with declining versus persisting clones. We show an interesting long-term course of a patient who relapsed after allogeneic stem cell transplantation (alloHCT) with SF3B1- and SRSF2-mutated AML and received selinexor as maintenance treatment for 4 years. Measurable residual disease (MRD) remained detectable for 2 weeks after donor lymphocyte infusion (DLI) in this patient and then remained negative under selinexor maintenance treatment. Selinexor was tolerated well and was stopped after 4 years of SAIL treatment. We present an exploratory study and identify subclonal patterns of patients treated with selinexor., (© 2022. The Author(s).)

Atrial fibrillation (AF) is the most common arrhythmia in adults but its impact on allogeneic stem cell transplantation (SCT) is not well characterized. We studied AF manifestation during the hospital stay for allogeneic SCT, referred to as AF in hospital (AFiH), and analyzed the incidence of, risk factors for, and clinical impact of AFiH on intensive care unit (ICU)-/hospital-/ and overall survival (OS), relapse-free survival (RFS), nonrelapse mortality (NRM), and graft-versus-host disease (GVHD)-free, relapse-free survival (GRFS). This retrospective matched cohort study comprised 553 consecutive SCT recipients at Hannover Medical School between January 2013 and October 2019. Patients with AFiH were compared with a non-AFiH control cohort matched for Hematopoietic Stem Cell-Specific Comorbidity Index (HCT-CI), European Group for Blood and Marrow Transplantation (EBMT) risk score, disease, conditioning regimen and availability of molecular genetic data for patients with myeloid diseases. AFiH occurred in 46 patients (8%) at a median of 2 days (interquartile range, 0 to 8 days) after SCT. Patient history of AF and elevated NT-proBNP and high-sensitivity troponin T levels, but not conventional echocardiographic parameters, were predictive for AFiH. AFiH occurred more often in patients with mutations in DNMT3A, TET2, and ASXL1 genes related to clonal hematopoiesis compared with patients with wild-type alleles, with the greatest impact from DMT3A mutations. ICU admission was significantly higher in the AFiH cohort (46% versus 7%), without significant differences in ICU or post-ICU hospital survival (62% versus 40% and 52% versus 40%, respectively). The main cause of death was sepsis. In terms of long-term outcomes, the incidences of relapse and grade II-IV acute GVHD did not differ significantly between the AFiH and the non-AFiH groups; however, OS was significantly shorter in the AFiH group (1 year OS, 39% versus 65%), owing to late noncardiac NRM (1 year NRM, 49% versus 27%). Although the underlying cellular and molecular mechanisms remain to be characterized in further detail, these data clearly demonstrate the impact of inpatient AF manifestation/AFiH on long-term outcomes of SCT recipients., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Next-generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in patients with acute myeloid leukemia (AML) is widely applicable and prognostic prior to allogeneic hematopoietic cell transplantation (alloHCT). We evaluated the prognostic role of clonal hematopoiesis-associated DNMT3A, TET2, and ASXL1 (DTA) and non-DTA mutations for MRD monitoring post-alloHCT to refine MRD marker selection. Of 154 patients with AML, 138 (90%) had at least one mutation at diagnosis, which were retrospectively monitored by amplicon-based error-corrected NGS on day 90 and/or day 180 post-alloHCT. MRD was detected in 34 patients on day 90 and/or day 180 (25%). The rate of MRD positivity was similar when DTA and non-DTA mutations were considered separately (17.6% vs 19.8%). DTA mutations had no prognostic impact on cumulative incidence of relapse, relapse-free survival, or overall survival in our study and were removed from further analysis. In the remaining 131 patients with at least 1 non-DTA mutation, clinical and transplantation-associated characteristics were similarly distributed between MRD-positive and MRD-negative patients. In multivariate analysis, MRD positivity was an independent adverse predictor of cumulative incidence of relapse, relapse-free survival, and overall survival but not of nonrelapse mortality. The prognostic effect was independent of different cutoffs (above limit of detection, 0.1% and 1% variant allele frequency). MRD log-reduction between diagnosis and post-alloHCT assessment had no prognostic value. MRD status post-alloHCT had the strongest impact in patients who were MRD positive prior to alloHCT. In conclusion, non-DTA mutations are prognostic NGS-MRD markers post-alloHCT, whereas the prognostic role of DTA mutations in the posttransplant setting remains open., (© 2021 by The American Society of Hematology.)

Clonal hematopoiesis of indeterminate potential (CHIP) is linked to leukemia gene mutations and associates with an increased risk for coronary artery disease and poor prognosis in ischemic cardiomyopathy. Two recurrently mutated genes in CHIP and adult acute myeloid leukemia (AML) encode for isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). Global expression of mutant IDH2 in transgenic mice-induced dilated cardiomyopathy and muscular dystrophy. In this retrospective observational study, we investigated whether mutant IDH1/2 predisposes to cardiovascular disease in AML patients. Among 363 AML patients, IDH1 and IDH2 mutations were detected in 26 (7.2%) and 39 patients (10.7%), respectively. Mutant IDH1 patients exhibited a significantly higher prevalence of coronary artery disease (26.1% vs. 6.4%, p = 0.002). Applying inverse probability-weighting analysis, patients with IDH1/2 mutations had a higher risk for a declining cardiac function during AML treatment compared to IDH1/2 wild type patients [left ventricular ejection fraction pretreatment compared to 10 months after diagnosis: 59.2% to 41.9% (p < 0.001) vs 58.5% to 55.4% (p = 0.27), respectively]. Mechanistically, RNA sequencing and immunostaining in hiPS-derived cardiomyocytes indicated that the oncometabolite R-2HG exacerbated doxorubicin mediated cardiotoxicity. Evaluation of IDH1/2 mutation status may therefore help identifying AML patients at risk for cardiovascular complications during cytotoxic treatment.

Isolated myeloid sarcoma (MS) is a rare malignancy in which myeloid blast forms tumors at various locations while the bone marrow (BM) remains cytomorphologically free from disease. We analyzed isolated MS from four patients and their BMs at initial diagnosis and follow-up, using a custom next-generation sequencing (NGS) panel. We observed possible clonal evolution and a clonal hematopoiesis of indeterminate potential (CHIP)-like finding in the BM of one of three cases with detectable mutations. Clinical presentation of one patient suggested extramedullary confined homing of blasts to distal sites in the relapse situation still sparing the BM. In summary, our findings shall motivate future work regarding signals of extramedullary blast trafficking and clonal evolution in MS.

Mutant IDH1 (mIDH1) inhibitors have shown single-agent activity in relapsed/refractory AML, though most patients eventually relapse. We evaluated the efficacy and molecular mechanism of the combination treatment with azacitidine, which is currently the standard of care in older AML patients, and mIDH1 inhibitor BAY1436032. Both compounds were evaluated in vivo as single agents and in combination with sequential (azacitidine, followed by BAY1436032) or simultaneous application in two human IDH1 mutated AML xenograft models. Combination treatment significantly prolonged survival compared to single agent or control treatment (P<.005). The sequential combination treatment depleted leukemia stem cells (LSC) by 470-fold. Interestingly, the simultaneous combination treatment depleted LSCs by 33,150-fold compared to control mice. This strong synergy is mediated through inhibition of MAPK/ERK and RB/E2F signaling. Our data strongly argues for the concurrent application of mIDH1 inhibitors and azacitidine and predicts improved outcome of this regimen in IDH1 mutated AML patients.

To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.

NUP98-NSD1-positive acute myeloid leukemia (AML) is a poor prognostic subgroup that is frequently diagnosed in pediatric cytogenetically normal AML. NUP98-NSD1-positive AML often carries additional mutations in genes including FLT3, NRAS, WT1, and MYC . The purpose of our study was to characterize the cooperative potential of the fusion and its associated Neuroblastoma rat sarcoma (NRAS) mutation. By constitutively expressing NUP98-NSD1 and NRASG12D in a syngeneic mouse model and using a patient-derived xenograft (PDX) model from a NUP98-NSD1-positive AML patient, we evaluated the functional role of these genes and tested a novel siRNA formulation that inhibits the oncogenic driver NUP98-NSD1. NUP98-NSD1 transformed murine bone marrow (BM) cells in vitro and induced AML in vivo. While NRASG12D expression was insufficient to transform cells alone, co-expression of NUP98-NSD1 and NRASG12D enhanced the leukemogenicity of NUP98-NSD1. We developed a NUP98-NSD1-targeting siRNA/lipid nanoparticle formulation that significantly prolonged the survival of the PDX mice. Our study demonstrates that mutated NRAS cooperates with NUP98-NSD1 and shows that direct targeting of the fusion can be exploited as a novel treatment strategy in NUP98-NSD1-positive AML patients.

Mixed lineage leukemia ( MLL/KMT2A ) rearrangements (MLL-r) are one of the most frequent chromosomal aberrations in acute myeloid leukemia. We evaluated the function of Meningioma 1 (MN1), a co-factor of HOXA9 and MEIS1, in human and murine MLL-rearranged leukemia by CRISPR-Cas9 mediated deletion of MN1. MN1 was required for in vivo leukemogenicity of MLL positive murine and human leukemia cells. Loss of MN1 inhibited cell cycle and proliferation, promoted apoptosis and induced differentiation of MLL-rearranged cells. Expression analysis and chromatin immunoprecipitation with sequencing from previously reported data sets demonstrated that MN1 primarily maintains active transcription of HOXA9 and HOXA10, which are critical downstream genes of MLL, and their target genes like BCL2, MCL1 and Survivin. Treatment of MLL-rearranged primary leukemia cells with anti-MN1 siRNA significantly reduced their clonogenic potential in contrast to normal CD34 + hematopoietic progenitor cells, suggesting a therapeutic window for MN1 targeting. In summary, our findings demonstrate that MN1 plays an essential role in MLL fusion leukemias and serve as a therapeutic target in MLL-rearranged acute myeloid leukemia., (Copyright© 2020 Ferrata Storti Foundation.)