Murine models of myeloid neoplasia show how leukemia infiltration alters the hematopoietic stem cell (HSC) niche to reinforce malignancy at the expense of healthy hematopoiesis. However, little is known about the bone marrow architecture in humans and its impact on clinical outcome. Here, we dissect the bone marrow niche in patients with acute myeloid leukemia (AML) at first diagnosis. We combined immunohistochemical stainings with global gene expression analyses from these AML patients and correlated them with clinical features. Mesenchymal stem and progenitor cells (MSPCs) lost quiescence and significantly expanded in the bone marrow of AML patients. Strikingly, their HSC- and niche-regulating capacities were impaired with significant inhibition of osteogenesis and bone formation in a cell contact–dependent manner through inhibition of cytoplasmic β-catenin. Assessment of bone metabolism by quantifying peripheral blood osteocalcin levels revealed 30% lower expression in AML patients at first diagnosis than in non-leukemic donors. Furthermore, patients with osteocalcin levels ≤11 ng/mL showed inferior overall survival with a 1-year survival rate of 38.7% whereas patients with higher osteocalcin levels reached a survival rate of 66.8%. These novel insights into the human AML bone marrow microenvironment help translate findings from preclinical models and detect new targets which might pave the way for niche-targeted therapies in AML patients.

New methods like panel-based RNA fusion sequencing (RNA-FS) promise improved diagnostics in various malignancies. We here analyzed the impact of RNA-FS on the initial diagnostics of 241 cases with pediatric acute myeloid leukemia (AML). We show that, compared to classical cytogenetics (CCG), RNA-FS reliably detected risk-relevant fusion genes in pediatric AML. In addition, RNA-FS strongly improved the detection of cryptic fusion genes like NUP98::NSD1, KMT2A::MLLT10 and CBFA2T3::GLIS2 and thereby resulted in an improved risk stratification in 25 patients (10.4%). Validation of additionally detected non-risk-relevant high confidence fusion calls identified PIM3::BRD1, C22orf34::BRD1, PSPC1::ZMYM2 and ARHGAP26::NR3C1 as common genetic variants and MYB::GATA1 as recurrent aberration, which we here describe in AML subtypes M0 and M7 for the first time. However, it failed to detect rare cytogenetically confirmed fusion events like MNX1::ETV6 and other chromosome 12p-abnormalities. As add-on benefit, the proportion of patients for whom measurable residual disease (MRD) monitoring became possible was increased by RNA-FS from 44.4 to 75.5% as the information on the fusion transcripts’ sequence allowed the design of new MRD assays.

WT1 is a true chameleon, both acting as an oncogene and tumor suppressor. As its exact role in leukemogenesis is still ambiguous, research with model systems representing natural conditions surrounding the genetic alterations in WT1 is necessary. In a cohort of 59 leukemia/lymphoma cell lines, we showed aberrant expression for WT1 mRNA, which does not always translate into protein levels. We also analyzed the expression pattern of the four major WT1 protein isoforms in the cell lines and primary AML blasts with/without WT1 mutations and demonstrated that the presence of mutations does not influence these patterns. By introduction of key intronic and exonic sequences of WT1 into a lentiviral expression vector, we developed a unique tool that can stably overexpress the four WT1 isoforms at their naturally occurring tissue-dependent ratio. To develop better cellular model systems for WT1, we sequenced large parts of its gene locus and also other important myeloid risk factor genes and revealed previously unknown alterations. Functionally, inhibition of the nonsense-mediated mRNA decay machinery revealed that under natural conditions, the mutated WT1 alleles go through a robust degradation. These results offer new insights and model systems regarding the characteristics of WT1 in leukemia and lymphoma.

KMT2A rearrangements (KMT2A-r) are among the most common structural aberrations in pediatric acute myeloid leukemia (AML) and are very important for the risk group stratification of patients. Here, we report the outcome of 967 pediatric AML patients with a known KMT2A-r status. The large cohort was characterized by morphology, multicolor flow cytometry, classical cytogenetics and mutation analysis via panel sequencing. In total, the blasts of 241 patients (24.9%) showed KMT2A-r. KMT2A-r is associated with FAB M5, a high white blood cell count and younger age at diagnosis. When subgroups were combined, KMT2A-r had no impact on event-free survival (EFS) and overall survival (OS); however, various subgroups showed a different prognosis, ranging from a <50% OS for KMT2A/AFDN (n = 11) to a 100% chance of survival for patients harboring the rare translocation KMT2A/SEPTIN9 (n = 3, follow up of 3.7 to 9.6 years). A positive correlation of KMT2A-r with KRAS mutations (p < 0.001) existed, albeit without any prognostic impact. In addition, FLT3-ITDs were detected less frequently in AML with KMT2A-r (p < 0.001). Furthermore, KMT2A-r were mutually exclusive, with mutations in NPM1 (p = 0.002), KIT (p = 0.036), WT1 (p < 0.001) and CEBPA (p = 0.006), and translocations NUP98/NSD1 (p = 0.009), RUNX1/RUNX1T1 (p = 0.003) and CBFB/MYH11 (p = 0.006). In the 346 patients tested for CSPG4 expression, a correlation between CSPG4 expression and KMT2A-r was confirmed. However, CSPG4 expression also occurred in patients without KMT2A-r and had no significant prognostic impact on EFS and OS.