Acute myeloid leukemia with complex karyotype (CK-AML) is associated with poor prognosis, which is only in part explained by underlying TP53 mutations. Especially in the presence of complex chromosomal rearrangements, such as chromothripsis, the outcome of CK-AML is dismal. However, this degree of complexity of genomic rearrangements contributes to the leukemogenic phenotype and treatment resistance of CK-AML remains largely unknown. Applying an integrative workflow for the detection of structural variants (SVs) based on Oxford Nanopore (ONT) genomic DNA long-read sequencing (gDNA-LRS) and high-throughput chromosome confirmation capture (Hi-C) in a well-defined cohort of CK-AML identified regions with an extreme density of SVs. These rearrangements consisted to a large degree of focal amplifications enriched in the proximity of mammalian-wide interspersed repeat elements, which often result in oncogenic fusion transcripts, such as USP7::MVD, or the deregulation of oncogenic driver genes as confirmed by RNA-seq and ONT direct complementary DNA sequencing. We termed this novel phenomenon chromocataclysm. Thus, our integrative SV detection workflow combing gDNA-LRS and Hi-C enables to unravel complex genomic rearrangements at a very high resolution in regions hard to analyze by conventional sequencing technology, thereby providing an important tool to identify novel important drivers underlying cancer with complex karyotypic changes.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by high rate of relapse and mortality. Current chemotherapies whilst successful in eradicating blasts, are less effective in eliminating relapse-causing leukemic stem cells (LSCs). Although LSCs are usually identified as CD34+CD38- cells, there is significant heterogeneity in surface marker expression, and CD34- LSCs exist particularly in NPM1mut AMLs. By analyzing diagnostic primary DNMT3AmutNPM1mut AML samples, we suggest a novel flow cytometry sorting strategy particularly useful for CD34neg AML subtypes. To enrich for LSCs independently of CD34 status, positive selection for GPR56 and negative selection for NKG2D ligands are used. We show that the functional reconstitution capacity of CD34- and CD34+ LSCs as well as their transcriptomes are very similar which support phenotypic plasticity. Furthermore, we show that although CD34+ subpopulations can contain next to LSCs also normal and/or preleukemic hematopoietic stem cells (HSCs), this is not the case in CD34-GPR56+NKG2DL- enriched LSCs which thus can be isolated with high purity. Finally, we show that patients with AML, who retain at the time of diagnosis a reserve of normal and/or preleukemic HSCs in their bone marrow able to reconstitute immunocompromised mice, have significantly longer relapse-free and overall survival than patients with AML in whom functional HSCs are no longer detectable.