Long-term survival in patients with acute myeloid leukemia (AML) remains limited, primarily due to the persistence of leukemic stem cells (LSCs), which drive disease initiation and progression. In this study, using a previously generated single-cell transcriptomic atlas of AML-LSCs, we identified LIMS1 as a key regulatory gene in AML and showed that AML-LSCs display higher LIMS1 expression than AML blasts and healthy hematopoietic stem and progenitor cells (HSPCs).Our findings show that disrupting LIMS1 (via CRISPR- and shRNA-based methods) in AML cell lines, patient-derived xenografts (PDX), and primary human samples significantly impairs proliferation and clonogenic potential in vitro. In vivo, LIMS1 targeting reduces engraftment capacity and delays disease progression upon AML transplantation into immunocompromised mice. Limiting-dilution transplantation assays further reveal that LSC frequency strongly depends on LIMS1 expression. Of note, healthy HSPCs do not appear to rely on LIMS1 expression in in vitro assays. RNA-seq analysis indicated that LIMS1 deficiency alters gene expression patterns associated with cell migration and adhesion. Functional assays confirmed that loss of LIMS1 reduces AML cell migration and invasion in vitro and impairs their homing and colonization capacity of distant niches in vivo. Additionally, LIMS1 disruption appears to deregulate both AML cell motility and fibronectin-mediated adhesion.These findings align with the established role of LIMS1, an adaptor protein positioned at the nexus of integrins and growth factor receptor signaling, in mediating cell adhesion and migration processes. Our study highlights a critical role of LIMS1 in controlling AML-LSC functions through modulation of cell adhesion and migration processes, thereby unveiling potential new therapeutic avenues for AML.

Acute myeloid leukemia (AML) with complex karyotype is characterized by high genomic complexity, including frequent TP53 mutations and chromothripsis. Genomic rearrangements can reposition active enhancers near proto-oncogenes, leading to their aberrant expression; however, a comprehensive understanding of these events in AML is still incomplete. To facilitate the discovery of such "enhancer hijacking" events, we developed Pyjacker, a computational tool, and applied it to 39 AML samples with complex karyotype. Pyjacker identified several enhancer hijacking events in AML patient samples, including aberrant expression of MNX1, which can result from del(7)(q22q36) and is associated with hijacking of a CDK6 enhancer. MNX1 activation occurred in 1.4% of patients with AML and showed significant co-occurrence with BCOR mutations. Through a xenograft mouse model, we demonstrated that MNX1 is required for leukemia cell fitness. Pyjacker is an easy-to-use, accurate, and broadly applicable tool for identifying consequences of genomic events driving tumorigenesis, especially when germline genomic data are missing. SIGNIFICANCE:This study examines the consequences of structural alterations in AML and demonstrates that proto-oncogene activation by enhancer hijacking is an understudied pathomechanism. MNX1 overexpression demonstrates that deletions on chromosome 7q can not only lead to haploinsufficiency but also to activation of oncogenes by enhancer hijacking.

Current therapy strategies for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are mainly based on polychemotherapy. ABL-class-fusion-positive (+) BCP-ALL is associated with poor outcomes and is additionally treated with tyrosine kinase inhibitors such as Imatinib. Yet, Imatinib combined with chemotherapy can cause excessive toxicity and is ineffective in a substantial proportion of cases. Therefore, new treatment strategies are needed. Immunotherapy targeting IL-7Rα (CD127) employing research-grade antibodies has shown promising results in preclinical models of BCP-ALL, including BCR::ABL1+ patient-derived xenograft (PDX) models (Abdelrasoul et al., 2020). Moreover, we recently showed anti-leukemic efficacy of the CD127-targeting IgG4 clinical-grade antibody Lusvertikimab (LUSV) in a wide range of BCP-ALL samples, which was mainly driven by antibody-dependent phagocytosis (ADCP) and highly dependent on CD127 surface expression. Importantly, LUSV showed a good safety profile in healthy volunteers (Poirier et al., 2022) and has recently demonstrated a favorable safety and tolerability profile accompanied by positive efficacy results in a phase-2 trial of ulcerative colitis (NCT04882007). Therefore, we aimed to evaluate LUSV for treating ABL-class-fusion+ BCP-ALL in preclinical models. First, to determine the number of pediatric ABL-class-fusion+ patients that may potentially benefit from CD127-immunotherapy, CD127 surface expression was prospectively measured via flow cytometry in diagnostic BCP-ALL samples in accordance with EuroFlow and iBFM Flow guidelines. CD127-positivity (defined as ≥10% CD127+ ALL cells) was detected in 16/24 (66.6%) of cases, of which 6 (25%) were CD127high (≥ 50% CD127+ blasts), indicating that a substantial number of ABL-class-fusion+ patients could be eligible for LUSV therapy. Next, we tested the in vivo efficacy of LUSV in a preclinical phase2-like PDX study employing 7 ABL-class-fusion+ PDX samples (3x pediatric or adult BCR::ABL1+, 1x EBF::PDGFRB+, and 2x ETV6::ABL1+ PDX, including one bearing a CRISPR-Cas9-mediated CD19 knockout). Two mice per patient were transplanted with PDX cells and randomly assigned into treatment groups. LUSV therapy was initiated when mice showed 1% blasts in the peripheral blood (PB). When one of the two mice (control or LUSV therapy) developed clinical signs of leukemia, the peripheral blood of both PDX mice was analyzed for BCP-ALL cells. A blast reduction was observed in 7/7 (100%) of LUSV-treated PDX animals. This translated into a survival prolongation of LUSV-treated animals as compared to the control group (p=0.082). We next investigated the effect of Imatinib on CD127 expression in ABL-class-fusion+ cell lines and PDX cells in vitro. Pretreatment with sublethal doses of Imatinib (1µM) enhanced CD127 expression in ABL-class-fusion+ cell lines and PDX cells. This effect was more pronounced in CD127low than in CD127high BCP-ALL PDX samples. Importantly, both Imatinib and LUSV enhanced macrophage-mediated phagocytosis in ABL-class fusion+ cell lines and PDX cells as monotherapies. The strongest in vitro ADCP induction was observed when Imatinib and LUSV were combined. To verify this effect in vivo, we conducted a phase2-like PDX study employing 7 ABL-class-fusion+ PDX samples, testing the efficacy of LUSV, Imatinib, or the combination of both in an overt leukemia setting. Imatinib induced a reduction in median PB blast counts in treated mice compared to control animals (72.6% in the control group vs 58.1% in Imatinib-treated animals, not significant). LUSV treatment significantly reduced PB blasts as compared to control (17.15% PB blasts, p<0.0001). This effect was even more pronounced when comparing the control group with the Imatinib/LUSV combination (11.4% PB blasts, p<0.0001). Imatinib slightly prolonged the median survival of PDX-mice as compared to control (77 days vs. 70 days, not significant). LUSV and LUSV/Imatinib both led to a survival prolongation as compared to the controls (93 days in the LUSV- and 93 days in the LUSV/Imatinib group, p=0.0805 and p=0.0389, respectively). Animals treated with the combination showed no signs of enhanced toxicity as compared to the monotherapy groups. Overall, our data suggest LUSV as a potential immunotherapeutic agent for the treatment of ABL-class fusion+ BCP-ALL, warranting clinical investigation in pediatric and adult patients.

Acute myeloid leukemia with complex karyotype (ckAML) is characterized by high genomic complexity, including frequent TP53 mutations and chromothripsis. We hypothesized that the numerous genomic rearrangements could reposition active enhancers near proto-oncogenes, leading to their aberrant expression. We developed pyjacker, a computational tool for the detection of enhancer hijacking events, and applied it to a cohort of 39 ckAML samples. Pyjacker identified motor neuron and pancreas homeobox 1 ( MNX1 ), a gene aberrantly expressed in 1.4% of AML patients, often as a result of del([7][1])(q22q36) associated with hijacking of a CDK6 enhancer. MNX1 -activated cases show significant co-occurrence with BCOR mutations and a gene signature shared with t(7;12)(q36;p13) pediatric AML. We demonstrated that MNX1 is a dependency gene, as its knockdown in a xenograft model reduces leukemia cell fitness. In conclusion, enhancer hijacking is a frequent mechanism for oncogene activation in AML. Statement of significance This study examines the consequences of structural alterations and demonstrates that proto-oncogene activation by enhancer hijacking is an overlooked pathomechanism in AML. MNX1 overexpression demonstrates that deletions on chromosome 7q can not only lead to haploinsufficiency, but also to activation of oncogenes by enhancer hijacking, providing a novel leukemogenic mechanism. ### Competing Interest Statement UHT is currently employed at Oxford Nanopore Technologies. EJ is currently employed at AstraZeneca. LB has received honoraria from AbbVie, Amgen, Astellas, BristolMyers Squibb, Celgene, Daiichi Sankyo, Gilead, Hexal, Janssen, Jazz Pharmaceuticals, Menarini, Novartis, Pfizer, Roche, and Sanofi, as well as research support from Bayer and Jazz Pharmaceuticals. DBL received honoraria from Infectopharm GmbH. All other authors declared no conflict of interest. [1]: #ref-7

Acute myeloid leukemia (AML) long-term survival remains particularly poor due to the inability to eliminate leukemic stem cells (LSCs) capable of initiating and maintaining the disease. In this study, leveraging a previously generated single-cell transcriptomic atlas of AML-LSCs, we have identified LIMS1 as an important regulatory gene in AML-LSCs.Our findings demonstrate that functional targeting of LIMS1 (via CRISPR- and shRNA-mediated approaches) disrupts the proliferation and clonogenicity of AML cell lines and primary human samples in vitro, while also diminishing engraftment capacity and impeding disease progression in vivo upon transplantation into immunocompromised mice. Moreover, the absence of LIMS1 markedly attenuates the migration and invasion capacity of AML cells in vitro and compromises their ability to home to the bone marrow (BM) niche in in vivo homing assays. Notably, reduced colonization of distant sites is observed upon direct transplantation of AML cells into the BM. RNA-seq analysis further suggests a gene expression signature indicative of altered migration and cell adhesion in LIMS1-deficient AML cells.These findings align with the established role of LIMS1, an adaptor protein positioned at the nexus of integrins and growth factor receptors signaling, in mediating cell adhesion and migration processes. Our study elucidates a critical role for LIMS1 in governing AML-LSC function through modulation of cell adhesion and migration processes, thereby unveiling potential new therapeutic avenues for AML.

BACKGROUND:Acute leukemias represent deadly malignancies that require better treatment. As a challenge, treatment is counteracted by a microenvironment protecting dormant leukemia stem cells.METHODS:To identify responsible surface proteins, we performed deep proteome profiling on minute numbers of dormant patient-derived xenograft (PDX) leukemia stem cells isolated from mice. Candidates were functionally screened by establishing a comprehensive CRISPR‒Cas9 pipeline in PDX models in vivo.RESULTS:A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as an essential vulnerability required for the survival and growth of different types of acute leukemias in vivo, and reconstitution assays in PDX models confirmed the relevance of its sheddase activity. Of translational importance, molecular or pharmacological targeting of ADAM10 reduced PDX leukemia burden, cell homing to the murine bone marrow and stem cell frequency, and increased leukemia response to conventional chemotherapy in vivo.CONCLUSIONS:These findings identify ADAM10 as an attractive therapeutic target for the future treatment of acute leukemias.

Acute leukemias require more accurate and effective treatments, especially upon disease relapse. In search for novel therapeutic targets for acute leukemias, we established a pipeline for CRISPR-Cas9 mediated functional genomic screens which harbor the ability to elegantly increase our knowledge about vulnerabilities and gene dependencies. For a highly patient-related setting, we performed CRISPR knockout (KO) dropout screens in patient-derived xenograft (PDX) models in vivo, combining the advantages of studying an individual patient´s tumor cell in the physiologic in vivo bone marrow microenvironment.

High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients' leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.