A human genome editing–based <i>MLL</i>::<i>AF4</i> ALL model recapitulates key cellular and molecular leukemogenic features

Bueno, Clara; Torres-Ruiz, Raul; Velasco-Hernandez, Talia; Molina, Oscar; Petazzi, Paolo; Martinez, Alba; Rodriguez, Virginia; Vinyoles, Meritxell; Cantilena, Sandra; Williams, Owen; Vega-Garcia, Nerea; Rodriguez-Perales, Sandra; Segovia, Jose C.; Quintana-Bustamante, Oscar; Roy, Anindita; Meyer, Claus; Marschalek, Rolf; Smith, Alastair L.; Milne, Thomas A.; Fraga, Mario F.; Tejedor, Juan Ramón; Menéndez, Pablo

doi:10.1182/blood.2023020858

Cited by 4 publications

References 21 publications

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The fetal specific geneLIN28Bis essential for human fetal B-lymphopoiesis and initiation of KMT2A::AFF1 infant leukemia

Ling,

Jackson,

Elliott

et al. 2024

Preprint

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Infant ALL (iALL) is initiated in utero, most often by rearrangement of the KMT2A gene (KMT2Ar). It carries a very poor prognosis despite a lack of additional oncogenic driver mutations common in childhood ALL. Here, we aimed to identify specific properties of human fetal hematopoietic stem/progenitor cells (HSPC) that promote leukemic transformation in KMT2Ar iALL using molecular, functional and in vivo assays. First, by comparing transcriptomes of human fetal HSPC to adult HSPC we derived a fetal-specific gene signature and identified the fetal oncogene LIN28B and its downstream effectors among the top hits. These genes were also expressed in iALL. Functional assays revealed that LIN28B was essential in human fetal liver (FL) CD34+ cells to maintain proliferation and stemness, and support B- and NK-lymphopoiesis. To interrogate the role of LIN28B in iALL, we utilised a human FL-derived CRISPR-Cas9 KMT2A::AFF1 model. In this model, LIN28B-expressing leukemias were more proliferative in vitro and in vivo, with this advantage being lost upon LIN28B knockdown. Mechanistic studies showed that LIN28B acts by stabilizing key early B-lymphoid genes, epigenetic regulators, and cell cycle and anti-apoptotic genes. Finally, In the absence of LIN28B, human FL CD34+ cells fail to transform upon induction of KMT2A::AFF1 translocation. Thus, LIN28B has an essential role in normal human fetal B-lymphopoiesis, and is necessary for the initiation of KMT2A::AFF1 iALL in fetal cells in the absence of co-operating mutations. It has a role in making leukemias more aggressive, suggesting it is a potential target in LIN28B-expressing leukemias.

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The fetal specific geneLIN28Bis essential for human fetal B-lymphopoiesis and initiation of KMT2A::AFF1 infant leukemia

Ling,

Jackson,

Elliott

et al. 2024

Preprint

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NSGS mice humanized with cord blood mononuclear cells show sustained and functional myeloid–lymphoid representation with limited graft-versus-host disease

Panisello,

Aschero,

Martinez-Moreno

et al. 2024

J Immunother Cancer

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Humanized immunodeficient mice serve as critical models for investigating the functional interplay between transplanted human cells and a pre-reconstituted human immune system. These models facilitate the study of molecular and cellular pathogenic mechanisms and enable the evaluation of the efficacy and toxicity of immunotherapies, thereby accelerating their preclinical and clinical development. Current strategies rely on inefficient, long-term/delayed hematopoietic reconstitution by CD34+ hematopoietic progenitors or short-term reconstitution with peripheral blood mononuclear cells (PB-MNCs) associated with high rates of graft-versus-host disease (GvHD) and an inefficient representation of immune cell populations. Here, we hypothesized that immunologically naïve cord blood mononuclear cells (CB-MNCs) could serve as a superior alternative, providing long-lasting and functionally effective immune reconstitution. We conducted a comprehensive comparison between the non-obese diabetic (NOD).Cg-Prkdc∧ˆscid-IL2rg∧ˆtm1Wjl/SzJ (NSG) and NSG-Tg(CMV-IL3,CSF2,KITLG)∧ˆ1Eav/MloySzJ (NSGS) immunodeficient mouse models following humanization with either PB-MNCs or CB-MNCs. We assessed the engraftment dynamics of various human immune cells over time and monitored the development of GvHD in both models. For the most promising model, we extensively evaluated immune cell functionalityin vitroandin vivousing sarcoma and leukemia xenografts. Humanizing NSGS mice with CB-MNCs results in a rapid, robust, and sustained representation of a diverse range of functional human lymphoid and myeloid cell populations while minimizing GvHD incidence. In this model, human immune cell populations significantly impair the growth and engraftment of sarcoma and B-cell acute lymphoblastic leukemia cells, with a significant inverse correlation between immune cell levels and tumor growth. This study establishes a fast, efficient, and reliablein vivoplatform for various applications in cancer immunotherapy, particularly for exploring the complex interactions between cancer cells, immune cells, and the tumor microenvironmentin vivo, prior to clinical development.

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NG2 is a target gene of MLL-AF4 and underlies glucocorticoid resistance in MLLr B-ALL by regulating NR3C1 expression

Lopez-Millan,

Rubio-Gayarre,

Vinyoles

et al. 2024

Blood

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B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, with long-term overall survival rates of ~85%. However, B-ALL harboring rearrangements of the MLL gene (also known as KMT2A), referred to as MLLr B-ALL, is common in infants and is associated with poor 5-year survival (<30%), frequent relapses, and refractoriness to glucocorticoids (GCs). GCs are an essential part of the treatment backbone for B-ALL and GC resistance is a major clinical predictor of poor outcome. Elucidating the mechanisms of GC resistance in MLLr B-ALL is, therefore, critical to guide therapeutic strategies that deepen the response after induction therapy. Neuron-glial antigen-2 (NG2) expression is a hallmark of MLLr B-ALL and is minimally expressed in healthy hematopoietic cells. We recently reported that NG2 expression is associated with poor prognosis and that anti-NG2 immunotherapy strongly reduces/delays relapse in MLLr B-ALL xenograft models. Despite its contribution to MLLr B-ALL pathogenesis and its diagnostic utility, the role of NG2 in MLLr-mediated leukemogenesis/chemoresistance remains elusive. Here we show that NG2 is an epigenetically regulated direct target gene of the leukemic MLL-AF4 fusion protein. NG2 negatively regulates the expression of the GC receptor NR3C1 and confers GC resistance to MLLr B-ALL cells in vitro and in vivo. Mechanistically, NG2 interacts with FLT3 to render ligand-independent activation of FLT3 signaling (a hallmark of MLLr B-ALL) and downregulation of NR3C1 via AP-1-mediated trans-repression. Collectively, our study elucidates the role of NG2 in GC resistance in MLLr B-ALL through FLT3/AP-1-mediated downregulation of NR3C1, providing novel therapeutic avenues for MLLr B-ALL.

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A human genome editing–based MLL::AF4 ALL model recapitulates key cellular and molecular leukemogenic features

Cited by 4 publications

References 21 publications

The fetal specific geneLIN28Bis essential for human fetal B-lymphopoiesis and initiation of KMT2A::AFF1 infant leukemia

The fetal specific geneLIN28Bis essential for human fetal B-lymphopoiesis and initiation of KMT2A::AFF1 infant leukemia

NSGS mice humanized with cord blood mononuclear cells show sustained and functional myeloid–lymphoid representation with limited graft-versus-host disease

NG2 is a target gene of MLL-AF4 and underlies glucocorticoid resistance in MLLr B-ALL by regulating NR3C1 expression

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