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Defined human primary cell model systems with growth dependence on oncogenes are highly requested to investigate tumor pathogenesis and to validate pharmacological inhibitors that specifically target oncoproteins and their executing protein complex partners. In acute myeloid leukemia (AML), transcription factors such as RUNX1 and MLL1, which are important for normal blood cell development, frequently harbor mutations including chromosomal translocations with other coding genes, resulting in tumor-promoting gain-of-function fusion proteins. These oncoproteins completely modify transcriptional programs, thereby inducing malignant cell phenotypes. A common theme of the chimeric gene products is their physical interaction with a variety of chromatin-modifying effector molecules, including histone acetyltransferases (HATs) and histone deacetylases (HDACs). These aberrant multiprotein machineries disturb gene expression and promote malignant cell growth. In this chapter, we briefly summarize the current understanding regarding AML-associated oncogene-driven human CD34+ blood progenitor cell expansion in ex vivo liquid cultures. We provide a step-by-step protocol to establish oncogene-induced human CD34+ blood progenitor cell cultures suitable to analyze the impact of transcriptional repressor/HDAC activity in these human AML cell models.

GATA2 zinc-finger (ZF) mutations are associated with distinct entities of myeloid malignancies. The specific distribution of these mutations points toward different mechanisms of leukemogenesis depending on the ZF domain affected. In this study, we compared recurring somatic mutations in ZF1 and ZF2. All tested ZF mutants disrupted DNA binding in vitro. In transcription assays, co-expression of FOG1 counteracted GATA2-dependent transcriptional activation, while a variable response to FOG1-mediated repression was observed for individual GATA2 mutants. In primary murine bone marrow cells, GATA2 wild-type (WT) expression inhibited colony formation, while this effect was reduced for both mutants A318T (ZF1) and L359V (ZF2) with a shift toward granulopoiesis. In primary human CD34

The cytokine receptor colony stimulating factor 3 (CSF3R) controls differentiation, proliferation and function of granulocytes as well as mobilization of hematopoietic stem cells upon activation via G-CSF. An oncogenic role of CSF3R mutations has been mainly described in chronic neutrophilic leukemia (CNL) and, more rarely, in atypical chronic myeloid leukemia and pediatric acute myeloid leukemia (AML) (Maxson et al., 2013, N Engl J Med; Maxson et al.,2016, Blood). CSF3R mutations are classified in two distinct categories: truncating mutations and proximal-membrane domain mutations. While truncating mutations lead to a higher sensitivity toward the ligand, the more common proximal-membrane mutations are ligand-independent in activating downstream signaling. Recently, we found CSF3R recurrently mutated in 5% (13/292) of patients with adult core-binding factor (CBF) leukemia (Opatz et al., 2020, Leukemia). Both CBF leukemia subgroups, characterized by either an inversion of chromosome 16 (inv(16)) or translocation t(8;21), were affected. The most prevalent mutation hotspot was T618 in the t(8;21) subgroup, localizing to the proximal-membrane region, recurrently affected also in CNL. We set out to study the role of CSF3R mutations in AML t(8;21). Using CD34+ hematopoietic stem cells from adult healthy donors, we observed that co-expression of the CSF3R mutant T618I (CSF3R T618I) but not CSF3R wildtype (WT)together with the t(8;21) related RUNX1-RUNX1T1 fusion led to a competitive outgrowth of cells (Figure 1A,B). In contrast, over-expression of either CSF3R WT or CSF3R T618Ialone did not lead to a competitive advantage. Furthermore, in a culture composed of >95% RUNX1-RUNX1T1 positive expanded cells, super-infection with the retroviral CSF3R T618I construct led to complete outgrowth of double positive cells over a period of 60 days. These data suggest an oncogenic cooperation between RUNX1-RUNX1T1 and CSF3R T618I. Comparing the cytomorphology of RUNX1-RUNX1T1 single positive cells and RUNX1-RUNX1T1/CSF3R T618I double positive cells, the single positive cells were heterogeneous with a mixture of mature and immature cells (Figure 1C), whereas double positive cells remained mostly undifferentiated, featuring a homogeneous blast-like morphology (Figure 1D). In accordance with these findings, limiting and serial dilution experiments clearly showed an increased self-renewal potential for double oncogene expressing cells compared to cells expressing RUNX1-RUNX1T1 only. This further supports a transforming potential of CSF3R T618I in combination with RUNX1-RUNX1T1. RNA sequencing of outgrown double oncogene expressing cells revealed upregulation of distinct pathways related to myeloid differentiation, in particular neutrophil cell activation and migration. In addition, we found GLI2 as potential downstream effector and putative pharmacological target in AML t(8;21) with CSF3R T618I (Figure 1E). GLI2 expression is a negative prognostic marker in AML, associated with significantly shorter event free and overall survival (Wellbrock et al., 2015, Clin Cancer Res). Acting in the hedgehog-signaling pathway, GLI2 is not expressed in healthy adult tissues. Of note, patient data and mouse models support a specific collaboration of upregulated GLI2 and FLT3-ITD in AML (Wellbrock et al., 2015, Clin Cancer Res; Lim et al., 2015, Sci Transl Med). Conducting further experiments in the granulocyte-macrophage colony stimulating factor (GM-CSF) dependent t(8;21)-positive cell line SKNO-1, we observed cytokine independent growth in the presence of CSF3R T618I but not CSF3R WT. GM-CSF withdrawal of SKNO-1 native cells, vector control or CSF3R WTexpressing cells led to cell cycle arrest that could be overcome by expression of CSF3R T618I. Subsequently, we investigated pharmacological counteraction of CSF3R T618I through inhibition of putative effectors. CSF3R T618I expressing SKNO-1 cells were significantly more sensitive towards treatment with the GLI inhibitor GANT61, as compared to vector or CSF3R WT controls (Figure 1F). Taken together, our results suggest a specific oncogenic collaboration between RUNX1-RUNX1T1 and CSF3R T618I with potential therapeutic implications. Figure 1 Figure 1. Disclosures Redondo Monte: Minaris Regenerative Medicine: Current Employment. Greif: AstraZeneca: Honoraria.

Long-term ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells (HSPCs) proves to be unfeasible as cellular differentiation occurs when HSPCs are detached from their supporting bone marrow stem cell niche. This issue renders it difficult to make use of the proliferation capacity of HSPCs to subsequently produce functional blood cells in relevant numbers, e.g. for cell therapy approaches. To circumvent this challenge, leukemia-associated chimeric transcription factors, including MLL fusion proteins, can be exploited for their pronounced ability to propel cell proliferation while preserving cell immaturity. By designing the protein's activity controllable, the immature state can be abolished at an arbitrary point in time enabling terminal differentiation. In this study, we employed the fusion gene mixed lineage leukemia/eleven nineteen leukemia (MLL-ENL) for engineering an inducible protein switch. For this purpose, we fused the coding sequence of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the transcription factor MLL-ENL and subsequently expressed the protein switch (DD-MLL-ENL) in human CD34+ HSPCs derived from adult healthy donors. In the presence of the specific ligand Shield1, DD-mediated protein degradation is prevented leading to massive and long-term expansion of HSPC-derived late monocytic precursors in the presence of IL-3, IL-6, SCF, FLT3-L, TPO and GM-CSF. The cells do not exhibit additional driver mutations, feature a normal karyotype and telomere length, and sustain immaturity that is strictly dependent on Shield1 supplementation every other day even after two years of ex vivo culture. Upon Shield1 deprivation, the cells completely lost self-renewal and colony-forming properties and spontaneously differentiated. By changing the cytokines to GM-CSF in combination with IFN-γ and LPS we differentiated the progenitor cells into macrophages (MΦ) (Fig. 1 A, B). Immunophenotypic characterization revealed upregulation of the monocyte/macrophage-associated surface markers CD14, CD80, CD86, CD163 and MHC class I and II, concordant with monocytic morphology as judged by cytospin preparations. Analysis of the transcription of selected inflammatory genes, including IL-6 and IL-10, revealed overlapping M1 and M2 macrophage characteristics. Furthermore, mRNA expression profiles using nCounter Systems technology covering a total of 770 myeloid innate immunity-related genes proves the cells' identity as differentiated phagocytes shown by upregulation of gene clusters involved in Fc receptor signaling, TLR signaling, antigen presentation and T cell activation. In functional assays, we demonstrated the ability of the obtained cells to migrate towards the chemokine CCL2 in a 3D chemotaxis assay, attach to VCAM-1 under flow and shear stress and produce reactive oxygen species. Regarding the cells' phagocytic capability, we could verify the uptake of bacterial particles as well as apoptotic cells in efferocytosis assays. Finally, we demonstrated IgG Fc region recognition and binding by the expressed Fcγ receptors enabling phagocytosis of lymphoblastic tumor cells, including Daudi, Raji and patient-derived MCL cells in an antibody-dependent manner using rituximab (RTX), daratumumab (Dara) and trastuzumab (Trast) as a negative control (Fig. 1C). Overall, we could demonstrate the conversion of a harmful leukemic transcription factor into a useful molecular tool for large-scale ex vivo production of functional blood cells. Such engineered controllable protein switches might have the potential to be employed as molecular tools to produce functional immune cells for cell-based immunotherapeutic approaches. Figure 1 Figure 1. Disclosures Redondo Monte: Minaris Regenerative Medicine: Current Employment. Beier: Alexion: Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Other: Travel reembursement. Weigert: Janssen: Speakers Bureau; Epizyme: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding. Greif: AstraZeneca: Honoraria.

Chromosomal translocations represent frequent events in leukemia. In t(8;21)+ acute myeloid leukemia, RUNX1 is fused to nearly the entire ETO protein, which contains four conserved nervy homology regions, NHR1-4. Furthermore RUNX1/ETO interacts with ETO-homologous proteins via NHR2, thereby multiplying NHR domain contacts. As shown recently, RUNX1/ETO retains oncogenic activity upon either deletion of the NHR3 + 4 N-CoR/SMRT interaction domain or substitution of the NHR2 tetramer domain. Thus, we aimed to clarify the specificities of the NHR domains. A C-terminally NHR3 + 4 truncated RUNX1/ETO containing a heterologous, structurally highly related non-NHR2 tetramer interface translocated into the nucleus and bound to RUNX1 consensus motifs. However, it failed to interact with ETO-homologues, repress RUNX1 targets, and transform progenitors. Surprisingly, transforming capacity was fully restored by C-terminal fusion with ETO's NHR4 zinc-finger or the repressor domain 3 of N-CoR, while other repression domains failed. With an inducible protein assembly system, we further demonstrated that NHR4 domain activity is critically required early in the establishment of progenitor cultures expressing the NHR2 exchanged truncated RUNX1/ETO. Together, we can show that NHR2 and NHR4 domains can be replaced by heterologous protein domains conferring tetramerization and repressor functions, thus showing that the NHR2 and NHR4 domain structures do not have irreplaceable functions concerning RUNX1/ETO activity for the establishment of human CD34+ cell expansion. We could resemble the function of RUNX1/ETO through modular recomposition with protein domains from RUNX1, ETO, BCR and N-CoR without any NHR2 and NHR4 sequences. As most transcriptional repressor proteins do not comprise tetramerization domains, our results provide a possible explanation as to the reason that RUNX1 is recurrently found translocated to ETO family members, which all contain tetramer together with transcriptional repressor moieties.

Numerous cell–cell and cell–matrix interactions within the bone marrow microenvironment enable the controlled lifelong self-renewal and progeny of hematopoietic stem and progenitor cells (HSPCs). On the cellular level, this highly mutual interaction is granted by cell adhesion molecules (CAMs) integrating differentiation, proliferation, and pro-survival signals from the surrounding microenvironment to the inner cell. However, cell–cell and cell–matrix interactions are also critically involved during malignant transformation of hematopoietic stem/progenitor cells. It has become increasingly apparent that leukemia-associated gene products, such as activated tyrosine kinases and fusion proteins resulting from chromosomal translocations, directly regulate the activation status of adhesion molecules, thereby directing the leukemic phenotype. These observations imply that interference with adhesion molecule function represents a promising treatment strategy to target pre-leukemic and leukemic lesions within the bone marrow niche. Focusing on myeloid leukemia, we provide a current overview of the mechanisms by which leukemogenic gene products hijack control of cellular adhesion to subsequently disturb normal hematopoiesis and promote leukemia development.