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Human bone marrow failure (BMF) syndromes result from the loss of hematopoietic stem and progenitor cells (HSPC), and this loss has been attributed to cell death; however, the cell death triggers, and mechanisms remain unknown. During BMF, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) increase. These ligands are known to induce necroptosis, an inflammatory form of cell death mediated by RIPK1, RIPK3, and MLKL. We previously discovered that mice with a hematopoietic RIPK1 deficiency (Ripk1HEM KO) exhibit inflammation, HSPC loss, and BMF, which is partially ameliorated by a RIPK3 deficiency; however, whether RIPK3 exerts its effects through its function in mediating necroptosis or other forms of cell death remains unclear. Here, we demonstrate that similar to a RIPK3 deficiency, an MLKL deficiency significantly extends survival and like Ripk3 deficiency partially restores hematopoiesis in Ripk1HEM KO mice revealing that both necroptosis and apoptosis contribute to BMF in these mice. Using mouse models, we show that the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) is up-regulated in mouse RIPK1-deficient bone marrow cells and that ZBPl's function in endogenous nucleic acid sensing is necessary for HSPC death and contributes to BMF. We also provide evidence that IFNγ mediates HSPC death in Ripk1HEM KO mice, as ablation of IFNγ but not TNFα receptor signaling significantly extends survival of these mice. Together, these data suggest that RIPK1 maintains hematopoietic homeostasis by preventing ZBP1 activation and induction of HSPC death. [ABSTRACT FROM AUTHOR]

Polymorphonuclear leukocytes are essential for host defense to infectious diseases. CCAAT/enhancer binding protein [Epsilon] (C/EBP[Epsilon]) is preferentially expressed in granulocytes and lymphoid cells. Mice with a null mutation in C/EBP[Epsilon] develop normally and are fertile but fail to generate functional neutrophils and eosinophils. Opportunistic infections and tissue destruction lead to death by 3-5 months of age. Furthermore, end-stage mice develop myelodysplasia, characterized by proliferation of atypical granulocytes that efface the bone marrow and result in severe tissue destruction. Thus, C/EBP[Epsilon] is essential for terminal differentiation and functional maturation of committed granulocyte progenitor cells.

The CBFB-MYH11 gene was introduced into mouse genome by inserting the 3' part of the human MYH11 cDNA into the end of exon 5 of the mouse Cbfb gene through homologous recombination. This led to the creation of a half genomic (Cbfb), half cDNA (MYH11) fusion gene, which, in turn, resulted in the generation of a Cbf(beta)-SMMHC fusion protein under the control of the Cbfb promoter. Cbf(beta)-SMMHC is shown to dominantly suppress the normal function of Cbf gene products and inhibits the differentiation of hematopoietic stem cells.

Transposon Tn3 was transposed from the plasmid to the chromosomes of two independent populations of Escherichia coli grown in glucose-limited conditions after several generations. The E. coli populations contained a single clone of plasmid pBR322 derivative that carried the transposable DNA sequences. The transposed DNA fragments were similar in sizes but differed in their insertion sites in the two bacterial populations. The transpositions conferred to an increase in relative fitness in the environment inhabited by the E. coli populations.

Comments on the ability of the fusion gene Cbfb-MYH11 to block myeloid differentiation and predispose mice to acute myelomonocytic leukemia. Necessity of alteration of other critical genes to trigger leukemogenesis; Development of acute myleomonocytic leukemia in N-ethyl-N-nitrosourea-treated chimeras.

The gene encoding the RUNX1 transcription factor is mutated in a subset of T-cell acute lymphoblastic leukemia (T-ALL) patients, and RUNX1 mutations are associated with a poor prognosis. These mutations cluster in the DNA-binding Runt domain and are thought to represent loss-of-function mutations, indicating that RUNX1 suppresses T-cell transformation. RUNX1 has been proposed to have tumor suppressor roles in T-cell leukemia homeobox 1/3-transformed human T-ALL cell lines and NOTCH1 T-ALL mouse models. Yet, retroviral insertional mutagenesis screens identify RUNX genes as collaborating oncogenes in MYC-driven leukemia mouse models. To elucidate RUNX1 function(s) in leukemogenesis, we generated Tal1/Lmo2/Rosa26-CreERT2Runx1f/f mice and examined leukemia progression in the presence of vehicle or tamoxifen. We found that Runx1 deletion inhibits mouse leukemic growth in vivo and that RUNX silencing in human T-ALL cells triggers apoptosis. We demonstrate that a small molecule inhibitor, designed to interfere with CBFβ binding to RUNX proteins, impairs the growth of human T-ALL cell lines and primary patient samples. We demonstrate that a RUNX1 deficiency alters the expression of a crucial subset of TAL1- and NOTCH1-regulated genes, including the MYB and MYC oncogenes, respectively. These studies provide genetic and pharmacologic evidence that RUNX1 has oncogenic roles and reveal RUNX1 as a novel therapeutic target in T-ALL. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) results from the activity of driver mutations that deregulate proliferation and survival of hematopoietic stem cells (HSCs). The fusion protein CBFβ-SMMHC impairs differentiation in hematopoietic stem and progenitor cells and induces AML in cooperation with other mutations. However, the combined function of CBFp-SMMHC and cooperating mutations in preleukemic expansion is not known. Here, we used NrasLSL-G12D; Cbfb56M knock-in mice to show that allelic expression of oncogenic NrasG12D and cbfβ-SMMHC increases survival of preleukemic short-term HSCs and myeloid progenitor cells and maintains the differentiation block induced by the fusion protein. NrasG12D and Cbfp-SMMHC synergize to induce leukemia in mice in a cell-autonomous manner, with a shorter median latency and higher leukemia-initiating cell activity than that of mice expressing Cbfp-SMMHC. Furthermore, NrasLSL-G12D; Cbfb56M leukemic cells were sensitive to pharmacologic inhibition of the MEK/ERK signaling pathway, increasing apoptosis and Bim protein levels. These studies demonstrate that Cbfp-SMMHC and NrasG12D promote the survival of preleukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inversion(16) AML-targeted therapies. [ABSTRACT FROM AUTHOR]

Mice harboring three mouse mammary tumor virus (MMTV) variants develop pregnancy-dependent (PD) tumors that progress to pregnancy-independent (PI) behavior through successive passages. Herein, we identified 10 predominant insertions in PI transplants from 8 independent tumor lines. These mutations were also detected in small cell populations in the early PD passages. In addition, we identified a new viral insertion upstream of the gene Rspo3, which is overexpressed in three of the eight independent tumor lines and codes for a protein very similar to the recently described protein encoded by Int7. This study suggests that during progression towards hormone independence, clonal expansion of cells with specific mutations might be more relevant than the occurrence of new MMTV insertions. [ABSTRACT FROM AUTHOR]

The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancy (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 was shown to regulate inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to epigenetically repress two inflammatory signaling pathways in neutrophils: Toll-like receptor 4 (TLR4) and type I interferon (IFN) signaling. RUNX1 loss in GMPs augments neutrophils' inflammatory response to the TLR4 ligand lipopolysaccharide through increased expression of the TLR4 coreceptor CD14. RUNX1 binds Cd14 and other genes encoding proteins in the TLR4 and type I IFN signaling pathways whose chromatin accessibility increases when RUNX1 is deleted. Transcription factor footprints for the effectors of type I IFN signaling-the signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRFs)-were enriched in chromatin that gained accessibility in both GMPs and neutrophils when RUNX1 was lost. STAT1::STAT2 and IRF motifs were also enriched in the chromatin of retrotransposons that were derepressed in RUNX1-deficient GMPs and neutrophils. We conclude that a major direct effect of RUNX1 loss in GMPs is the derepression of type I IFN and TLR4 signaling, resulting in a state of fixed maladaptive innate immunity., (© 2023 Zezulin et al.; Published by Cold Spring Harbor Laboratory Press.)

Acute myeloid leukemia (AML) is characterized by recurrent chromosomal rearrangements that encode for fusion proteins which drive leukemia initiation and maintenance. The inv(16) (p13q22) rearrangement is a founding mutation and the associated CBFβ-SMMHC fusion protein is essential for the survival of inv(16) AML cells. This Chapter will discuss our understanding of the function of this fusion protein in disrupting hematopoietic homeostasis and creating pre-leukemic blasts, in its cooperation with other co-occurring mutations during leukemia initiation, and in leukemia maintenance. In addition, this chapter will discuss the current approaches used for the treatment of inv(16) AML and the recent development of AI-10-49, a selective targeted inhibitor of CBFβ-SMMHC/RUNX1 binding, the first candidate targeted therapy for inv(16) AML.

The gene encoding for core-binding factor beta (CBFbeta) is altered in acute myeloid leukemia samples with an inversion in chromosome 16, expressing the fusion protein CBFbeta-SMMHC. Previous studies have shown that this oncoprotein interferes with hematopoietic differentiation and proliferation and participates in leukemia development. In this study, we provide evidence that Cbfbeta modulates the oncogenic function of this fusion protein. We show that Cbfbeta plays an important role in proliferation of hematopoietic progenitors expressing Cbfbeta-SMMHC in vitro. In addition, Cbfbeta-SMMHC-mediated leukemia development is accelerated in the absence of Cbfbeta. These results indicate that the balance between Cbfbeta and Cbfbeta-SMMHC directly affects leukemia development, and suggest that CBF-specific therapeutic molecules should target CBFbeta-SMMHC function while maintaining CBFbeta activity.

Mouse mammary tumor virus (LA) induces pregnancy-dependent mammary tumors that progress toward autonomy. Here we show that in virgin females, pregnancy-dependent tumor transplants are able to remain dormant for up to 300 days. During that period, these tumors synthesize DNA, express high levels of estrogen and progesterone receptors (ER+PR+) and are able to resume growth after hormone stimulation. Surprisingly, in a subsequent transplant generation, all these tumors are fully able to grow in virgin females, they express low levels of ER and PR (ER-PR-) and have a monoclonal origin; i.e., show all of the features we have described previously in pregnancy-independent tumors. Histologically, mouse mammary tumor virus (LA)-induced tumors are morphologically similar to genetically engineered mouse (GEM) mammary tumors that overexpress genes belonging to the Wnt pathway. Interestingly, in the virus-induced neoplasias, pregnancy-independent passages arising after a dormant phase usually display a lower level of glandular differentiation together with epithelial cell trans-differentiation, a specific feature associated to Wnt pathway activation. In addition, dormancy can lead to the specific selection of Int2/Fgf3 mutated and overexpressing cells. Therefore, our results indicate that during hormone-dependent tumor dormancy, relevant changes in cell population occur, allowing rapid progression after changes in the animal internal milieu.