ALBERT

Description: Key Points The CEBPA locus harbors 14 enhancers of which distinct combinations are active in different CEBPA-expressing tissues. A +42-kb enhancer is required for myeloid-lineage priming to drive adequate CEBPA expression levels necessary for neutrophilic maturation.

Description: Key Points miR-139-3p and miR-199a-3p, induced by ICL-induced damage, respectively, cause a loss and gain of hematopoietic progenitors. miR-199a-3p is an onco-microRNA (onco-miR) causing AML in a Cebpa-deficient mouse model. Target genes of miR-199a-3p include PRDX6, RUNX1, and SUZ12.

Description: Severe congenital neutropenia (SCN) is characterized by a maturation arrest at the promyelocyte stage and consequently a severe reduction of peripheral neutrophils. Administration of colony stimulating factor 3 (CSF3) restores neutrophil levels in over 90% of SCN patients, leading to an improved survival rate. SCN patients have an increased risk to develop secondary MDS or AML. Leukemic progression of SCN frequently involves the acquisition of a mutation in the gene encoding CSF3 receptor (CSF3R) in the neutropenic phase, followed by a mutation in runt-related transcription factor 1 (RUNX1) prior to transformation to MDS/AML (Skokowa et al, Blood, 2014). CSF3R mutations in SCN truncate the CSF3R C-terminus. RUNX1 mutations in SCN/AML are predominantly single nucleotide variations in the Runt-homology domain (RHD). Frameshift/nonsense mutations in the transactivation domain (TAD) also occur, albeit less frequently. To investigate how the combination of CSF3R and RUNX1 mutations affects myelopoiesis, we isolated hematopoietic stem and progenitor cells (HSPCs) from mice expressing either wild-type (WT) or a truncated (d715) CSF3R, retrovirally transduced them with TAD (S291fsX9) or RHD (D171N) mutants of RUNX1, or an empty vector control (EV), and cultured the cells in the presence or absence of CSF3. FACS analysis was performed to assess numbers of hematopoietic stem cells (LSKs, Lin- Sca1+ Kit+), early myeloid progenitors (LKs, Lin- Sca1- Kit+), common myeloid progenitors (CMPs, LK CD34+ CD16/32low), granulocyte-monocyte progenitors (GMPs, LK CD34+ CD16/32hi), immature (CD11b+, Gr-1lo) and mature (CD11b+, Gr-1hi) neutrophils. Whereas a normal differentiation pattern was observed in the CSF3R-WT background, activation of CSF3R-d715 led to defective neutrophil differentiation. Introduction of the RUNX1 mutants further aggravated the differentiation block and resulted in a selective expansion of HSPC subsets. While the EV and CSF3R-WT controls showed a relatively equal distribution of LK and LSK cells over time, the combination of CSF3R-d715 and RUNX1-TAD led to a 10-fold increase in absolute LSK numbers relative to EV (LSKs day 9: TAD: 1.23x107, EV: 1.27x106, RHD: 4.86x105, n=3, p=0.05) and a 7:1 ratio of LSK over LK. In contrast, the combination of CSF3R-d715 and RUNX1-RHD predominantly expanded LKs (LKs day 9: RHD: 8.77x106, EV: 2.77x106, TAD: 1.69x106, n=3, p=0.027) resulting in a 18:1 ratio of LK over LSK. Further analysis showed that these LKs were blocked in differentiation at the CMP to GMP transition stage (CMP to GMP ratio: RHD: 111.5, EV: 9.3, TAD: 1.8). To interrogate which mechanisms are responsible for the distinct differentiation defects caused by the RUNX1-RHD and -TAD mutants, we performed RNA-Seq on FACS purified LSK and LK populations. CMP to GMP transition is controlled by CCAAT/enhancer-binding proteins (C/EBPs), mainly C/EBPα and C/EBPβ. The combination of CSF3R-d715 and RUNX1-RHD, blocking the transition from CMP to GMP, induced the expression of C/EBPγ, a strong antagonist of C/EBP transcriptional activity (FPKM: day 2: 107.2, day 5: 424.3, day 9: 801.3). A major function of CEBPs in driving CMP to GMP transition is to suppress E2F- and Myc-driven transcription of genes involved in cell cycling. Consistent with this, single sample gene set enrichment analysis with output of hallmark pathways (Broad Institute) showed de-repression of E2F (p=0.002) and Myc (p=0.036) pathways in CSF3R-d715/RUNX1-RHD LKs relative to CSF3R-d715/EV. Interestingly, the RUNX1-TAD mutant that selectively expanded LSKs in combination with CSF3R-d715 did not alter C/EBP expression and function but affected genes involved in ribosomal biogenesis. We conclude that RUNX1-TAD and RHD mutants differentially cooperate with the CSF3R truncating mutations that are frequently acquired in the neutropenic phase of SCN and take alternative routes for LSK versus LK expansion. How this affects the leukemogenic nature of the RUNX1 mutations in combination with CSF3R truncation and how we can functionally interfere with these mechanisms is currently under investigation in in vivo transplantation settings. Disclosures No relevant conflicts of interest to declare.

Description: Primary alterations of the mesenchymal niche can induce myelodysplasia and acute myeloid leukemia in mouse models, introducing a concept of niche-driven leukemogenesis (Raaijmakers et al, Nature 2010). The molecular mechanisms and human relevance of this concept, however, have remained elusive. We addressed these key questions by modelling Shwachman-Diamond-Syndrome (SDS), a human monogenic congenital disorder caused by loss-of function mutation in the SBDS gene and characterized by skeletal defects, bone marrow failure and a striking propensity for leukemic evolution. Targeted Sbds deletion from mesenchymal progenitor cells (MPCs) in mice (OsxCre/+Sbdsf/f; OCSf/f) resulted in bone abnormalities faithfully recapitulating human disease, including short stature and early-onset osteoporosis. Skeletal defects were associated with genotoxic stress in hematopoietic stem and progenitor cells (HSPCs) as demonstrated by mitochondrial membrane hyperpolarization, oxidative stress, DNA damage and cell cycle checkpoint activation (transcriptional modulation of DNA damage response/repair pathways and G0-G1 cell cycle arrest). DNA damage could be partially rescued by in vivo administration of the ROS scavenger N-acetylcysteine supporting the notion of niche induced DNA damage in HSPCs induced by mitochondria-derived superoxide radicals. Mechanistically, Sbds deficiency caused activation of the p53 tumor suppressorpathway in MPCs (upregulation of P53 and transcriptional activation of downstream targets (GSEA). Genetic deletion of Trp53 from MPCs (Osxcre/+Sbdsf/fTrp53f/f mice) rescued the skeletal phenotype and genotoxic stress in HSPCs. Comparison of the transcriptome of MPCs from OCSf/f mice to their highly FACS-purified mesenchymal (CD45-CD235-7AAD-CD31-CD271+CD105+) human equivalents from SDS patients (RNAseq; n=5) demonstrated a striking overlap in disrupted gene programs (GSEA), including ribosome biogenesis and significant overexpression of the proinflammatory molecules such as S100A8 and S100A9, bona fide p53 downstream targets. Activation of p53 and inflammatory molecules was an MPC-autonomous consequence of Sbds depletion as demonstrated by ex vivo knockdown of the gene in OP9 cells. S100A8/A9 overexpression and secretion from MPCs from OCSf/f mice was confirmed by FCM and serum ELISA. Exposure of HSPCs to recombinant murine S100A8/9 resulted in increased DNA damage and apoptosis associated with transcriptional activation of TLR4 downstream signaling, a bona fide S100A8A9 receptor. In vivo TLR4 blockade by neutralizing antibodies resulted in reduced γH2AX foci in HSPCs from OCSf/f mice, in support of the existence of a Tpr53-S100A8/A9-TLR4 axis driving genotoxic stress. Formal demonstration that niche-derived S100A8/9 is sufficient to drive genotoxic stress in HSPCs was provided by transplantation of wild-type hematopoietic cells into recipient S100A8/A9 transgenic mice (Cheng et al., 2008) resulting in accumulation of mitochondrial superoxide radicals and DNA-damage in wild-type HSPCs. Finally, to further define the clinical relevance of this inflammatory MPC-HSPC axis to human disease, we performed massive parallel RNA-sequencing of FACS purified mesenchymal cells from homogeneously treated low-risk MDS patients (n=45). Overexpression of S100A8 and S100A9 in MPCs(confirmed by IHC) was found in a considerable subset of patients (17/45; 38%). S100A8/9+ mesenchymal cells displayed transcriptional activation of p53 and TLR programs, in line with findings in the mouse model. Strikingly, patients in the niche-S100A8/9+ group displayed a higher frequency of leukemia evolution (29.4% vs. 14.2%) with significantly shorter evolution time (average 3.4 (1-7.5) vs 18.5 (7-40); p=.03) and progression-free survival (median 11.5 vs. 53 months, p=.03), independent of established prognostic factors and risk classification systems. Collectively, the data define niche-HSPC inflammatory signaling through the p53-S100A8/A9-TLR axis as an actionable determinant of genotoxic stress and disease outcome in human preleukemia, opening the way to niche-instructed, therapeutic targeting to attenuate leukemic evolution. Disclosures No relevant conflicts of interest to declare.