ALBERT

Description: Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have reduced numbers of neutrophils in peripheral blood (

Description: Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome. We recently reported a high frequency of cooperating RUNX1 and CSF3R mutations in CN patients that developed AML or MDS. To study the mechanism of leukemia development in CN, we established a model for step-wise leukemia progression in CN using iPSC-based hematopoietic differentiation in combination with CRISPR/Cas9-mediated gene editing of iPSCs. Using this model, we confirmed that co-acquisition of CSF3R and RUNX1 mutations is necessary and sufficient to induce leukemia in CN. We also identified BAALC (brain and acute leukemia, cytoplasmic) upregulation as a key leukemogenic event downstream of RUNX1 and CSF3R mutations. BAALC mRNA was upregulated in CN/AML blasts (n = 5) and in CD34+ HSPCs generated from CN/AML iPSCs of two patients. Importantly, CRISPR/Cas9-mediated knockout of BAALC in CN/AML-iPSCs reversed defective myeloid differentiation of CN/AML blasts to the levels observed in healthy donor hematopoietic stem cells. We further investigate the mechanism of BAALC up-regulation. In silico analysis of the BAALC gene promoter in combination to publicly available ChIP-Seq data revealed three putative RUNX1 binding sites that were validated using ChIP assay in lysates of NB4 cells. Interestingly, transduction of healthy donor CD34+ cells with lentiviral constructs expressing WT RUNX1 led to inhibition of BAALC mRNA expression, whereas transduction with two RUNX1 RUNT domain mutants resulted in the marked BAALC up-regulation, as compared to the control BFP transduced cells. These data suggest that mutated RUNX1 failed to inhibit BAALC expression in CD34+ HSPCs. To evaluate the mechanism of leukemogenic transformation in CN, we performed RNA-Seq analysis of CD34+ cells derived from CN and CN/AML iPSC clones. GSEA revealed that changes in gene expression between CN- and CN/AML-HSPCs were strongly correlated with gene expression signatures of "Wierenga STAT5 targets" and "reactome ATF4 targets", an observation in line with the markedly elevated levels of STAT5 and ATF4 in CN/AML-HSPCs. Importantly, gene expression differences between CN/AML-HSPCs and CN-HSPCs were correlated to "Valk AML" targets in GSEA, suggesting that HSPCs generated from CN/AML-iPSCs possess characteristics of AML cells. Strong support for the leukemogenic role of upregulated BAALC in CN/AML was provided by further GSEA analysis of the BAALC KO CN/AML-HSPCs. We observed a reversal in the expression of a majority of genes in the studied leukemia-associated pathways in CN/AML-HSPCs after BAALC knockout compared with CN/AML-HSPCs. Since there are no direct inhibitors for BAALC available and protein structure is not solved yet, BAALC effects can be targeted only indirectly. Morita et al., Leukemia, 2015 showed that BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and Klf4. We treated CD45+ cells generated from CN/AML or healthy donor (HD) iPSCs with MEK1/2- or MEK1-specific inhibitors or vehicle control and evaluated cell proliferation and differentiation (CFU assay). We were able to induce ~ 40-60 % cell death of CN/AML cells upon treatment with each of inhibitors, whereas HD CD45+ cells were unaffected. Moreover, treatment of CN/AML cells with MEK1/2 inhibitor led to an increase in CFU-G formation, as compared to vehicle control cells. Using connectivity Map analysis of RNA-Seq data of CD34+ cells generated from CN/AML iPSCs vs CN/AML BAALC KO iPSCs, we identified small molecule p38/MAPK14 inhibitor that could possibly reverse BAALC-mediated leukemogenic gene expression signature. We treated CN/AML iPSC-generated CD34+ cells for 7 days with this inhibitor and subsequently performed CFU assay. We found an increase in CFU-GM formation. In summary, using CN/AML-iPSC-model, we confirmed the major role of BAALC in leukemia development downstream of CSF3R and RUNX1 mutations in CN. Inhibition of MAPK/ERK-pathway downstream of BAALC reduced proliferation and partially induced myeloid differentiation of CN/AML-derived hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

Description: Key Points LMO2 is deacetylated by the NAMPT/SIRT2 pathway. LMO2 deacetylation is essential for LIM domain binding 1 binding and TAL1 complex activation during hematopoiesis and T-ALL leukemogenesis.

Description: Patients with pre-leukemic bone marrow failure syndrome, severe congenital neutropenia (CN) have ~ 20% risk of developing acute myeloid leukemia (AML) (CN-AML). More than 70 % of CN-AML patients co-acquire CSF3R and RUNX1 mutations as shown by our group (Skokowa et al 2014), indicating a cooperative role of the mutations in these two genes in the development of AML in CN patients. In order to investigate the interaction between these mutations we conducted in vitro experiments on lineage negative (lin-) bone marrow mononuclear cells (BMCs) from C57BL/6-d715csf3r mice (d715-mice). These mice carry homozygous d715G CSF3R mutations, but do not develop AML. We isolated lin- BMCs from d715 mice and transduced these cells with four different lentivirus vectors carrying BFP only (CTRL), RUNX1-Wild type BFP (RUNX1-WT), RUNX1-R139G BFP (RUNX1-MUT1) and RUNX1-R174L BFP (RUNX1-MUT2). These RUNX1 mutations where found in CN-AML patients. 72 hours after transduction, we sorted BFP+ cells and compared G-CSF triggered myeloid differentiation in vitro. We found that cells transduced with each RUNX1 mutants exhibited reduced percentages of myeloid CD11b+, Gr-1+ and double positive cells compared to RUNX1-WT. We also conducted CFU re-plating experiments with transduced cells and found that cell transduced with each of RUNX1 mutants showed 7- (RUNX1-MUT1) and 8- (RUNX1-MUT2) times higher re-plating capacity than RUNX1-WT and CTRL transduced cells. To identify signaling pathways that are deregulated in G-CSFR-mutated HSCs clones after co-acquisition of RUNX1 mutations, we performed microarray study. We starved transduced and sorted lin- BMCs for 24 hours and treated cells with G-CSF for 48 hours before mRNA was collected. Expression profiles where generated by microarray (GeneChip Mouse Gene 2.0 ST Array). Pathway Analysis was conducted using IPA Software and Motif activity response analysis (MARA) was performed using ISMARA web tool. Reported Transcription factors and targets have Z-value ≥ 2 or ≤-2 and p ≤ 0,05 and are thus considered statistically significant. Interestingly, ISMARA analysis showed, that the highest active motif in RUNX1-Mutants was Irf2_Irf1_Irf8_Irf9_Irf7 motif which is essential for the regulation of the interferon pathway genes. The corresponding transcription factors are amongst others regulated by Sp1 and Stat2 that were also active. Correspondingly, IPA Pathway analysis showed, that Interferon Signaling was highly upregulated in cells transduced with each of two RUNX1 mutants, compared to overexpressed RUNX1-WT (Z = 2). Additionally, pathway analysis showed the upregulation and activation of IL-6, IL-8-, Toll like Receptor- and TREM1 signaling pathways. This data suggests that the mutated RUNX1 may cause activation of the pro-inflammatory cell state propagating proliferation, which may be emerging as a cause of clonal hematopoiesis (CH) and consequently may lead to MDS/AML (Hemmati et al 2017). Another active motif is Spi1/PU.1 mainly known as an essential transcription factor for monocytic differentiation, but also as a maintenance factor of the pre-leukemia initiating cells (pre-LICs) or even leukemia initiating cells (LICs) (Staber et al 2014). Interestingly, we recently described elevated expression of PU.1 in hematopoietic cells of CN patients. These data together with our in vitro finding indicating that RUNX1 mutation causes differentiation block and clonal proliferation of HSCs, supporting the hypothesis, that the RUNX1 mutations are the driving factor in leukemic transformation in CN. Additionally, ISMARA revealed an upregulation of the Metyl-CpG Binding Protein 2 (Mecp2) motif. Mecp2 is a proto-oncogene that represses transcription through interaction with the corepressor SIN3A and histone deacetylases. Thus, remodeling the transcriptional profile and inhibiting differentiation. Taken together, our data shows that RUNX1 mutations in combination with CSF3R mutations may cause (1) increased proliferation through the induction of a proinflammatory cell state, (2) induce self-renewal through expression of essential proteins for LIC maintenance and (3) dimineshed myeloid differentiation through demethylation inhibition and down regulation of hematopoietic differentiation pathways. We are currently validating the model of the leukemogenic transformation in CN patients using functional studies in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

Description: Severe congenital neutropenia (CN) is a monogenic bone marrow failure syndrome with the frequency of 1:200,000 and is characterized by an absolute neutrophil count below 500 cells per microliter. Patients with CN suffer from severe life-threatening bacterial infections starting early after birth due to the absent or very low numbers of neutrophils in peripheral blood. While CN is a heterogeneous disease caused by many different gene mutations, autosomal-dominant ELANE mutations are the most common cause of CN. Although the majority of CN patients respond to daily treatment with granulocyte colony-stimulating factor (G-CSF), approximately 15 % do not respond at doses up to 20 μg/kg/day and approximately 20 % of G-CSF treated patients develop myelodysplasia (MDS) or acute myeloid leukemia (AML). In the present study, we first established an efficient gene-editing platform for induced pluripotent stem cells (iPSC) of CN patients using CRISPR/Cas9 technology. The platform uses ribonucleoprotein form of CRISPR/Cas9 making the editing approach safer as it is virus- or DNA free. Also, any further selection step or introducing extra modifications in the genome of edited cells such as silent mutation are not required. We generated and characterized iPSCs from ELANE-CN patients harboring p.A57V, p.C151Y, and p.G214R mutations, that are more severe hot-spot mutations associated with G-CSF non-response or MDS/AML. We corrected each mutation followed by EB-based hematopoietic differentiation, to evaluate and compare granulocytic differentiation of CN-patient specific iPSCs, with or without ELANE mutation, in an isogenic model. To study granulocytic differentiation, we performed live cell counts, flow cytometry analysis of myeloid-specific surface marker expression, CFU assay, cell morphology of cytospin preparations and neutrophil functional tests. Our isogenic model showed that correction of ELANE mutations led to fully normalized granulocytic differentiation. We have recently shown that CRISPR/Cas9 mediated ELANE knockout (KO) enables neutrophilic maturation of primary HSPCs and iPSCs of CN patients. We observed that granulocytic differentiation of ELANE KO iPSCs and primary HSPCs were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO neutrophils were also normal. To model CN in silico and to reveal the key driving pathomechanisms, we designed an isogenic patient-specific disease modeling system by comparing RNA-sequencing results of CN-ELANE corrected- or CN-ELANE KO hematopoietic stem and progenitor cells (HSPCs) to the original CN-ELANE patient cells. HSPCs were derived from iPSC lines. Our analysis showed a degree of similarity in enriched pathways upon ELANE correction or ELANE KO in a patient-specific manner. Thus, upon correction of p.C151Y mutation, TNF, IL4 and IL13 signaling pathways as well as MAPK signaling, PD-1 signaling and IL10 signaling were down-regulated. Interestingly, the same pathways were down-regulated upon ELANE KO in HSPCs of the same CN patient. Correction of p.A57V mutation led to down-regulation of IL12 expression which activates STAT family. Upon ELANE KO in the cells from the same patient, IL12, IL18, and IL1-beta expression were down-regulated. We also identified common pathways enriched in most of the isogenic samples upon ELANE correction or ELANE KO like down-regulation of MAPK or IFN α/β signaling as well as down-regulation of the Rap-1 signaling pathway leading to the Erk pathway activation. Analysis of putative transcription factor binding sites (TFBSs) that are enriched in the differentially expressed gene list upon ELANE mutation correction or ELANE KO showed that transcription factors GKLF (KLF4), MAZ, Kaiso (ZBTB33) and CHURCHILL are highly enriched in UP-regulated genes, for both, correction and KO samples. Taken together, we established a safe and efficient CRISPR/Cas9-RNP based ELANE gene-correction/knockout platform of iPSCs of ELANE-CN patients that may be used to establish an isogenic disease modeling system or provide novel stem cell-based therapy for CN patients with a high risk of leukemia development as well as for G-CSF-non-responsive patients. This platform could be also applied for other monogenic bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

Description: Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have markedly reduced numbers of neutrophils in peripheral blood (12x1012 viral copies per ml). Our gene-editing protocol produced on average 79,7 % (± 8,62 %) of total editing (TE) in healthy donor HSPCs (n=6). When we transduced healthy donor HSPCs with rAAV6 containing the template at MOI 105 after electroporation with CRISPR/Cas9 RNP, we achieved 38,1 % (± 1,3 %) knock-in (KI) efficiency and 82,3 % (± 8,2 %) TE (n=2). We further applied this approach to primary HSPCs from 5 CN patients harboring the p.W44X HAX1 mutation. We achieved 84,4 % (± 4,2 %) TE and 65,8 % (± 7,12 %) KI. Too proof, that our editing reintroduced HAX1 protein expression, we performed Western Blot analysis of edited cells (n=2) and were able to detect relevant amounts of HAX1 protein. To assess the effect of HAX1 correction on the neutropenic phenotype in vitro, we performed a liquid culture differentiation assay of edited HSPCs to neutrophils. HSPCs from the same patients that were edited in the AAVS1 safe harbor were used as isogenic controls. In the AAVS1 locus the editing efficiency was 76,74 % (± 17,07 %) total indels. By morphological assessment of Wright-Giemsa stained cytospins of edited cells derived on day 14 of differentiation revealed significant (p = 0,005) increases of mature neutrophils for all five edited HAX1-CN patient samples, as compared to the respective controls. This phenotype correction was also observed in flow cytometry by a significant (p = 0,011) increase of mature CD34-CD45+ CD15+CD16+ neutrophils (n=5). To investigate if the HAX1 mutation correction and reinforced expression of HAX1 protein improved the sensitivity of HSPCs to oxidative stress as described by Klein et al. 2007, we performed live-cell imaging of caspase3/7 activation. Live-cell imaging revealed a substantial reduction of H2O2-induced apoptosis in corrected HAX1-CN patients derived HSPCs (n=3). Furthermore, the corrected differentiated cells were investigated for functional hallmarks of granulocytes. We could observe that HAX1 gene-edited HSPCs showed comparable chemotaxis, phagocytosis and no defects in ROS production to isogenic control edited cells. Taken together, we established a protocol for efficient selection-free correction of HAX1 p.W44X mutation in primary HSPCs using CRISPR/Cas9 and rAVV6 HDR repair templates. Our gene-editing reintroduced HAX1 protein expression in primary HSPCs from HAX1-CN patients. Neutrophils derived from corrected cells showed functional improvements in survival to oxidative stress and general neutrophil functions. We believe that these results are enticing to be investigated further for potential clinical translation as an autologous stem cell therapy for HAX1-CN patients. Disclosures No relevant conflicts of interest to declare.