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Over the last decades, rapid technological and scientific advances have led to a merge of molecular sciences and clinical medicine, resulting in a better understanding of disease mechanisms and the development of novel therapies that exploit specific molecular lesions or profiles driving disease. Precision oncology is here used as an example, illustrating the potential of precision/personalized medicine that also holds great promise in other medical fields. Real-world implementation can only be achieved by dedicated healthcare connected centers which amass and build up interdisciplinary expertise reflecting the complexity of precision medicine. Networks of such centers are ideally suited for a nation-wide outreach offering access to precision medicine to patients independent of their place of residence. Two of these multicentric initiatives, Genomic Medicine Sweden (GMS) and the Centers for Personalized Medicine (ZPM) initiative in Germany have teamed up to present and share their views on core concepts, potentials, challenges, and future developments in precision medicine. Together with other initiatives worldwide, GMS and ZPM aim at providing a robust and sustainable framework, covering all components from technology development to clinical trials, ethical and legal aspects as well as involvement of all relevant stakeholders, including patients and policymakers in the field.

Background: Analysis of measurable residual disease (MRD) is increasingly being implemented in the clinical care of children and adults with acute myeloid leukemia (AML). However, MRD methodologies differ and discordances in results lead to difficulties in interpretation and clinical decision-making. The aim of this study was to compare results from reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiparameter flow cytometry (MFC) in childhood AML and describe the kinetics of residual leukemic burden during induction treatment. Methods: In 15 children who were treated in the NOPHO-AML 2004 trial and had fusion transcripts quantified by RT-qPCR, we compared MFC with RT-qPCR for analysis of MRD during (day 15) and after induction therapy. Eight children had RUNX1::RUNX1T1, one CBFB::MYH11 and six KMT2A::LLT3. Results: When ≥0.1% was used as cut-off for positivity, 10 of 22 samples were discordant. The majority (9/10) were MRD positive with RT-qPCR but MRD negative with MFC, and several such cases showed presence of mature myeloid cells. Fusion transcript expression was verified in mature cells as well as in CD34 expressing cells sorted from diagnostic samples. Conclusions: Measurement with RT-qPCR suggests slower response kinetics than indicated from MFC, presumably due to the presence of mature cells expressing fusion transcript. The prognostic impact of early measurements with RT-qPCR remains to be determined.

Overcoming drug-resistance and the subsequent relapse that often occurs with monotherapy is crucial in the treatment of acute myeloid leukemia. We here demonstrate that therapy-resistant leukemia initiating cells can be targeted using a novel inhibitor of mitochondrial transcription (IMT). The compound inhibits mitochondrial RNA polymerase activity and sensitizes the resistant population to the induction of apoptosis. In vitro studies on acute myeloid leukemia cells demonstrate that IMT prevents cell proliferation, and together with a selective BCL-2 inhibitor, venetoclax, induce apoptosis and suppress oxidative phosphorylation (OXPHOS) synergistically. AML mouse models treated with IMT in combination with venetoclax show prolonged survival in venetoclax-resistant models. Our findings suggest that certain therapy-resistant leukemia cell populations display a unique dependency on mitochondrial transcription and can be targeted with IMT.TeaserLeukemia cells resistant to standard treatment are sensitive to a recently developed inhibitor of mitochondrial gene expression.

Certain subtypes of acute myeloid leukemia (AML) in children have still inferior outcome. One of these AML subtypes has a translocation t(7;12)(q36;p13), always leading to high expression of MNX1 and often to MNX1::ETV6 fusion expression. Here we identified the transforming event in this AML and possible ways to target them. Only MNX1 was able to induce AML in mice, and this was observed using hematopoietic stem and progenitor cells derived from fetal origin but not from adult bone marrow. The restriction in the transforming capacity to cells from fetal liver origin is in concordance with the fact that t(7;12)(q36;p13) AML is mostly restricted to infants. Ectopic expression of MNX1 led to increase of H3K4me1, H3K4me2 and H3K4me3, reduction in H3K27me3, accompanied with changes in genome-wide chromatin accessibility and genome expression, likely mediated through MNX1 interaction with the methionine cycle and different methyltransferases. MNX1 expression resulted in increased DNA damage, depletion of the Lin-/Sca1+/c-Kit+ population and skewing toward the myeloid lineage. These effects, together with leukemia development, was prevented by the S-adenosylmethionine analog Sinefungin. In conclusion, we have shown the importance of MNX1 in leukemia development in AML with t(7;12), supporting a rationale for targeting MNX1 and downstream pathways.

Certain subtypes of acute myeloid leukemia (AML) in children have inferior outcome. One of these has a translocation t(7;12)(q36;p13) leading to aMNX1::ETV6fusion along with high expression of MNX1. Here we identified the transforming event in this AML and possible ways of treatment. Only MNX1 was able to induce AML in mice, with similar gene expression and pathway enrichment to t(7;12) AML patient data. Importantly, this leukemia was only induced in immune incompetent mice using fetal but not adult hematopoietic stem and progenitor cells. The restriction in transforming capacity to cells from fetal liver is in alignment with t(7;12)(q36;p13) AML being mostly seen in infants. Expression of MNX1 led to increased histone 3 lysine 4 mono-, di- and trimethylation, reduction in H3K27me3, accompanied with changes in genome-wide chromatin accessibility and genome expression, likely mediated through MNX1 interaction with the methionine cycle and methyltransferases. MNX1 expression increased DNA damage, depletion of the Lin-/Sca1+/c-Kit+ population and skewing toward the myeloid lineage. These effects, together with leukemia development, was prevented by pretreatment with the S-adenosylmethionine analog Sinefungin. In conclusion, we have shown the importance of MNX1 in development of AML with t(7;12), supporting a rationale for targeting MNX1 and downstream pathways.

Acute myeloid leukemia (AML) results from aberrant hematopoietic processes and these changes are frequently initiated by chromosomal translocations. One particular subtype, AML with translocation t(7;12)(q36;p13), is found in children diagnosed before 2 years of age. The mechanisms for leukemogenesis induced by t(7;12) is not understood, in part because of the lack of efficient methods to reconstruct the leukemia-associated genetic aberration with correct genomic architecture and regulatory elements. We therefore created induced pluripotent stem cell (iPSC) lines that carry the translocation t(7;12) using CRISPR/Cas9. These t(7;12) iPSC showed propensity to differentiate into all three germ layers, confirming retained stem cell properties. The potential for differentiation into hematopoietic stem and progenitor cells (HSPC) was shown by expression of CD34, CD43 and CD45. Compared with the parental iPSC line, a significant decrease in cells expressing CD235a and CD41a was seen in the t(7;12) iPSC-derived HSPC (iHSPC), suggesting a block in differentiation. Moreover, colony formation assay showed an accumulation of cells at the erythroid and myeloid progenitor stages. Gene expression analysis revealed significant down-regulation of genes associated with megakaryocyte differentiation and up-regulation of genes associated with myeloid pathways but also genes typically seen in AML cases with t(7;12). Thus, this iPSC t(7;12) leukemia model of the t(7;12) AML subtype constitutes a valuable tool for further studies of the mechanisms for leukemia development and to find new treatment options.

Whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS), with the ability to provide comprehensive genomic information, have become the focal point of research interest as novel techniques that can support precision diagnostics in routine clinical care of patients with various cancer types, including hematological malignancies. This national multi-center study, led by Genomic Medicine Sweden, aims to evaluate whether combined application of WGS and WTS (WGTS) is technically feasible and can be implemented as an efficient diagnostic tool in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In addition to clinical impact assessment, a health-economic evaluation of such strategy will be performed.The study comprises four phases (i.e., retrospective, prospective, real-time validation, and follow-up) including approximately 700 adult and pediatric Swedish AML and ALL patients. Results of WGS for tumor (90×) and normal/germline (30×) samples as well as WTS for tumors only will be compared to current standard of care diagnostics. Primary study endpoints are diagnostic efficiency and improved diagnostic yield. Secondary endpoints are technical and clinical feasibility for routine implementation, clinical utility, and health-economic impact.Data from this national multi-center study will be used to evaluate clinical performance of the integrated WGTS diagnostic workflow compared with standard of care. The study will also elucidate clinical and health-economic impacts of a combined WGTS strategy when implemented in routine clinical care.[https://doi.org/10.1186/ISRCTN66987142], identifier [ISRCTN66987142].

Polycythaemia vera (PV) patients have an overall comparatively favourable prognosis, but disease progression is very heterogeneous and life-threatening thrombosis and bleedings are frequent complications in untreated disease. Moreover, transformation to more severe secondary myelofibrosis and acute myeloid leukaemia can occur. The aim of this study was to identify gene mutations that could be used together with clinical data as prognostic markers to guide treatment decisions in PV patients. A well-characterized WHO-defined cohort of PV patients was used. Clinical data and blood values were evaluated and a myeloid sequencing panel was used to screen for additional mutations other than the diagnostic JAK2 V617F and JAK2 exon 12 mutations. In 78% of the PV patients, at least one mutation additional to JAK2 V617F was detected. Additional mutations in genes coding for epigenetic modifiers, like TET2, DNMT3A and ASXL1, were most frequent. When correlated to overall survival, mutations in ASXL1 were significantly associated with inferior survival. In an attempt to obtain prognostic guidance in a larger number of patients, the presence of ASXL1 mutations was combined with age and vascular complications prior to diagnosis. Based on these data we were able to define three risk groups that predicted survival.

MicroRNAs (miRNAs) are commonly deregulated in acute myeloid leukemia (AML), affecting critical genes not only through direct targeting, but also through modulation of downstream effectors. Homeobox (Hox) genes balance self-renewal, proliferation, cell death, and differentiation in many tissues and aberrant Hox gene expression can create a predisposition to leukemogenesis in hematopoietic cells. However, possible linkages between the regulatory pathways of Hox genes and miRNAs are not yet fully resolved. We identified miR-708 to be upregulated in Hoxa9/Meis1 AML inducing cell lines as well as in AML patients. We further showed Meis1 directly targeting miR-708 and modulating its expression through epigenetic transcriptional regulation. CRISPR/Cas9 mediated knockout of miR-708 in Hoxa9/Meis1 cells delayed disease onset in vivo, demonstrating for the first time a pro-leukemic contribution of miR-708 in this context. Overexpression of miR-708 however strongly impeded Hoxa9 mediated transformation and homing capacity in vivo through modulation of adhesion factors and induction of myeloid differentiation. Taken together, we reveal miR-708, a putative tumor suppressor miRNA and direct target of Meis1, as a potent antagonist of the Hoxa9 phenotype but an effector of transformation in Hoxa9/Meis1. This unexpected finding highlights the yet unexplored role of miRNAs as indirect regulators of the Hox program during normal and aberrant hematopoiesis.

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease composed of clinically relevant subtypes defined by recurrent cytogenetic aberrations. The majority of the aberrations used in risk grouping for treatment decisions are extensively studied, but still a large proportion of pediatric AML patients remain cytogenetically undefined and would therefore benefit from additional molecular investigation. As aberrant epigenetic regulation has been widely observed during leukemogenesis, we hypothesized that DNA methylation signatures could be used to predict molecular subtypes and identify signatures with prognostic impact in AML. To study genome-wide DNA methylation, we analyzed 123 diagnostic and 19 relapse AML samples on Illumina 450k DNA methylation arrays. We designed and validated DNA methylation-based classifiers for AML cytogenetic subtype, resulting in an overall test accuracy of 91%. Furthermore, we identified methylation signatures associated with outcome in t(8, 21)/RUNX1-RUNX1T1, normal karyotype, and MLL/KMT2A-rearranged subgroups (p &lt, 0.01). Overall, these results further underscore the clinical value of DNA methylation analysis in AML.

Minimal residual disease (MRD) in acute myeloid leukemia (AML) is of major prognostic importance. The genetic landscape of AML is characterized by numerous somatic mutations, which constitute potential MRD markers. Leukemia-specific mutations can be identified with exome sequencing at diagnosis and assessed during follow-up at low frequencies by using targeted deep sequencing. Our aim was to further validate this patient-tailored assay for substitution mutations. By applying a statistical model, which corrects for position-specific errors, a limit of detection for single nucleotide variations of variant allele frequency (VAF) of 0.02% was achieved. The assay was linear in MRD range (0.03% to 1%) with good precision [CV, 4.1% (2.2% to 5.7%) at VAF 1% and 13.3% (8.8% to 19.4%) at VAF 0.1%], and low relative bias [7.9% (2.5% to 15.3%) at VAF 1%]. When applied to six childhood AML cases and compared with multiparameter flow cytometry for MRD analysis, deep sequencing showed concordance and superior sensitivity. Further high concordance was found with expression of fusion transcripts RUNX1-RUNX1T1 and KMT2A-MLLT10. The deep sequencing assay also detected mutations in blood when VAF in bone marrow exceeded 0.1% (n = 19). In conclusion, deep sequencing enables reliable detection of low levels of residual leukemic cells. Introduction of this method in patient care will allow for highly sensitive MRD surveillance in virtually every patient with AML.

Endothelin receptor type A (EDNRA) is known as a mediator of cell proliferation and survival. Aberrant regulation of EDNRA has been shown to play a role in tumor growth and metastasis. Using a global gene expression screen, we found that expression of Ednra was upregulated in murine leukemia inducing cells co-expressing Hoxa9 and Meis1 compared to cells only expressing Hoxa9. The aim of this study was to explore the role of Ednra in leukemogenesis further. In a murine bone marrow transplantation model, mice transplanted with cells overexpressing Ednra and Hoxa9 succumbed to leukemia significantly earlier than mice transplanted with cells overexpressing Hoxa9 only. Furthermore, overexpression of Ednra led to increased proliferation and resistance to apoptosis of bone marrow cells in vitro. We could also show that Meis1 binds to the Ednra promoter region, suggesting a regulatory role for Meis1 in Ednra expression. Taken together, our results suggest a role for Ednra in Hoxa9/Meis1-driven leukemogenesis.

BACKGROUND Cord blood units (CBUs) are processed, frozen, and thawed before use in hematopoietic stem cell (HSC) transplantation. The manipulations affect HSC functionality, that is, induce apoptosis and reduce viability. HSC content, commonly expressed as CBU potency, that is, the expected ability of a CBU to restore hematopoiesis, is traditionally approximated through viable CD34+ cells and the colony-forming unit (CFU) cell cultivation assay. Alternative approaches, for example, the aldehyde dehydrogenase (ALDH) enzyme-based assay, are also forthcoming. We hypothesized that the ALDH assay might exclude apoptotic cells since it is based on enzyme activity. To investigate this, we designed a protocol for simultaneous staining of viable and apoptotic CD34+ and ALDH+ cells using 7-aminoactinomycin (7-AAD) and annexin V, in frozen-thawed CBUs. Results were correlated with results from the colony-forming unit-granulocyte/macrophage (CFU-GM) assay. STUDY DESIGN AND METHODS Samples from 57 CBUs were thawed and simultaneously analyzed for CD34+ cells, ALDH+ cells, viability (7-AAD), and apoptosis (annexin V) using flow cytometry. Enumeration of CFUs was also performed. RESULTS No nonviable and few apoptotic cells (mean 0.7%) were identified in the ALDH+ population compared to the viable CD34+ population (mean 3.6%). The total number of ALDH+ cells correlated better than viable CD34+ cells (r = 0. 72 vs. r = 0.66; p

Micro-ribonucleic acid-155 (miR-155) is one of the first described oncogenic miRNAs. Although multiple direct targets of miR-155 have been identified, it is not clear how it contributes to the pathogenesis of acute myeloid leukemia. We found miR-155 to be a direct target of Meis1 in murine Hoxa9/Meis1 induced acute myeloid leukemia. The additional overexpression of miR-155 accelerated the formation of acute myeloid leukemia in Hoxa9 as well as in Hoxa9/Meis1 cells in vivo. However, in the absence or following the removal of miR-155, leukemia onset and progression were unaffected. Although miR-155 accelerated growth and homing in addition to impairing differentiation, our data underscore the pathophysiological relevance of miR-155 as an accelerator rather than a driver of leukemogenesis. This further highlights the complexity of the oncogenic program of Meis1 to compensate for the loss of a potent oncogene such as miR-155. These findings are highly relevant to current and developing approaches for targeting miR-155 in acute myeloid leukemia.

Pediatric acute myeloid leukemia (AML) with the translocation t(7;12)(q36;p13) is a form of childhood leukemia associated with inferior outcome. In the current investigation we have studied the gene alterations that are induced by this chromosomal translocation, a gene fusion MNX1-ETV6 but also ectopic expression of the homeobox gene MNX1. In a murine bone marrow transplantation model, our results showed that ectopic expression of MNX1 was able to induce AML, but not the MNX1-ETV6 gene fusion. However, this transformation to AML was only seen using hematopoietic progenitor cells from fetal liver and not adult bone marrow cells. This was accompanied with an alteration of cell differentiation towards myeloid lineage, increased proliferation and colony formation capacity induced by MNX1. Our data also showed that ectopic expression of MNX1, and to a lower extend MNX1-ETV6 fusion, induced DNA damage and cell cycle arrest, where we saw an increased expression and binding of the MNX1 and the MNX1-ETV6 fusion to the gene CEP164 that has a critical role in cell cycle regulation and DNA damage response, and the transcriptional pioneer factor gene PBX1 that is important in development and differentiation. Furthermore, global histone modifications, specifically H3K4me3 and H3K27me, were induced by MNX1 but also the MNX1-ETV6 fusion. We could also see an enrichment of methylation/methionine proteins from mass spectrometry analysis of proteins associated with MNX1. In conclusion, these results suggest a possible mechanism by which MNX1 mediates its transforming effect through abnormal methylation of proteins that result in changed histone modifications and subsequent dysregulation of gene expression. Our finding that only fetal liver cells were able to transform into AML is also in concordance with the clinical finding that this AML subtype is only found in very young children.

IF 2.845; International audience; External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay setup and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Introduction The translocation t(12;21)(p13;q22) resulting in the fusion gene ETV6-RUNX1, is the most frequent gene fusion in childhood B lymphoblastic leukemia. In the Nordic Society of Paediatric Haematology and Oncology ALL-2008 treatment protocol, treatment stratification in B-lineage ALL is based on results of minimal residual disease (MRD) analysis with fluorescence-activated cell sorting (FACS). In this study, we determined whether RT-qPCR of the ETV6-RUNX1 fusion transcript can be a reliable alternative for MRD analysis. Methods Seventy-eight bone marrow samples from 29 children at diagnosis and day 15, 29, and 78 during treatment were analyzed for MRD with FACS and with quantitative reverse transcription polymerase chain reaction (RT-qPCR). Fusion transcript MRD was defined as the ETV6-RUNX1/GUSB ratio at the follow-up time point (day 15/29/78) divided with the ETV6-RUNX1/GUSB ratio at diagnosis (%). Results MRD analysis with FACS and with RT-qPCR of ETV6-RUNX1 fusion transcript showed strong correlation. All cases showed concordant results at the treatment stratifying time points day 29 and day 78, when comparing the two methods with a cutoff set to 0.1%. Conclusion RT-qPCR is a valuable addition and could also be an alternative to FACS in cases where FACS is not achievable for MRD analysis.

HOXA9, MEIS1 and FLT3 are genes frequently upregulated in human acute myeloid leukemia. Hoxa9 and Meis1 also cooperate to induce aggressive AML with high Flt3 expression in mice, suggesting an important role for Flt3 in Hoxa9/Meis1-induced leukemogenesis. To define the role of Flt3 in AML with high Hoxa9/Meis1, we treated mice with Hoxa9/Meis1-induced AML with the Flt3 inhibitor AC220, used an Flt3-ligand (FL-/-) knockout model, and investigated whether overexpression of Flt3 could induce leukemia together with overexpression of Hoxa9. Flt3 inhibition by AC220 did not delay AML development in mice transplanted with bone marrow cells overexpressing Hoxa9 and Meis1. In addition, Hoxa9/Meis1 cells induced AML in FL-/- mice as rapid as in wild-type mice. However, FL-/- mice had reduced organ infiltration compared with wild-type mice, suggesting some Flt3-dependent effect on leukemic invasiveness. Interestingly, leukemic Hoxa9/Meis1 cells from sick mice expressed high levels of Flt3 regardless of presence of its ligand, showing that Flt3 is a passive marker on these cells. In line with this, combined engineered overexpression of Flt3 and Hoxa9 did not accelerate the progression to AML. We conclude that the Hoxa9- and Meis1-associated upregulation of Flt3 is not a requirement for leukemic progression induced by Hoxa9 and Meis1.

The prognosis of children with acute myeloid leukemia (AML) is improving but still unsatisfactory. We and others have shown that measurable residual disease (MRD) as measured with multiparameter flow cytometry (MFC) is a very strong prognostic factor, however this technique cannot reliably identify low-risk cases. An alternative approach for MRD analysis is quantification of leukemia-specific transcripts using reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR). This approach has shown strong prognostic value in adult AML but its significance in childhood AML is less studied. In this retrospective study, we evaluated early treatment response with RT-qPCR and MFC in parallel to determine treatment kinetics and the relationship between results of the two methods. The study included 15 children (7 females, 8 males, median age 6 years (range 1-16)), diagnosed 2004-2011 with de novo AML with a quantifiable fusion transcript (8 with RUNX1-RUNX1T1, one with CBFB-MYH11 and 6 with KMT2A-MLLT3), all treated in the NOPHO AML-2004 trial. MRD analyses were performed on samples from day 15 from start of the first induction course and just before the first consolidation course. MRD analysis with MFC was performed in bone marrow samples by identifying leukemia-associated immunophenotypes (LAIP), and reported as percent of viable cells. Fusion transcripts were analyzed with RT-qPCR in bone marrow and blood according to the Europe against cancer program, and reported as percentage of diagnostic level in bone marrow. Results of all MRD analyses were reported to clinicians but not used for treatment decisions. All patients achieved complete remission. 9 patients relapsed (median time from diagnosis 12 months, range 8-16); 5 with RUNX1-RUNX1T1 and 4 with KMT2A-MLLT3. When we compared results of RT-qPCR in blood and bone marrow day 15 and before consolidation, there was a high correlation; 0.93, p0.1%. These analyses confirmed the absence of immature cells with LAIP and detected either more mature myeloid cells or immature cells without LAIP. We hypothesize that fusion transcripts can be present in mature cells not detected as MRD using MFC. This was supported by analyzing sorted immature and mature cells from bone marrow samples at diagnosis of AML (n=4, adults). In CD34-CD117+ cells, leukemic transcripts (RUNX1-RUNX1T1/CBFB-MYH11/mutated NPM1) were 67±10% (mean±SEM) of the level in the most immature cells, CD34+CD117+, from the same patients and in mature granulocytes (CD34-CD117-, high SSC) 88±9%. In conclusion, RT-qPCR and MFC provide different information during induction treatment. RT-qPCR has a stronger ability to detect remaining leukemic burden, but whether this burden is clinically relevant remains to be shown. The impact of fusion transcript levels on relapse risk is currently investigated in the NOPHO-DBH AML-2012 trial in which MFC is used for risk stratification. Disclosures No relevant conflicts of interest to declare.

MicroRNAs (miRNAs) are commonly deregulated in acute myeloid leukemia (AML), affecting critical genes not only through direct targeting, but also through modulation of downstream effectors. Homeobox (Hox) genes balance self-renewal, proliferation, cell death, and differentiation in many tissues and aberrant Hox gene expression can create a predisposition to leukemogenesis in hematopoietic cells. However, possible linkages between the regulatory pathways of Hox genes and miRNAs are not yet fully resolved. We identified miR-708 to be upregulated in Hoxa9/Meis1 AML inducing cell lines as well as in AML patients. We further showed Meis1 directly targeting miR-708 and modulating its expression through epigenetic transcriptional regulation. CRISPR/Cas9 mediated knockout of miR-708 in Hoxa9/Meis1 cells delayed disease onset in vivo, demonstrating for the first time a pro-leukemic contribution of miR-708 in this context. Overexpression of miR-708 however strongly impeded Hoxa9 mediated transformation and homing capacity in vivo through modulation of adhesion factors and induction of myeloid differentiation. Taken together, we reveal miR-708, a putative tumor suppressor miRNA and direct target of Meis1, as a potent antagonist of the Hoxa9 phenotype but an effector of transformation in Hoxa9/Meis1. This unexpected finding highlights the yet unexplored role of miRNAs as indirect regulators of the Hox program during normal and aberrant hematopoiesis.

BACKGROUND The stem cell content in cord blood (CB) units is routinely assessed regarding nucleated cells, CD34+ cell count, and number of colony-forming units (CFUs). Efforts are made toward finding better ways of defining stemness of CB units. Side population (SP) phenotype and activity of aldehyde dehydrogenase (ALDH) are functional markers of stemness that can be assayed using flow cytometry. STUDY DESIGN AND METHODS We have developed a protocol for simultaneous determination of CD34+, SP, and ALDH+ populations in relation to immature white blood cells (CD45dim) in CB. Viable nucleated cells were consecutively stained for SP and ALDH activity and with antibodies against the CD45, CD34, and CD117 antigens. RESULTS The SP and ALDH+ populations could reliably be measured simultaneously. The median sizes of the SP and the ALDH+ populations were 0.85 and 3.3% of CD45dim cells, respectively. There was no overlap between the SP and ALDH+ populations. Cells that were ALDH+ expressed CD34 and CD117, but SP cells were negative for these markers. The ALDH+ cell content correlated with CD34+ cell content (p

The t(7;12)(q36;p13) (MNX1/ETV6) is not included in the WHO classification but has been described in up to 30% of acute myeloid leukemia (AML) in children2 years and associated with a poor prognosis. We present the clinical and cytogenetics characteristics of AML cases with t(7;12)(p36;p13). A literature review identified 35 patients with this translocation, published between 2000 and 2015. Outcome data were available in 22 cases. The NOPHO-AML (Nordic Society for Pediatric Hematology and Oncology) database contained 651 patients with AML from 1993 to 2014 and seven (1.1%) had the translocation. The t(7;12) was only present in patients2 years of age (median age 6 months) but none was diagnosed as newborn. These patients constituted 4.3% of the patients2 years of age. There was a strong association with trisomy 19 (literature: 86%, NOPHO: 100%) and +8 (literature: 19%, NOPHO: 14%). Seventeen of 22 patients from the literature with t(7;12) and four of seven patients from the NOPHO database suffered from relapse. The patients with t(7;12) had a 3-year event free survival of 24% (literature) vs. 43% (NOPHO) and a 3-year overall survival of 42% (literature) vs. 100% (NOPHO). None of the NOPHO patients was treated with hematopoietic stem cell transplantation (HSCT) in first complete remission. Relapse was frequent but the salvage rate using HSCT was high. We conclude that t(7;12)(q36;13) is a unique subgroup of childhood AML with presentation before 2 years of age with most cases being associated with +19.

Successful hematopoietic stem and progenitor cell (HSPC) transplantation rests upon reliable methods for their enumeration in sources such as cord blood (CB). Methods used today are costly, time consuming and exhaust the limited number of cells needed for transplantation. The aim of this study was to analyze if surplus plasma from CB contains biomarkers that can predict HSPC content in CB. Frozen, surplus plasma from 95 CB units was divided into two groups based on CD34+ cell concentration. Birth weight, gestation age, gender, mode of delivery, collection volume, nucleated cell count and colony forming unit assay results were available. Samples were analyzed with a proximity ligation assay covering 92 different proteins. Two-group t-test with p-values adjusted for false discovery rate (FDR) identified 5 proteins that significantly differed between the two groups. CDCP1 was the most significant (FDR adjusted p-value 0.006). Correlation with CDCP1 concentration was most significant for CD34+ concentration and nucleated cell count. Multivariate analysis showed that CD34 and gender seemed to influence the level of CDCP1. In conclusion, CDCP1 was identified as a potential biomarker of HSPC content in CB. The finding also warrants further investigation for a possible role of CDCP1 in regulating HSPC presence in CB. Keywords: Hematopoietic stem and progenitor cells, Cord blood, Proximity ligation assay, CDCP1

Introduction: Non-random cytogenetic aberrations are often involved in the development of AML in children and several aberrations can serve as diagnostic markers, prognosis predictors and impact choice of therapy. In infant AML, a chromosomal translocation t(7;12)(q36;p13) has been found in up to 20-30 % of the cases, making it the second most common genetic aberration in this age group after KMT2A (MLL) rearrangements. Previous studies indicate that this patient group has a dismal prognosis with virtually no event-free survival. Limiting the chances to improve this is the lack of understanding how the t(7;12)(q36;p13) is involved in leukemia development. The translocation leads to a gene fusion MNX1-ETV6 but also to increased MNX1 gene expression. Although both ETV6 and MNX1 are expressed in normal hematopoietic tissues, the role of the fusion protein MNX1-ETV6in the development of AML is not established. Also unclear is whether the driver of leukemogenesis is the fusion itself or the simultaneous overexpression of MNX1. The aim of this study was to assess the transformation capacity and the molecular mechanism of the MNX1-ETV6 fusion and the overexpressed MNX1in vitro and in vivo using murine transplantation models. Material and methods: In a liquid culture system, we introduced the MNX1-ETV6 fusion, MNX1 overexpression, or empty vector into primary murine (C57BL/6) hematopoietic progenitor cells with retroviral transfection. Cells were isolated from either adult bone marrow after 5-FU stimulation, or from fetal liver at E14.5. After enrichment by fluorescence activated cell sorting based on vector co-expressed green/yellow fluorescence protein, transfected cells were used for in vitro experiments and for transplantation into lethally irradiated immunocompetent C57BL/6 mice or sub-lethally irradiated immunocompromised NSGW41 mice. In vitro, cells were assessed with RNA sequencing for gene expression, gamma H2AX assay for DNA double strand brakes, flow cytometry for lineage marker expression, apoptosis and proliferation, and with colony forming unit assay. Results: Upon transplantation, only fetal liver cells transduced with MNX1 or with MNX1-ETV6 fusion were able to induce leukemia in immunocompromised (NSGW41) mice. When MNX1 or MNX1-ETV6 transduced cells were transplanted into immunocompetent mice (C57BL/6) mice, no leukemia development was seen, when either fetal liver or adult bone marrow cells were used for transduction. However, when immunocompromised mice were transplanted with MNX1 or MNX1-ETV6 fusion expressing cells they typically developed signs of disease after 1-2 months and exhibited leukocytosis and elevated blast cells in blood and bone marrow, severe anemia, and enlarged spleens with infiltration of leukemic cells. The cells showed expression of predominantly myeloid markers. In vitro, cells with overexpression of MNX1 or MNX1-ETV6 fusion expression also showed altered lineage differentiation in favor of myeloid differentiation. In addition, MNX1 overexpressing cells, but not MNX1-ETV6 expressing cells, exhibited increased proliferation and colony formation capacity. Both MNX1 overexpressing and MNX1-ETV6 fusion expressing cells showed increased DNA damage as evident from an increased gamma-phosphorylated H2AX in fetal liver and adult bone marrow transduced cells respectively, accompanied with G1 arrest, compared to cells transduced with empty vectors. Both MNX1 and MNX1-ETV6 also led to increased apoptosis in adult bone marrow (3-fold) and to a lesser extent in fetal liver cells (1.5-fold). Results from transcriptome sequencing showed enrichment for specific pathways in G2/M transition of cell cycle in cells transduced by either MNX1or the MNX1-ETV6 fusion. Further investigations to elucidate the molecular mechanisms and pathways through which MNX1 and/or MNX1-ETV6 induce leukemia is ongoing. Conclusions: MNX1 overexpression and MNX1-ETV6 fusion, both characteristics of infant AML with t(7;12)(q36;p13), induced leukemogenic effects in both fetal liver cells and adult bone marrow cells, but could cause a myeloid leukemia only under immunocompromised conditions. This may be of importance for the exclusive prevalence of this AML subtype in young children, with the highest peak during the first six months of life when the immune system is less developed. Disclosures No relevant conflicts of interest to declare.

Introduction Acute myeloid leukemia (AML) is the result of aberrant hematopoietic processes, such as enhanced proliferation, blocked differentiation, and dysregulated apoptosis of hematopoietic stem and progenitor cells, and frequently these changes are initiated by chromosomal translocations in leukemia. Efficient methods for modelling leukemia and to recreate leukemia-associated genetic aberrations, such as chromosome translocations, are therefore crucial for investigating how leukemia is initiated. Today, most such models are murine and usually based on introduction of fusion gene transcripts of interest under the control of a constitutive active promoter using lenti- or retroviral transduction rather than the chromosomal translocation itself. The aim of the current project was to create a human cellular model of a chromosomal translocation that is typically found in AML in children under 24 months of age, the translocation t(7;12)(q36;p13). This translocation has been associated with poor prognosis, and leads to a gene fusion MNX1-ETV6 but also aberrant MNX1 gene expression. Its mechanism for leukemia initiation is so far unknown, mainly due to lack of a suitable experimental model. Material and methods CRISPR/Cas9 was used to reconstruct the genetics of the t(7;12)(q36;p13) rearrangement in human induced pluripotent stem cells (iPSC) while maintaining the genomic architecture and regulatory elements. Ribonucleoprotein (RNP) complex was delivered by lipofection (Nucleofection, Amaxa 4D system) into undifferentiated iPSC (ChiPSC 22, Cellartis). An ATTO550 tag on tracrRNA/RNP complex was used to sort out positive cells by flow cytometry and then seeded as single-cells in 96-well plates. Genomic DNA from the single-cell derived iPSC clones were screened by PCR for the presence of the translocation and positive clones were verified with a FISH probe specific for t(7;12)(q36;p13) (Double Fusion Break Apart probe, Metasystem). RT-qPCR was used to detect and quantify the expression of MNX1-ETV6 fusion and MNX1 transcripts. Differentiation potential was tested with the Trilineage Differentiation and Hematopoietic Kits (STEMdiff, STEMCELL Technologies). Results Using CRISPR/Cas9, we could successfully generate iPSC with the t(7;12)(q36;p13) translocation. The translocation was confirmed using conventional karyotyping and FISH and the mRNA expression of the fusion was confirmed with RT-qPCR. No additional chromosomal aberrations were seen. The t(7;12)(q36;p13) iPSC showed similar growth and differentiation properties as the parental iPSC. They showed propensity to differentiate into all three germ layers, confirming their pluripotent stem cell properties. The potential for differentiation into hematopoietic progenitor cells was shown by expression of CD34+, CD43+ and CD45+. In AML with t(7;12)(q36;p13), MNX1 mRNA expression is increased and this may play a role for leukemia development. In the t(7;12)(q36;p13) iPSC, RT-qPCR indeed showed increased expression of MNX1 expression compared with iPSC without the translocation. This increase of MNX1 was not seen in murine adult bone marrow or fetal liver cells transduced with retrovirus expressing the MNX1-ETV6 fusion. Further characterization of the t(7;12)(q36;p13) iPSC, e.g. whole exome and transcriptome sequencing and engraftment potential in immunocompromised mice (NSG-SGM), is ongoing. Conclusion In summary, we have using CRISPR/Cas9 successfully created a t(7;12)(q36;p13) iPSC line with potential to differentiate into hematopoietic progenitor cells and with gene expression pattern similar to what is seen in human AML samples with the t(7;12)(q36;p13). The introduction of the MNX1-ETV6 fusion in its correct genomic context could recapitulate local gene regulation, making it superior to models based on lenti- or retroviral introduction of fusion genes transcripts. In conclusion, this created cell line will be a valuable tool to study the mechanisms behind t(7;12)(q36;p13) AML, a severe form of AML associated with poor prognosis. Disclosures No relevant conflicts of interest to declare.

The present research approaches for finding new therapies of acute myeloid leukemia (AML) are much focused on targeting the different genetic alterations in AML but many of them fail, probably due to the heterogeneity of the leukemic cells to be targeted. Recently, mitochondrial dependency is shown to play an important role in the progression of AML. AML cells exhibit high expression of mitochondrial RNA polymerase (POLRMT) which indirectly leads to high oxidative phosphorylation. Based on its upregulation as well as unique structure, POLRMT would have a great potential as a therapeutic target in AML. Recently, a novel inhibitor of mitochondrial transcription (IMT) has been developed which exhibits an anti-cancer potential by specifically targeting POLRMT. Here, we investigated the effect of IMT on AML. First, we showed that AML cell lines are sensitive to IMT. We could see a significant decline in proliferation in all human AML cell lines in a concentration-dependent manner, acquired by measurement of ATP production. Next, we investigated the effect of IMT in vivo in immunodeficient mice which were transplanted with an AML cell line that harbors FLT3-ITD mutation. IMT was administrated for 21 days from two weeks post-transplantation and compared with mice either treated with AC220 (FLT inhibitor) or vehicle (n=10/group). Results show that IMT was well tolerated by the mice and there was no sign of toxicity in normal tissues despite a significant delay or prevention of the onset of leukemia. Median survival of IMT treated mice=114 days vs vehicle treated=45 days (p

Background In children, T-cell acute lymphoblastic leukemia (T-ALL) has inferior prognosis compared with B-cell precursor ALL. In order to improve survival, individualized treatment strategies and ...

CML cells induce PARP-1 dependent parthanatos in NK cells, and inhibition of PARP-1 maintains lymphocyte viability and function. NK cells and T cells are commonly dysfunctional in CML, and their status may determine the course of disease. We aimed to define the molecular mechanisms of leukemia-induced immunosuppression with focus on the role of ROS and the PARP-1 pathway of cell death. Malignant granulocytes from patients with BCR-ABL-positive CML expressed the oxygen radical-producing enzyme NOX, produced large amounts of ROS, and triggered extensive cell death in NK cells. Inhibition of PARP-1 maintained NK cell viability in cocultures with suppressive leukemic cells. Under conditions of oxidative stress, PARP-1 inhibition upheld the capacity of NK cells to kill myeloid leukemic cells, in addition to restoring the proliferation and cytokine production of NK cells and cytotoxic T cells. Our findings are suggestive of a novel pathway of relevance to immunosuppression in CML.

The innate immune system and, in particular, activation of the multi-protein complex known as the inflammasome complex are involved in ischemic injury in myocardial cells. The nucleotide-binding leucine-rich repeat-containing pyrin receptor 3 (NLRP3) inflammasome has been linked to inflammation and NLRP3 is especially important for increased inflammation in atherosclerosis, which may lead to myocardial infarction. Here we investigated how inflammasome molecules are affected in human ischemic heart tissue. Surprisingly the important member of the inflammasome complex, NLRP3, displayed markedly decreased levels in human ischemic heart tissue compared with non ischemic control heart tissue. However, subsequent gene analysis revealed mutations in NLRP3 in human ischemic heart tissues but not in non-ischemic control tissue. Gene polymorphisms in the NLRP3 inflammasome have been shown to be associated with increased IL-1β and IL-18 production and severe inflammation. The autoinflammatory disorder familial Mediterranean fever (FMF) is associated with decreased expression of the Mediterranean fever gene (MEFV) and increased inflammation. We also observed reduced expression of MEFV in ischemic versus non-ischemic heart tissue. Further analyses showed a mutation in MEFV in human ischemic heart tissue but not in non-ischemic control tissue. Our data show that defects in the inflammasome and associated proteins may be involved in promoting ischemic heart disease.

Summary The transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feedback loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk signaling induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia., Graphical Abstract, Highlights • Meis1 regulates expression and activation of Syk in Hoxa9-driven leukemia • Syk upregulation in Hoxa9/Meis1-driven leukemia is mediated by miR-146a • Increased Syk activity induces a Meis1 transcriptional program • In vivo, Meis1 sensitizes Hoxa9-driven leukemia to Syk inhibition, Mohr et al. identify a Meis1-dependent regulatory loop involving PU.1, miR-146a, and Syk, resulting in the addiction to Syk activity in Hoxa9/Meis1-transformed myeloid progenitor cells. Syk inhibition disrupts the regulatory loop and prolongs survival of mice with Hoxa9/Meis1-driven acute myeloid leukemia.

Next-generation sequencing techniques have revealed that leukemic cells in acute myeloid leukemia often are characterized by a limited number of somatic mutations. These mutations can be the basis for the detection of leukemic cells in follow-up samples. The aim of this study was to identify leukemia-specific mutations in cells from patients with acute myeloid leukemia and to use these mutations as markers for minimal residual disease. Leukemic cells and normal lymphocytes were simultaneously isolated at diagnosis from 17 patients with acute myeloid leukemia using fluorescence-activated cell sorting. Exome sequencing of these cells identified 240 leukemia-specific single nucleotide variations and 22 small insertions and deletions. Based on estimated allele frequencies and their accuracies, 191 of these mutations qualified as candidates for minimal residual disease analysis. Targeted deep sequencing with a significance threshold of 0.027% for single nucleotide variations and 0.006% for NPM1 type A mutation was developed for quantification of minimal residual disease. When tested on follow-up samples from a patient with acute myeloid leukemia, targeted deep sequencing of single nucleotide variations as well as NPM1 was more sensitive than minimal residual disease quantification with multiparameter flow cytometry. In conclusion, we here describe how exome sequencing can be used for identification of leukemia-specific mutations in samples already at diagnosis of acute myeloid leukemia. We also show that targeted deep sequencing of such mutations, including single nucleotide variations, can be used for high-sensitivity quantification of minimal residual disease in a patient-tailored manner.

PTEN acts as a phosphatase for PIP3 and negatively regulates the PI3K/AKT pathway, and p27(KIP1) is a cyclin-dependent kinase inhibitor that regulates the G1 to S-phase transition by binding to and regulating the activity of cyclin-dependent kinases. Genetic alterations of PTEN or CDKN1B (p27(KIP1)) are common in hematological malignancies. To better understand how mutations in these two genes might cooperate in leukemogenesis, we inactivated both genes in the hematological compartment in mice. Here, we show that the combined inactivation of Pten and Cdkn1b results in a more severe myeloproliferative neoplasm phenotype associated with lower hemoglobin, enlarged spleen and liver, and shorter lifespan compared to inactivation of Pten alone. More severe anemia and increased myeloid infiltration and destruction of the spleen contributed to the earlier death of these mice, and elevated p-AKT, cyclin D1, and cyclin D3 might contribute to the development of this phenotype. In conclusion, PTEN and p27(KIP1) cooperate in tumor suppression in the hematological compartment.