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Background: Precision medicine has the potential to improve cardiovascular disease (CVD) risk prediction in individuals with type 2 diabetes (T2D). Methods: We conducted a systematic review and meta-analysis of longitudinal studies to identify potentially novel prognostic factors that may improve CVD risk prediction in T2D. Out of 9380 studies identified, 416 studies met inclusion criteria. Outcomes were reported for 321 biomarker studies, 48 genetic marker studies, and 47 risk score/model studies. Results: Out of all evaluated biomarkers, only 13 showed improvement in prediction performance. Results of pooled meta-analyses, non-pooled analyses, and assessments of improvement in prediction performance and risk of bias, yielded the highest predictive utility for N-terminal pro b-type natriuretic peptide (NT-proBNP) (high-evidence), troponin-T (TnT) (moderate-evidence), triglyceride-glucose (TyG) index (moderate-evidence), Genetic Risk Score for Coronary Heart Disease (GRS-CHD) (moderate-evidence); moderate predictive utility for coronary computed tomography angiography (low-evidence), single-photon emission computed tomography (low-evidence), pulse wave velocity (moderate-evidence); and low predictive utility for C-reactive protein (moderate-evidence), coronary artery calcium score (low-evidence), galectin-3 (low-evidence), troponin-I (low-evidence), carotid plaque (low-evidence), and growth differentiation factor-15 (low-evidence). Risk scores showed modest discrimination on internal validation, with lower performance on external validation. Conclusions: Despite high interest in this topic, very few studies conducted rigorous analyses to demonstrate incremental predictive utility beyond established CVD risk factors for T2D. The most promising markers identified were NT-proBNP, TnT, TyG and GRS-CHD, with the highest strength of evidence for NT-proBNP. Further research is needed to determine their clinical utility in risk stratification and management of CVD in T2D.

Maf transcription factors are critical regulators of beta-cell function. We have previously shown that reduced MafA expression in human and mouse islets is associated with a pro-inflammatory gene signature. Here, we investigate if the loss of Maf transcription factors induced autoimmune processes in the pancreas. Transcriptomics analysis showed expression of pro-inflammatory as well as immune cell marker genes. However, clusters of CD4+ T and B220+ B cells were associated primarily with adult MafA−/−MafB+/−, but not MafA−/− islets. MafA expression was detected in the thymus, lymph nodes and bone marrow suggesting a novel role of MafA in regulating immune-cell function. Analysis of pancreatic lymph node cells showed activation of CD4+ T cells, but lack of CD8+ T cell activation which also coincided with an enrichment of naïve CD8+ T cells. Further analysis of T cell marker genes revealed a reduction of T cell receptor signaling gene expression in CD8, but not in CD4+ T cells, which was accompanied with a defect in early T cell receptor signaling in mutant CD8+ T cells. These results suggest that loss of MafA impairs both beta- and T cell function affecting the balance of peripheral immune responses against islet autoantigens, resulting in local inflammation in pancreatic islets.

Summary A gradual restriction in lineage potential of multipotent stem/progenitor cells is a hallmark of adult hematopoiesis, but the underlying molecular events governing these processes remain incompletely understood. Here, we identified robust expression of the leukemia-associated transcription factor hepatic leukemia factor (Hlf) in normal multipotent hematopoietic progenitors, which was rapidly downregulated upon differentiation. Interference with its normal downregulation revealed Hlf as a strong negative regulator of lymphoid development, while remaining compatible with myeloid fates. Reciprocally, we observed rapid lymphoid commitment upon reduced Hlf activity. The arising phenotypes resulted from Hlf binding to active enhancers of myeloid-competent cells, transcriptional induction of myeloid, and ablation of lymphoid gene programs, with Hlf induction of nuclear factor I C (Nfic) as a functionally relevant target gene. Thereby, our studies establish Hlf as a key regulator of the earliest lineage-commitment events at the transition from multipotency to lineage-restricted progeny, with implications for both normal and malignant hematopoiesis., Graphical Abstract, Highlights • During hematopoiesis, Hlf is sharply downregulated upon exit from multipotency • Prolonged Hlf expression instructs GMLPs to adopt myeloid fates • Failure to downregulate Hlf is incompatible with appropriate B and T lymphopoiesis • Hlf governs molecular programs driving myelopoiesis and inhibiting lymphopoiesis, Regulators of early blood cell formation are important in both health and disease. Wahlestedt et al. identify abrupt downregulation of the transcription factor Hlf during hematopoietic differentiation. Failure to downregulate Hlf leads to a drastically skewed output of mature blood cells, positioning Hlf as a critical regulator of hematopoiesis.

Translocations involving the mixed lineage leukemia-1 are recurrent events in acute leukemia and associate with lymphoid (ALL), myeloid (AML) or mixed lineage (MLL) subtypes. Despite an association with ALL in humans, murine MLL fusion models are persistently restricted to AML. We here explored this issue using an inducible mixed lineage leukemia-eleven nineteen leukemia (MLL-ENL) mouse model. Although multiple progenitor cell types with myeloid potential are potent AML leukemia-initiating cells, also the earliest lymphoid progenitors were capable of initiating AML. This ability to evoke a latent myeloid potential in the earliest lymphoid progenitors was lost upon further lymphoid commitment. At the same time, more downstream/committed lymphoid precursors also failed to initiate lymphoid leukemia. Co-expression of MLL-ENL with a constitutively active RAS allele, the most common co-mutation in MLL fusion leukemias, could influence on both disease latency and lineage assignment of developing leukemia in what appears to be a mutation-order-dependent manner. Finally, CEBPB-mediated transdifferentation of committed and otherwise leukemia-incompetent B-cell progenitors imbued these cells with leukemic competence for AML. Therefore, apart from providing detailed insight into the differential responsiveness of candidate target cells to a first-hit MLL fusion event, our data warrants caution to therapeutic approaches based on the concept of transdifferentiation.

Using a conditional model of MLL-ENL–driven AML, Dudenhöffer-Pfeifer and Bryder show that NK cell and adaptive immunity influences little on leukemia initiation from normal cells. The data argue against immune escape as a universal primary transformation event in AML., Although it is firmly established that endogenous immunity can prevent cancer outgrowth, with a range of immunomodulatory strategies reaching clinical use, most studies on the topic have been restricted to solid cancers. This applies in particular to cancer initiation, where model constraints have precluded investigations of immunosurveillance and immunoediting during the multistep progression into acute myeloid leukemia (AML). Here, we used a mouse model where the chimeric transcription factor MLL-ENL can be conditionally activated in vivo as a leukemic “first-hit,” which is followed by spontaneous transformation into AML. We observed similar disease kinetics regardless of whether AML developed in WT or immunocompromised hosts, despite more permissive preleukemic environments in the latter. When assessing transformed AML cells from either primary immunocompetent or immunocompromised hosts, AML cells from all sources could be targets of endogenous immunity. Our data argue against immunoediting in response to selective pressure from endogenous immunity as a universal primary transformation event in AML.

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13-4 and Munc13-1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13-1 and Munc13-4, are functionally redundant in murine CTLs.

Missense mutations and other chromosomal perturbations underlie the heterogeneity of acute leukemia. While Mixed lineage leukemia-1 (MLL1/KMT2A) translocations are recurrent in human acute leukemia, most if not all MLL fusions are permissive for either acute myeloid (AML), acute lymphoid (ALL) or mixed/biphenotypic leukemia (MLL). At the same time, previous work have suggested that MLL rearranged leukemias require few additional mutations to induce transformation(1, 2). The MLL-ENL translocation is apart from being associated with AML also found in human acute T cell leukemia (T-ALL). Despite this, but consistent with most previous observations in mice, we failed to observe development of ALL even when inducing MLL-ENL in multiple early hematopoietic progenitors(3). We hypothesized that this could be caused by a compromised T cell generation induced by the MLL-ENL translocation product. Culturing bone marrow Granulocyte-Monocyte-Lymphoid Progenitor (GMLPs) on OP9-DL1 stromal cells revealed that MLL-ENL blocked differentiation at a stage that phenotypically corresponds to a primitive DN1 stage. In vivo, short-term induction of MLL-ENL led to deregulation of T cell differentiation, with an evident block at the DN2/3 stage. As such a compromise in T cell generation could omit T-ALL development from immature T cell progenitors in the bone marrow, we next assessed the leukemic capacity of defined lymphoid progenitor cells at different stages of development. The latent myeloid potential of DN1 cells and early B lymphoid progenitors (BLPs), a property lost upon further differentiation of these cells, was sufficient to confer potent leukemia initiating activity for AML. By contrast, mice transplanted with of later stages of T cell and B cell progenitors failed to associate with development of disease. The discrepancy of lineage assignment between MLL translocations in established human leukemia and attempts to mimic these in the mouse might be due to a requirement of necessary co-mutations. Although MLL fusions typically associate with few secondary mutations, previous studies have established that activating somatic mutations in the RAS pathway are frequently co-occurring with MLL rearranged leukemias. Therefore, we next investigated whether mutation order might influence on the developing murine leukemia. Co-expression of KRASG12D in GMLPs led to a significant reduction in disease latency compared to MLL-ENL alone. However, disease was still restricted to the myeloid lineage. In strikingly contrast, but in agreement with previous reports, mice receiving KRASG12D expressing GMLPs in the absence of MLL-ENL expression developed T-ALL with a median latency of 143 days and with roughly a 30% penetrance. The onset of MLL-ENL expression 5 weeks post transplantation in KRASG12D expressing GMLPs also gave rise to T-ALL, but with a shorter mean latency (111 days) and higher (50%) penetrance. The sequence of acquisition of somatic mutations therefore can influence not only on disease latency, but also on the lineage assignment of the developing leukemia. Finally, to identify novel somatic mutations occurring during MLL-ENL induced leukemogenesis, we generated paired leukemic samples by transplanting leukemia initiating cells from individual donor mice into separate cohorts of recipients, followed by whole genome and exome sequencing upon development of AML. While MLL-ENL induced AML associated with very few secondary mutations, targeted re-sequencing of a panel of variants validated a few single nucleotide variants from leukemic samples, including amongst others driver mutations in PTPN11 and RAS known from studies of human leukemia. Further studies are now undertaken to obtain functional insights into the mode of action of the identified somatic mutations. References 1. Andersson AK, Ma J, Wang J, Chen X, Gedman AL, Dang J, et al. The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias. Nature genetics. 2015;47(4):330-7. 2. Cancer Genome Atlas Research N. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England journal of medicine. 2013;368(22):2059-74. 3. Ugale A, Norddahl GL, Wahlestedt M, Sawen P, Jaako P, Pronk CJ, et al. Hematopoietic stem cells are intrinsically protected against MLL-ENL-mediated transformation. Cell reports. 2014;9(4):1246-55. Disclosures No relevant conflicts of interest to declare.

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13-4 and Munc13-1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13-1 and Munc13-4, are functionally redundant in murine CTLs.

Cytotoxic T lymphocytes (CTLs) are an important part of the immune system and kill cells which are infected by pathogens like viruses or bacteria (named antigen-presenting-cell or APC). Two different killing mechanisms are being used by CTLs - (1) exocytosis of lytic granules which contain cytotoxic substances and (2) the so called Fas/FasL pathway which relies on agonist/receptor interaction and induces apoptosis in the APC. So far the quantitative contribution of either pathway to the total killing capacity of mouse CTL was not known. To address this question we used a knock out mouse strain (Jinx) which lacks the SNARE associated protein Munc13-4. As a result CTLs from these mice display a severe defect in the secretion of cytotoxic substances, as we could demonstrate by a Granzyme B release assay (reduction of 85% ± 4%) and a FACS based degranulation assay (reduction of 70% ± 9%). Interestingly we found that despite the lytic granule release failure CTLs from Munc13-4 -/- have a remaining killing capacity of about 43%, arguing for a significant contribution of the Fas/FasL pathway. To strengthen this finding we established a single cell assay in which the increase of intracellular Ca2+ in the APC was used as a measure of apoptosis and found that about 50% of killing was induced by the Fas/FasL pathway alone. We conclude from these data that both killing mechanisms are equally important and function synergistically to maintain the cytoxic activity of a mouse CTL.