DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escape mismatch repair. Moreover, doublenucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old. microbial evolution | horizontal gene transfer | DNA degradation | early life | anachronistic evolution

It is unknown, why only a minority of chronic myeloid leukemia (CML) patients sustains treatment free remission (TFR) after discontinuation of tyrosine kinase inhibitor (TKI) therapy in deep molecular remission (MR). Here we studied, whether expression of the T-cell inhibitory receptor (CTLA-4)-ligand CD86 (B7.2) on plasmacytoid dendritic cells (pDC) affects relapse risk after TKI cessation. CML patients in MR displayed significantly higher CD86pDC frequencies than normal donors (P<0.0024), whereas TFR patients had consistently low CD86pDC (n=12). This suggested that low CD86pDC might be predictive of TFR. Indeed, in a prospective analysis of 122 patients discontinuing their TKI within the EURO-SKI trial, the one-year relapse-free survival (RFS) was 30.1% (95% CI 15.6-47.9) for patients with >95 CD86pDC per 10 lymphocytes, but 70.0% (95% CI 59.3-78.3) for patients with <95 CD86pDC (hazard ratio (HR) 3.4, 95%CI: 1.9-6.0; P<0.0001). Moreover, only patients with <95 CD86pDC derived a significant benefit from longer (>8 years) TKI exposure before discontinuation (HR 0.3, 95% CI 0.1-0.8; P=0.0263). High CD86pDC counts significantly correlated with leukemia-specific CD8 Tcell exhaustion (Spearman correlation: 0.74, 95%-CI: 0.21-0.92; P=0.0098). Our data demonstrate that CML patients with high CD86pDC counts have a higher risk of relapse after TKI discontinuation.

Despite the curative potential of allogeneic stem cell transplantation (SCT), therapeutic options for patients relapsing after transplantation are limited and the prognosis is poor.

A minority of chronic myeloid leukemia (CML) patients is capable of successfully discontinuing imatinib. Treatment modalities to increase this proportion are currently unknown. Here, we assessed the role of interferon alpha 2a (IFN) on therapy discontinuation in a previously reported cohort of 20 chronic phase CML patients who were treated upfront with IFN alpha plus imatinib followed by IFN monotherapy to maintain cytogenetic or molecular remission (MR) after imatinib discontinuation. After a median follow-up of 7.9 years (range, 5.2-12.2), relapse-free survival was 73% (8/11 patients) and 84% (5/6 patients) for patients who discontinued imatinib in major MR (MMR) and MR4/MR4.5, respectively. Ten patients discontinued IFN after a median of 4.5 years (range, 0.24-9.3). After a median of 2.8 years (range, 0.7-5.1), nine of them remain in ongoing treatment-free remission with MR5 (n=6) and MR4.5 (n=3). The four patients who still administer IFN are in stable MR5, MR4.5, MR4, and MMR, respectively. In conclusion, an IFN/imatinib induction treatment followed by a temporary IFN maintenance therapy may enable a high rate of treatment discontinuation in CML patients in at least MMR when stopping imatinib.

Internal tandem duplications (ITD) in the Fms-related tyrosine kinase 3 receptor (FLT3) are associated with a dismal prognosis in acute myeloid leukemia (AML). FLT3 inhibitors such as sorafenib may improve outcome, but only few patients display long-term responses, prompting the search for underlying resistance mechanisms and therapeutic strategies to overcome them. Here we identified that the nuclear factor of activated T cells, NFATc1, is frequently overexpressed in FLT3-ITD-positive (FLT3-ITD+) AML. NFATc1 knockdown using inducible short hairpin RNA or pharmacological NFAT inhibition with cyclosporine A (CsA) or VIVIT significantly augmented sorafenib-induced apoptosis of FLT3-ITD+ cells. CsA also potently overcame sorafenib resistance in FLT3-ITD+ cell lines and primary AML. Vice versa, de novo expression of a constitutively nuclear NFATc1-mutant mediated instant and robust sorafenib resistance in vitro. Intriguingly, FLT3-ITD+ AML patients (n=26) who received CsA as part of their rescue chemotherapy displayed a superior outcome when compared with wild-type FLT3 (FLT3-WT) AML patients. Our data unveil NFATc1 as a novel mediator of sorafenib resistance in FLT3-ITD+ AML. CsA counteracts sorafenib resistance and may improve treatment outcome in AML by means of inhibiting NFAT.

Plasmacytoid dendritic cells (pDC) are the main producers of a key T-cell-stimulatory cytokine, IFNα, and critical regulators of antiviral immunity. Chronic myeloid leukemia (CML) is caused by BCR-ABL, which is an oncogenic tyrosine kinase that can be effectively inhibited with ABL-selective tyrosine kinase inhibitors (TKI). BCR-ABL-induced suppression of the transcription factor interferon regulatory factor 8 was previously proposed to block pDC development and compromise immune surveillance in CML. Here, we demonstrate that pDCs in newly diagnosed CML (CML-pDC) develop quantitatively normal and are frequently positive for the costimulatory antigen CD86. They originate from low-level -expressing precursors. CML-pDCs also retain their competence to maturate and to secrete IFN. RNA sequencing reveals a strong inflammatory gene expression signature in CML-pDCs. Patients with high CML-pDC counts at diagnosis achieve inferior rates of deep molecular remission (MR) under nilotinib, unless nilotinib therapy is combined with IFN, which strongly suppresses circulating pDC counts. Although most pDCs are-negative in MR, a substantial proportion of CML-pDCs persists under TKI treatment. This could be of relevance, because CML-pDCs elicit CD8 T cells, which protect wild-type mice from CML. Together, pDCs are identified as novel functional DC population in CML, regulating antileukemic immunity and treatment outcome in CML. CML-pDC originates from low-level BCR-ABL expressing stem cells into a functional immunogenic DC-population regulating antileukemic immunity and treatment outcome in CML. .

Background Chronic myeloid leukemia (CML) stem cells are inherently insensitive to tyrosine-kinase inhibitors (TKI). However, an important minority of CML patients was shown to discontinue TKI without experiencing molecular relapse. Underlying mechanisms are currently unknown. Plasmacytoid dendritic cells (pDCs) are critical regulators of immune responses. Following activation, pDC upregulate MHC-class II and other DC activation markers such as CD86 (also known as B7.2). CD86 is a co-stimulatory molecule during T-cell activation, but also ligand of the inhibitory immune checkpoint receptor CTLA-4, which counteracts T-cell activation. The origin and function of pDC in CML biology is unknown. Within a sub-study of the EUROSKI TKI discontinuation trial we prospectively tested the hypothesis that pDC counts and CD86 expression status govern relapse risk following TKI discontinuation. Methods: Using flow cytometry, cell sorting and fluorescence in situ hybridization (FISH), CD86 expression and BCR-ABL status were analyzed in PDCA-2+/CD123+ peripheral blood (pB) pDC of untreated CML patients (CML pDC), normal donors and 123 patients, who had stopped TKI therapy in deep molecular remission within the international EUROSKI study (EUDRACT 2011-000440-22). All 123 EUROSKI patients had given written informed consent to participate in the immunological sub-study of the EUROSKI trial. Fresh samples from 19 EUROSKI centers in Germany were centrally analyzed prior, as well as 1, 2, 3 and 6 months after TKI discontinuation. PB CD86+ pDC counts were calculated per 105 cells in the lymphocyte gate. Decision trees and 10-fold cross validation were employed to establish relapse prediction accuracy for this value. Results CML pDC were BCR-ABL-FISH positive (median: 81%; range, 57 to 100%). In contrast, the proportion of CD86+ CML pDC varied substantially (median: 25.9%, range 3.2% to 82.4%), suggesting that CD86 expression on CML pDC was not a direct consequence of oncogenic BCR-ABL signaling. This was confirmed experimentally in a murine CML model. In contrast to CML pDC, remission pDC were always BCR-ABL FISH negative (n=10), but still displayed a comparable high proportion of CD86 positive pDC (median: 21%; range, 2.2% to 62%). In contrast, normal donor pDC were rarely CD86 positive (median: 6.8%; range, 4.2% to 17%), reinforcing the aberrant, and BCR-ABL-independent nature of CD86 expression on CML and remission pDC. As a result, healthy donors displayed only between 26 to 84 CD86+ pDC per 105 lymphocytes, whereas EUROSKI remission patients exhibited between 6 to 309 CD86+ pDC per 105 lymphocytes. Based on the important role of CD86 as a high affinity ligand of the inhibitory immune checkpoint receptor CTLA-4, we next asked, whether CD86+ pDC counts are associated with relapse risk after TKI discontinuation. Strikingly, statistical models suggested that a CD86+ pDC count below or above 95 CD86+ pDC/105 lymphocytes optimally separated two relapse categories of EUROSKI patients. Whereas relapse free survival (RFS) (loss of MMR) for patients with more than 95 CD86+ pDC/105 lymphocytes was 30% (n=32), RFS was 69% for patients (n=91) with less than 95 CD86+ pDC/105 lymphocytes (p<0.0001) (Figure 1). Patients with more than 95 CD86+ pDC/105 lymphocytes also displayed significantly more PD1+ (exhausted) CD8+ T-cells (p=0.03) than patients with less than 95 CD86+ pDC/105 lymphocytes. This result supports the notion that chronically higher CD86+ pDC counts trigger a T-cell exhaustion phenotype and thus inhibit anti-CML immune responses upon TKI discontinuation. Conclusion: Our data suggest for the first time a mechanism that governs relapse biology after TKI discontinuation in CML. It is proposed that a chronic engagement of the T-cell inhibitory immune checkpoint receptor CTLA-4 by abundant levels of CD86+ on remission pDC impedes a T-cell-dependent control of CML stem cell survival after stopping TKI. CTLA-4 blocking antibodies such as ipilimumab might therefore prevent molecular relapse and overcome stem cell persistence in CML especially in patients with high CD86+ pDC counts. Figure 1. Relapse free survival after TKI discontinuation according to the number of CD86+ pDC at the time of stopping TKI in the EURO-SKI trial. Figure 1. Relapse free survival after TKI discontinuation according to the number of CD86+ pDC at the time of stopping TKI in the EURO-SKI trial. Disclosures Burchert: Bristol Myers Squibb: Honoraria. Saussele:Novartis Pharma: Honoraria, Other: Travel grant, Research Funding; BMS: Honoraria, Other: Travel grant, Research Funding; Pfizer: Honoraria, Other: Travel grant; ARIAD: Honoraria. Müller:Ariad: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding. Lange:Novartis: Research Funding. Hochhaus:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mahon:NOVARTIS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy, Honoraria.

Cloning and characterization of a novel druggable fusion kinase in acute myeloid leukemia.Rearrangements of the genes for the platelet derived growth factor receptor alpha (PDGFR(alpha)) or the platelet derived growth factor receptor beta (PDGFR(beta)) have been identified in myeloid/lymphoid neoplasms with eosinophilia 1 . Fusions of PDGFR(beta), which encodes a type III receptor tyrosine kinase result in constitutive kinase activation and promotion of survival, proliferation and cell migration 2 . Since the description of ETV6-PDGFR(beta) in 1994 3 , more than 30 PDGFR(beta) fusion partners have been described, most of which have been identified in myeloproliferative neoplasms or myelodysplastic syndrome/ myeloproliferative neoplasm overlap, but only rarely in acute myeloid leukemia (AML) 1, 4, 5 .PDGFR(beta) fusions are commonly in-frame mutations that contain an N-terminal fusion partner with dimerization/oligomerization motifs, enabling PDGFR-ligandindependent receptor tyrosine kinase activation 1, 6 . PDGFR(beta)-rearranged chronic myeloproliferative neoplasms show a favorable response to imatinib with a 10-year overall survival rate of 90 % 7 . The zinc finger protein "Zinc finger and BTB (broadcomplex, tramtrack, bric-à-brac) domain containing 11" (ZBTB11) is a transcriptional repressor, which was originally identified as a regulator of metallothionein 2A and recently described as a master regulator of neutrophil development 8 .Here we report on the cloning, biological and biochemical characterization of a novel ZBTB11-PDGFR(beta) fusion gene, which occurred in a therapy-resistant AML relapsing only shortly after allogeneic stem cell transplantation (SCT). A 53-year old female presented in October 2015 with the primary diagnosis of an AML (nucleophosmin (NPM1)-mutated, FMS-like tyrosine kinase 3 (FLT3)-wildtype, DNA (cytosine-5)-methytransferase 3A (DNMT3A)-wildtype, normal karyotype, European Leukemia Net (ELN): favorable risk). The patient received induction chemotherapy and one cycle of consolidation chemotherapy. Due to persistence of NPM1-positive minimal residual disease (MRD), the patient underwent SCT in March 2016. Three months later, the patient relapsed.At the time of relapse, bone marrow cytology showed an increased content of blasts and the appearance of atypical eosinophil precursors (Figure 1 A), which were not present at diagnosis. Cytogenetic analysis revealed a translocation t(3;5)(q12;q32).FISH suggested that one of the fusion partners of this translocation was PDGFR(beta) (Figure 1 B). Targeted resequencing identified ZBTB11 as the 5' fusion partner of PDGFR(beta) (Figure 1C). Sanger sequencing confirmed the ZBTB11-PDGFR(beta) fusion gene (Figure 1D). Breakpoint regions were downstream of exon7 of ZBTB11 and upstream of exon 11 of PDGFR(beta). Thus, the fusion transcript retained the coding sequences for the transmembrane and kinase domains of PDGFR(beta), suggesting that these two important functional domains of PDGFR(beta) were intact in the putative ZBTB11-PDGFR(beta) fusion protein...