Collaborative effect of Csnk1a1 haploinsufficiency and mutant p53 in Myc induction can promote leukemic transformation.

ABSTRACT: It is still not fully understood how genetic haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) contributes to malignant transformation of hematopoietic stem cells. We asked how compound haploinsufficiency for Csnk1a1 and Egr1 in the common deleted region on chromosome 5 affects hematopoietic stem cells. Additionally, Trp53 was disrupted as the most frequently comutated gene in del(5q) MDS using CRISPR/Cas9 editing in hematopoietic progenitors of wild-type (WT), Csnk1a1-/+, Egr1-/+, Csnk1a1/Egr1-/+ mice. A transplantable acute leukemia only developed in the Csnk1a1-/+Trp53-edited recipient. Isolated blasts were indefinitely cultured ex vivo and gave rise to leukemia after transplantation, providing a tool to study disease mechanisms or perform drug screenings. In a small-scale drug screening, the collaborative effect of Csnk1a1 haploinsufficiency and Trp53 sensitized blasts to the CSNK1 inhibitor A51 relative to WT or Csnk1a1 haploinsufficient cells. In vivo, A51 treatment significantly reduced blast counts in Csnk1a1 haploinsufficient/Trp53 acute leukemias and restored hematopoiesis in the bone marrow. Transcriptomics on blasts and their normal counterparts showed that the derived leukemia was driven by MAPK and Myc upregulation downstream of Csnk1a1 haploinsufficiency cooperating with a downregulated p53 axis. A collaborative effect of Csnk1a1 haploinsufficiency and p53 loss on MAPK and Myc upregulation was confirmed on the protein level. Downregulation of Myc protein expression correlated with efficient elimination of blasts in A51 treatment. The "Myc signature" closely resembled the transcriptional profile of patients with del(5q) MDS with TP53 mutation.

Single-cell analysis of cultured bone marrow stromal cells reveals high similarity to fibroblasts in situ.

Within the heterogenous pool of bone marrow stromal cells, mesenchymal stromal cells (MSCs) are of particular interest because of their hematopoiesis-supporting capacities, contribution to disease progression, therapy resistance, and leukemic initiation. Cultured bone marrow-derived stromal cells (cBMSCs) are used for in vitro modeling of hematopoiesis-stroma interactions, validation of disease mechanisms, and screening for therapeutic targets. Here, we place cBMSCs (mouse and human) in a bone marrow tissue context by systematically comparing the transcriptome of plastic-adherent cells on a single-cell level with in vivo counterparts. Cultured BMSCs encompass a rather homogenous cell population, independent of the isolation method used and, although still possessing hematopoiesis-supporting capacity, are distinct from freshly isolated MSCs and more akin to in vivo fibroblast populations. Informed by combined cell trajectories and pathway analyses, we illustrate that TGFb inhibition in vitro can preserve a more "MSC"-like phenotype.

Genetic barcoding systematically compares genes in del(5q) MDS and reveals a central role for CSNK1A1 in clonal expansion.

How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSCs) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, we performed a systematic comparison on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single-clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1-/+ multipotent progenitors showed a proproliferative gene signature and HSCs showed a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1-/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. We therefore propose a crucial role for Csnk1a1 haploinsufficiency in the selective advantage of 5q-HSCs, implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.

Otoferlin Is Required for Proper Synapse Maturation and for Maintenance of Inner and Outer Hair Cells in Mouse Models for DFNB9.

Deficiency of otoferlin causes profound prelingual deafness in humans and animal models. Here, we closely analyzed developmental deficits and degenerative mechanisms in Otof knock-out (Otof -/-) mice over the course of 48 weeks. We found otoferlin to be required for proper synapse development in the immature rodent cochlea: In absence of otoferlin, synaptic pruning was delayed, and postsynaptic boutons appeared enlarged at 2 weeks of age. At postnatal day 14 (P14), we found on average ∼15 synapses per inner hair cell (IHC) in Otof -/- cochleae as well as in wild-type controls. Further on, the number of synapses in Otof -/- IHCs was reduced to ∼7 at 8 weeks of age and to ∼6 at 48 weeks of age. In the same period, the number of spiral ganglion neurons (SGNs) declined in Otof -/- animals. Importantly, we found an age-progressive loss of IHCs to an overall number of 75% of wildtype IHCs. The IHC loss more prominently but not exclusively affected the basal aspects of the cochlea. For outer hair cells (OHCs), we observed slightly accelerated age-dependent degeneration from base to apex. This was associated with a progressive decay in DPOAE amplitudes for high frequency stimuli, which could first be observed at the age of 24 weeks in Otof -/- mice. Our data will help to plan and predict the outcome of a gene therapy applied at various ages of DFNB9 patients.

Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis.

Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.

First-In-Class CD13-Targeted Tissue Factor tTF-NGR in Patients with Recurrent or Refractory Malignant Tumors: Results of a Phase I Dose-Escalation Study.

BACKGROUND: Aminopeptidase N (CD13) is present on tumor vasculature cells and some tumor cells. Truncated tissue factor (tTF) with a C-terminal NGR-peptide (tTF-NGR) binds to CD13 and causes tumor vascular thrombosis with infarction. METHODS: We treated 17 patients with advanced cancer beyond standard therapies in a phase I study with tTF-NGR (1-h infusion, central venous access, 5 consecutive days, and rest periods of 2 weeks). The study allowed intraindividual dose escalations between cycles and established Maximum Tolerated Dose (MTD) and Dose-Limiting Toxicity (DLT) by verification cohorts. RESULTS: MTD was 3 mg/m2 tTF-NGR/day × 5, q day 22. DLT was an isolated and reversible elevation of high sensitivity (hs) Troponin T hs without clinical sequelae. Three thromboembolic events (grade 2), tTF-NGR-related besides other relevant risk factors, were reversible upon anticoagulation. Imaging by contrast-enhanced ultrasound (CEUS) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) showed major tumor-specific reduction of blood flow in all measurable lesions as proof of principle for the mode of action of tTF-NGR. There were no responses as defined by Response Evaluation Criteria in Solid Tumors (RECIST), although some lesions showed intratumoral hemorrhage and necrosis after tTF-NGR application. Pharmacokinetic analysis showed a t1/2(terminal) of 8 to 9 h without accumulation in daily administrations. CONCLUSION: tTF-NGR is safely applicable with this regimen. Imaging showed selective reduction of tumor blood flow and intratumoral hemorrhage and necrosis.

Rps14, Csnk1a1 and miRNA145/miRNA146a deficiency cooperate in the clinical phenotype and activation of the innate immune system in the 5q- syndrome.

RPS14, CSNK1A1, and miR-145 are universally co-deleted in the 5q- syndrome, but mouse models of each gene deficiency recapitulate only a subset of the composite clinical features. We analyzed the combinatorial effect of haploinsufficiency for Rps14, Csnk1a1, and miRNA-145, using mice with genetically engineered, conditional heterozygous inactivation of Rps14 and Csnk1a1 and stable knockdown of miR-145/miR-146a. Combined Rps14/Csnk1a1/miR-145/146a deficiency recapitulated the cardinal features of the 5q- syndrome, including (1) more severe anemia with faster kinetics than Rps14 haploinsufficiency alone and (2) pathognomonic megakaryocyte morphology. Macrophages, regulatory cells of erythropoiesis and the innate immune response, were significantly increased in Rps14/Csnk1a1/miR-145/146a deficient mice as well as in 5q- syndrome patient bone marrows and showed activation of the innate immune response, reflected by increased expression of S100A8, and decreased phagocytic function. We demonstrate that Rps14/Csnk1a1/miR-145 and miR-146a deficient macrophages alter the microenvironment and induce S100A8 expression in the mesenchymal stem cell niche. The increased S100A8 expression in the mesenchymal niche was confirmed in 5q- syndrome patients. These data indicate that intrinsic defects of the 5q- syndrome hematopoietic stem cell directly alter the surrounding microenvironment, which in turn affects hematopoiesis as an extrinsic mechanism.

Aminopeptidase N (CD13): Expression, Prognostic Impact, and Use as Therapeutic Target for Tissue Factor Induced Tumor Vascular Infarction in Soft Tissue Sarcoma.

Aminopeptidase N (CD13) is expressed on tumor vasculature and tumor cells. It represents a candidate for targeted therapy, e.g., by truncated tissue factor (tTF)-NGR, binding to CD13, and causing tumor vascular thrombosis. We analyzed CD13 expression by immunohistochemistry in 97 patients with STS who were treated by wide resection and uniform chemo-radio-chemotherapy. Using a semiquantitative score with four intensity levels, CD13 was expressed by tumor vasculature, or tumor cells, or both (composite value, intensity scores 1-3) in 93.9% of the STS. In 49.5% tumor cells, in 48.5% vascular/perivascular cells, and in 58.8%, composite value showed strong intensity score 3 staining. Leiomyosarcoma and synovial sarcoma showed low expression; fibrosarcoma and undifferentiated pleomorphic sarcoma showed high expression. We found a significant prognostic impact of CD13, as high expression in tumor cells or vascular/perivascular cells correlated with better relapse-free survival and overall survival. CD13 retained prognostic significance in multivariable analyses. Systemic tTF-NGR resulted in significant growth reduction of CD13-positive human HT1080 sarcoma cell line xenografts. Our results recommend further investigation of tTF-NGR in STS patients. CD13 might be a suitable predictive biomarker for patient selection.