Rebekka K. Schneider

(R)-2-Hydroxyglutarate Is Sufficient to Promote Leukemogenesis and Its Effects Are Reversible

Focusing on the Right Metabolite A variety of human cancers, including acute leukemias and brain tumors, have mutations in the genes encoding isocitrate dehydrogenase 1 or 2 (IDH1, IDH2), which cause overproduction of a metabolite called 2-hydroxyglutarate (2HG). Losman et al. (p. 1621, published online 7 February) show that the R- but not the S-enantiomer of 2HG can transform cells and that R-2HG mediates transformation at least in part through effects on protein modifying EglN prolyl hydroxylases. Importantly, the transforming activity of R-2HG was reversible, suggesting that therapeutic strategies focusing on inhibition of R-2HG production or inhibition of EglN prolyl hydroxylases merit further investigation. A metabolite specific to certain cancers, and of therapeutic interest, exists in two forms, only one of which is oncogenic. Mutations in IDH1 and IDH2, the genes coding for isocitrate dehydrogenases 1 and 2, are common in several human cancers, including leukemias, and result in overproduction of the (R)-enantiomer of 2-hydroxyglutarate [(R)-2HG]. Elucidation of the role of IDH mutations and (R)-2HG in leukemogenesis has been hampered by a lack of appropriate cell-based models. Here, we show that a canonical IDH1 mutant, IDH1 R132H, promotes cytokine independence and blocks differentiation in hematopoietic cells. These effects can be recapitulated by (R)-2HG, but not (S)-2HG, despite the fact that (S)-2HG more potently inhibits enzymes, such as the 5′-methylcytosine hydroxylase TET2, that have previously been linked to the pathogenesis of IDH mutant tumors. We provide evidence that this paradox relates to the ability of (S)-2HG, but not (R)-2HG, to inhibit the EglN prolyl hydroxylases. Additionally, we show that transformation by (R)-2HG is reversible.

Perivascular Gli1+ Progenitors Are Key Contributors to Injury-Induced Organ Fibrosis

Mesenchymal stem cells (MSCs) reside in the perivascular niche of many organs, including kidney, lung, liver, and heart, although their roles in these tissues are poorly understood. Here, we demonstrate that Gli1 marks perivascular MSC-like cells that substantially contribute to organ fibrosis. In vitro, Gli1(+) cells express typical MSC markers, exhibit trilineage differentiation capacity, and possess colony-forming activity, despite constituting a small fraction of the platelet-derived growth factor-β (PDGFRβ)(+) cell population. Genetic lineage tracing analysis demonstrates that tissue-resident, but not circulating, Gli1(+) cells proliferate after kidney, lung, liver, or heart injury to generate myofibroblasts. Genetic ablation of these cells substantially ameliorates kidney and heart fibrosis and preserves ejection fraction in a model of induced heart failure. These findings implicate perivascular Gli1(+) MSC-like cells as a major cellular origin of organ fibrosis and demonstrate that these cells may be a relevant therapeutic target to prevent solid organ dysfunction after injury.

Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS.

Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4CRBN), resulting in CK1α degradation. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. We found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degradation of CK1α. We further demonstrate that minor side chain modifications in thalidomide and a novel analogue, CC-122, can modulate the spectrum of substrates targeted by CRL4CRBN. These findings have implications for the clinical activity of lenalidomide and related compounds, and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.

The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds.

Adult human mesenchymal stem cells from bone marrow (BM-MSC) represent a promising source for skeletal regeneration. Perinatal MSC from Whartons Jelly of the umbilical cord (UC-MSC) are expected to possess enhanced differentiation capacities due to partial expression of pluripotency markers. For bone tissue engineering, it is important to analyse in vitro behaviour of stem cell/biomaterial hybrids concerning in vivo integration into injured tissue via migration, matrix remodelling and differentiation. This study compares the cell-mediated remodelling of three-dimensional collagen I/III gels during osteogenic differentiation of both cell types. When activated through collagen contact and subjected to osteogenic differentiation, UC-MSC differ from BM-MSC in expression and synthesis of extracellular matrix (ECM) proteins as shown by histology, immunohistochemistry, Western Blot analysis and realtime-RT-PCR. The biosynthetic activity was accompanied in both cell types by the ultrastructural appearance of hydroxyapatite/calcium crystals and osteogenic gene induction. Following secretion of matrix metalloproteinases (MMP), both MSC types migrated into and colonised the collagenous matrix causing matrix strengthening and contraction. These results indicate that UC-MSC and BM-MSC display all features needed for effective bone fracture healing. The expression of ECM differs in both cell types considerably, suggesting different mechanisms for bone formation and significant impact for bone tissue engineering.

Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells

Both intrinsic cell state changes and variations in the composition of stem cell populations have been implicated as contributors to aging. We used single-cell RNA-seq to dissect variability in hematopoietic stem cell (HSC) and hematopoietic progenitor cell populations from young and old mice from two strains. We found that cell cycle dominates the variability within each population and that there is a lower frequency of cells in the G1 phase among old compared with young long-term HSCs, suggesting that they traverse through G1 faster. Moreover, transcriptional changes in HSCs during aging are inversely related to those upon HSC differentiation, such that old short-term (ST) HSCs resemble young long-term (LT-HSCs), suggesting that they exist in a less differentiated state. Our results indicate both compositional changes and intrinsic, population-wide changes with age and are consistent with a model where a relationship between cell cycle progression and self-renewal versus differentiation of HSCs is affected by aging and may contribute to the functional decline of old HSCs.

3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche.

Here, we propose a collagen-based three-dimensional (3D) environment for hematopoietic stem and progenitor cells (HPC) with mesenchymal stem cells (MSC) derived either from bone marrow (BM) or umbilical cord (UC), to recapitulate the main components of the BM niche. Mechanisms described for HPC homeostasis were systematically analyzed in comparison to the conventional liquid HPC culture. The 3D-cultivation allows dissecting two sub-populations of HPC: (I) HPC in suspension above the collagen gel and (II) migratory HPC in the collagen fibres of the collagen gel. The different sites represent distinct microenvironments with significant impact on HPC fate. HPC in niche I (suspension) are proliferative and a dynamic culture containing HPC (CD34(+)/CD38(-)), maturing myeloid cells (CD38(+), CD13(+), CAE(+)) and natural killer (NK) cells (CD56(+)). In contrast, HPC in niche II showed clonal growth with significant high levels of the primitive CD34(+)/CD38(-) phenotype with starting myeloid (CD13(+), CAE(+)) differentiation, resembling the endosteal part of the BM niche. In contrast, UC-MSC are not adequate for HSC expansion as they significantly enhance HPC proliferation and lineage commitment. In conclusion, the 3D-culture system using collagen and BM-MSC enables HPC expansion and provides a potential platform to dissect regulatory mechanisms in hematopoiesis.

Cord blood-hematopoietic stem cell expansion in 3D fibrin scaffolds with stromal support

Expansion of multipotent, undifferentiated and proliferating cord blood (CB)-hematopoietic stem cells (HSC) in vitro is limited and insufficient. Bone marrow (BM) engineering in vitro allows mimicking the main components of the hematopoietic niche compared to conventional expansion strategies. In this study, four different 3D biomaterial scaffolds (PCL, PLGA, fibrin and collagen) were tested for freshly isolated cord blood (CB)-CD34(+) cell expansion in presence of (i) efficient exogenous cytokine supplementation and (ii) umbilical cord (UC)-mesenchymal stem cells (MSC). Cell morphology, growth and proliferation were analyzed in vitro as well as multi-organ engraftment and multilineage differentiation in a murine transplantation model. All scaffolds, except 3D PLGA meshes, supported CB-CD34(+) cell expansion, which was additionally stimulated by UC-MSC support. CB-CD34(+) cells cultured on human-derived 3D fibrin scaffolds with UC-MSC support i) reached the highest overall growth (5 × 10(8)-fold expansion of total nuclear cells after fourteen days and 3 × 10(7)-fold expansion of CD34(+) cells after seven days, p < 0.001), ii) maintained a more primitive immunophenotype for more cell divisions, iii) exhibited superior morphological, migratory and adhesive properties, and iv) showed the significantly highest numbers of engraftment and multilineage differentiation (CD45, CD34, CD13, CD3 and CD19) in BM, spleen and peripheral blood in long-term transplanted NSG mice compared to the other 3D biomaterial scaffolds. Thus, the 3D fibrin scaffold based BM-mimicry strategy reveals optimal requirements for translation into clinical protocols for CB expansion and transplantation.

Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS.

The casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage, whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the nondeleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target.

Human adult germline stem cells in question

Arising from: S. Conrad et al. 456, 344–349 (2008)10.1038/nature07404; Conrad et al. replyConrad et al. have generated human adult germline stem cells (haGSCs) from human testicular tissue, which they claim have similar pluripotent properties to human embryonic stem cells (hESCs). Here we investigate the pluripotency of haGSCs by using global gene-expression analysis based on their gene array data and comparing the expression of pluripotency marker genes in haGSCs and hESCs, and in haGSCs and human fibroblast samples derived from different laboratories, including our own. We find that haGSCs and fibroblasts have a similar gene-expression profile, but that haGSCs and hESCs do not. The pluripotency of Conrad and colleagues’ haGSCs is therefore called into question.

Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation

UNLABELLED Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN), but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of patients with CALR-mutant MPN. We further show that the thrombopoietin receptor MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN. SIGNIFICANCE The mechanism by which CALR mutations induce MPN remains unknown. In this report, we show that the positive charge of the CALR mutant C-terminus is necessary to transform hematopoietic cells by enabling binding between mutant CALR and the thrombopoietin receptor MPL.