Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms.

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in patients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis.

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.

Sustained long-term disease correction in a murine model of MPSII following stem cell gene therapy.

Mucopolysaccharidosis type II (MPSII) is a pediatric lysosomal storage disease caused by deficiencies in the IDS (iduronate-2-sulfatase) gene resulting in accumulation of glycosaminoglycans, multisystem disease, and profound neurodegeneration in severe forms. Although enzyme replacement therapy is available for somatic forms of disease, the inability of native IDS to pass the blood-brain barrier renders it ineffective for the brain. We previously demonstrated the short-term efficacy of a brain-targeted hematopoietic stem cell gene therapy approach to treat MPSII mice using lentiviral IDS fused to the blood-brain-barrier-crossing peptide ApoEII (IDS.ApoEII) in comparison with a lentivirus expressing native IDS and an unmanipulated bone marrow transplant. Here we evaluated the longevity of disease correction for 12-16 months following treatment. We observed sustained IDS enzyme activity in organs of long-term IDS.ApoEII-treated MPSII mice, similar to those analyzed 6 months post-treatment, with continued clearance of storage material in the brain and peripheral organs, maintained correction of astrogliosis, microgliosis, and correction of altered cytokines and chemokines. IDS.ApoEII also significantly reduced retinal atrophy, characteristic of MPSII. Overall, IDS.ApoEII resulted in systemic prevention of the MPSII phenotype, with no observed toxicity following treatment. This provides evidence of the sustained efficacy and safety of this treatment ahead of a recently opened clinical trial.

Biological drivers of clinical phenotype in myelofibrosis.

Myelofibrosis (MF) is a myeloproliferative disorder that exhibits considerable biological and clinical heterogeneity. At the two ends of the disease spectrum are the myelodepletive or cytopenic phenotype and the myeloproliferative phenotype. The cytopenic phenotype has a high prevalence in primary MF (PMF) and is characterized by low blood counts. The myeloproliferative phenotype is typically associated with secondary MF (SMF), mild anemia, minimal need for transfusion support, and normal to mild thrombocytopenia. Differences in somatic driver mutations and allelic burden, as well as the acquisition of non-driver mutations further influences these phenotypic differences, prognosis, and response to therapies such as JAK2 inhibitors. The outcome of patients with the cytopenic phenotype are comparatively worse and frequently pose a challenge to treat given the inherent exacerbation of cytopenias. Recent data indicate that an innate immune deregulated state that hinges on the myddosome-IRAK-NFκB axis favors the cytopenic myelofibrosis phenotype and offers opportunity for novel treatment approaches. We will review the biological and clinical features of the MF disease spectrum and associated treatment considerations.

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.

Still a burning question: the interplay between inflammation and fibrosis in myeloproliferative neoplasms.

PURPOSE OF REVIEW: Bone marrow fibrosis is the progressive replacement of blood-forming cells by reticulin fibres, caused by the acquisition of somatic mutations in hematopoietic stem cells. The molecular and cellular mechanisms that drive the progression of bone marrow fibrosis remain unknown, yet chronic inflammation appears to be a conserved feature in most patients suffering from myeloproliferative neoplasms. RECENT FINDINGS: Here, we review recent literature pertaining to the role of inflammation in driving bone marrow fibrosis, and its effect on the various hematopoietic and nonhematopoietic cell populations. SUMMARY: Recent evidence suggests that the pathogenesis of MPN is primarily driven by the hematopoietic stem and progenitor cells, together with their mutated progeny, which in turn results in chronic inflammation that disrupts the bone marrow niche and perpetuates a disease-permissive environment. Emerging data suggests that specifically targeting stromal inflammation in combination with JAK inhibition may be the way forward to better treat MPNs, and bone marrow fibrosis specifically.

Isolation of human bone marrow stromal cells from bone marrow biopsies for single-cell RNA sequencing.

Bone marrow (BM) mesenchymal stromal cells play an important role in regulating stem cell quiescence and homeostasis; they are also key contributors to various hematological malignancies. However, human bone marrow stromal cells are difficult to isolate and prone to damage during isolation. This protocol describes a combination of mechanical and enzymatic isolation of BM stromal cells from human BM biopsies, followed by FACS sorting to separate stromal sub-populations including mesenchymal stromal cells, fibroblasts, and Schwann cells for single-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Leimkühler et al. (2020).

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.

Increased CXCL4 expression in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.

An Improved Adeno-Associated Virus Vector for Neurological Correction of the Mouse Model of Mucopolysaccharidosis IIIA.

Patients with the lysosomal storage disease mucopolysaccharidosis IIIA (MPSIIIA) lack the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), one of the many enzymes involved in degradation of heparan sulfate. Build-up of un-degraded heparan sulfate results in severe progressive neurodegeneration for which there is currently no treatment. Experimental gene therapies based on gene addition are currently being explored. Following preclinical evaluation in MPSIIIA mice, an adeno-associated virus vector of serotype rh10 designed to deliver SGSH and sulfatase modifying factor 1 (SAF301) was trialed in four MPSIIIA patients, showing good tolerance and absence of adverse events with some improvements in neurocognitive measures. This study aimed to improve SAF301 further by removing sulfatase modifying factor 1 (SUMF1) and assessing if expression of this gene is needed to increase the SGSH enzyme activity (SAF301b). Second, the murine phosphoglycerate kinase (PGK) promotor was exchanged with a chicken beta actin/CMV composite (CAG) promotor (SAF302) to see if SGSH expression levels could be boosted further. The three different vectors were administered to MPSIIIA mice via intracranial injection, and SGSH expression levels were compared 4 weeks post treatment. Removal of SUMF1 resulted in marginal reductions in enzyme activity. However, promotor exchange significantly increased the amount of SGSH expressed in the brain, leading to superior therapeutic correction with SAF302. Biodistribution of SAF302 was further assessed using green fluorescent protein (GFP), indicating that vector spread was limited to the area around the injection tract. Further modification of the injection strategy to a single depth with higher injection volume increased vector distribution, leading to more widespread GFP distribution and sustained expression, suggesting this approach should be adopted in future trials.

Macrophage enzyme and reduced inflammation drive brain correction of mucopolysaccharidosis IIIB by stem cell gene therapy.

Mucopolysaccharidosis IIIB is a paediatric lysosomal storage disease caused by deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU), involved in the degradation of the glycosaminoglycan heparan sulphate. Absence of NAGLU leads to accumulation of partially degraded heparan sulphate within lysosomes and the extracellular matrix, giving rise to severe CNS degeneration with progressive cognitive impairment and behavioural problems. There are no therapies. Haematopoietic stem cell transplant shows great efficacy in the related disease mucopolysaccharidosis I, where donor-derived monocytes can transmigrate into the brain following bone marrow engraftment, secrete the missing enzyme and cross-correct neighbouring cells. However, little neurological correction is achieved in patients with mucopolysaccharidosis IIIB. We have therefore developed an ex vivo haematopoietic stem cell gene therapy approach in a mouse model of mucopolysaccharidosis IIIB, using a high-titre lentiviral vector and the myeloid-specific CD11b promoter, driving the expression of NAGLU (LV.NAGLU). To understand the mechanism of correction we also compared this with a poorly secreted version of NAGLU containing a C-terminal fusion to IGFII (LV.NAGLU-IGFII). Mucopolysaccharidosis IIIB haematopoietic stem cells were transduced with vector, transplanted into myeloablated mucopolysaccharidosis IIIB mice and compared at 8 months of age with mice receiving a wild-type transplant. As the disease is characterized by increased inflammation, we also tested the anti-inflammatory steroidal agent prednisolone alone, or in combination with LV.NAGLU, to understand the importance of inflammation on behaviour. NAGLU enzyme was substantially increased in the brain of LV.NAGLU and LV.NAGLU-IGFII-treated mice, with little expression in wild-type bone marrow transplanted mice. LV.NAGLU treatment led to behavioural correction, normalization of heparan sulphate and sulphation patterning, reduced inflammatory cytokine expression and correction of astrocytosis, microgliosis and lysosomal compartment size throughout the brain. The addition of prednisolone improved inflammatory aspects further. Substantial correction of lysosomal storage in neurons and astrocytes was also achieved in LV.NAGLU-IGFII-treated mice, despite limited enzyme secretion from engrafted macrophages in the brain. Interestingly both wild-type bone marrow transplant and prednisolone treatment alone corrected behaviour, despite having little effect on brain neuropathology. This was attributed to a decrease in peripheral inflammatory cytokines. Here we show significant neurological disease correction is achieved using haematopoietic stem cell gene therapy, suggesting this therapy alone or in combination with anti-inflammatories may improve neurological function in patients.

Identification of age-dependent motor and neuropsychological behavioural abnormalities in a mouse model of Mucopolysaccharidosis Type II.

Severe mucopolysaccharidosis type II (MPS II) is a progressive lysosomal storage disease caused by mutations in the IDS gene, leading to a deficiency in the iduronate-2-sulfatase enzyme that is involved in heparan sulphate and dermatan sulphate catabolism. In constitutive form, MPS II is a multi-system disease characterised by progressive neurocognitive decline, severe skeletal abnormalities and hepatosplenomegaly. Although enzyme replacement therapy has been approved for treatment of peripheral organs, no therapy effectively treats the cognitive symptoms of the disease and novel therapies are in development to remediate this. Therapeutic efficacy and subsequent validation can be assessed using a variety of outcome measures that are translatable to clinical practice, such as behavioural measures. We sought to consolidate current knowledge of the cognitive, skeletal and motor abnormalities present in the MPS II mouse model by performing time course behavioural examinations of working memory, anxiety, activity levels, sociability and coordination and balance, up to 8 months of age. Cognitive decline associated with alterations in spatial working memory is detectable at 8 months of age in MPS II mice using spontaneous alternation, together with an altered response to novel environments and anxiolytic behaviour in the open-field. Coordination and balance on the accelerating rotarod were also significantly worse at 8 months, and may be associated with skeletal changes seen in MPS II mice. We demonstrate that the progressive nature of MPS II disease is also seen in the mouse model, and that cognitive and motor differences are detectable at 8 months of age using spontaneous alternation, the accelerating rotarod and the open-field tests. This study establishes neurological, motor and skeletal measures for use in pre-clinical studies to develop therapeutic approaches in MPS II.