Maria Askmyr

Dissociation of bone resorption and bone formation in adult mice with a non-functional V-ATPase in osteoclasts leads to increased bone strength

Osteopetrosis caused by defective acid secretion by the osteoclast, is characterized by defective bone resorption, increased osteoclast numbers, while bone formation is normal or increased. In contrast the bones are of poor quality, despite this uncoupling of formation from resorption. To shed light on the effect of uncoupling in adult mice with respect to bone strength, we transplanted irradiated three-month old normal mice with hematopoietic stem cells from control or oc/oc mice, which have defective acid secretion, and followed them for 12 to 28 weeks. Engraftment levels were assessed by flow cytometry of peripheral blood. Serum samples were collected every six weeks for measurement of bone turnover markers. At termination bones were collected for µCT and mechanical testing. An engraftment level of 98% was obtained. From week 6 until termination bone resorption was significantly reduced, while the osteoclast number was increased when comparing oc/oc to controls. Bone formation was elevated at week 6, normalized at week 12, and reduced onwards. µCT and mechanical analyses of femurs and vertebrae showed increased bone volume and bone strength of cortical and trabecular bone. In conclusion, these data show that attenuation of acid secretion in adult mice leads to uncoupling and improves bone strength.

Identification of ETV6-RUNX1 -like and DUX4 -rearranged subtypes in paediatric B-cell precursor acute lymphoblastic leukaemia

Fusion genes are potent driver mutations in cancer. In this study, we delineate the fusion gene landscape in a consecutive series of 195 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP ALL). Using RNA sequencing, we find in-frame fusion genes in 127 (65%) cases, including 27 novel fusions. We describe a subtype characterized by recurrent IGH-DUX4 or ERG-DUX4 fusions, representing 4% of cases, leading to overexpression of DUX4 and frequently co-occurring with intragenic ERG deletions. Furthermore, we identify a subtype characterized by an ETV6-RUNX1-like gene-expression profile and coexisting ETV6 and IKZF1 alterations. Thus, this study provides a detailed overview of fusion genes in paediatric BCP ALL and adds new pathogenetic insights, which may improve risk stratification and provide therapeutic options for this disease.

Selective killing of candidate AML stem cells by antibody targeting of IL1RAP

IL1RAP, a co-receptor for interleukin (IL)-1 and IL-33 receptors, was previously found to be highly upregulated on candidate chronic myeloid leukemia stem cells, allowing for leukemia-selective killing using IL1RAP-targeting antibodies. We analyzed IL1RAP expression in a consecutive series of 29 patients with acute myeloid leukemia (AML) and, based on the level of expression in mononuclear cells (MNCs), we divided the samples into 3 groups: IL1RAP low (n = 6), IL1RAP intermediate (n = 11), and IL1RAP high (n = 12). Within the CD34+CD38- population, the intermediate and high groups expressed higher levels of IL1RAP than did corresponding normal cells. With the aim to target AML stem cells, an anti-IL1RAP monoclonal antibody was generated followed by isotype switching for improved antibody-dependent, cell-mediated cytotoxicity activity. Using this antibody, we achieved selective killing of AML MNC, CD34+CD38+, and CD34+CD38- cells. Our findings demonstrate that IL1RAP is a promising new therapeutic target in AML.

Towards a better understanding and new therapeutics of osteopetrosis.

Lack of or dysfunction in osteoclasts result in osteopetrosis, a group of rare but often severe, genetic disorders affecting skeletal tissue. Increase in bone mass results in skeletal malformation and bone marrow failure that may be fatal. Many of the underlying defects have lately been characterized in humans and in animal models of the disease. In humans, these defects often involve mutations in genes expressing proteins involved in the acidification of the osteoclast resorption compartment, a process necessary for proper bone degradation. So far, the only cure for children with severe osteopetrosis is allogeneic hematopoietic stem cell (HSC) transplantation but without a matching donor this form of therapy is far from optimal. The characterization of the genetic defects opens up the possibility for gene replacement therapy as an alternative. Accordingly, HSC‐targeted gene therapy in a mouse model of infantile malignant osteopetrosis was recently shown to correct many aspects of the disease.

Antibodies targeting human IL1RAP (IL1R3) show therapeutic effects in xenograft models of acute myeloid leukemia

Significance Acute myeloid leukemia (AML) is a hematologic malignancy with poor survival. Current treatment with chemotherapy does not target the leukemic cells specifically and is associated with severe side effects. Here we demonstrate that antibodies directed at the cell surface molecule IL-1 receptor accessory protein (IL1RAP), expressed on immature AML cells, show strong antileukemic effects in mice transplanted with human AML cells and that the mechanism behind the cell killing is through recruitment of effector cells. Using antibodies against IL1RAP also capable of blocking IL-1 signaling, we show that the proliferation of human AML cells can be inhibited, providing a second mode of action of IL1RAP antibodies. These results provide critical evidence in support of a rapid clinical development of an antibody-based anti-IL1RAP therapy in AML. Acute myeloid leukemia (AML) is associated with a poor survival rate, and there is an urgent need for novel and more efficient therapies, ideally targeting AML stem cells that are essential for maintaining the disease. The interleukin 1 receptor accessory protein (IL1RAP; IL1R3) is expressed on candidate leukemic stem cells in the majority of AML patients, but not on normal hematopoietic stem cells. We show here that monoclonal antibodies targeting IL1RAP have strong antileukemic effects in xenograft models of human AML. We demonstrate that effector-cell–mediated killing is essential for the observed therapeutic effects and that natural killer cells constitute a critical human effector cell type. Because IL-1 signaling is important for the growth of AML cells, we generated an IL1RAP-targeting antibody capable of blocking IL-1 signaling and show that this antibody suppresses the proliferation of primary human AML cells. Hence, IL1RAP can be efficiently targeted with an anti-IL1RAP antibody capable of both achieving antibody-dependent cellular cytotoxicity and blocking of IL-1 signaling as modes of action. Collectively, these results provide important evidence in support of IL1RAP as a target for antibody-based treatment of AML.

Effects of the bone marrow microenvironment on hematopoietic malignancy

The bone marrow (BM) is contained within the bone cavity and is the main site of hematopoiesis, the continuous development of blood cells from immature hematopoietic stem and progenitor cells. The bone marrow consists of developing hematopoietic cells and non-hematopoietic cells, the latter collectively termed the bone marrow microenvironment. These non-hematopoietic cells include cells of the osteoblast lineage, adipocytes and endothelial cells. For many years these bone marrow microenvironment cells were predicted to play active roles in regulating hematopoiesis, and recent studies have confirmed such roles. Importantly, more recent data has indicated that cells of the BM microenvironment may also contribute to hematopoietic diseases. In this review we provide an overview of the roles of the data suggesting that the cells of the bone marrow microenvironment may play an active role in the initiation and progression of hematopoietic malignancy.

Myelosuppressive Therapies Significantly Increase Pro‐Inflammatory Cytokines and Directly Cause Bone Loss

Skeletal‐related events resulting from accelerated bone loss are common complications in patients treated for a range of cancers. However, the mechanisms and rate of bone loss after myelosuppression are unclear. We, therefore, investigated this in mice and humans. We treated mice with different myelosuppressive therapies (chemotherapy or irradiation with or without transplantation) and studied their effects on bone structure. Myelosuppression of mice rapidly caused an increase in bone resorption that was not matched by bone formation. The resultant significant and persistent bone loss early after therapy was associated with increased inflammatory cytokines, in particular, monocyte chemoattractant protein 1 (MCP1). Therapy‐induced bone loss was prevented with a single dose of the bisphosphonate zoledronic acid (ZA), administered before myelosuppression. Importantly, ZA treatment of mice did not impair hematopoiesis, including hematopoietic stem cell function. Furthermore, examination of serum from patients before and after autologous or allogeneic stem cell transplantion (SCT) revealed altered levels of bone turnover markers and elevated inflammatory cytokines. MCP1 levels in serum obtained between days 7 and 14 post‐SCT positively correlated with bone loss observed at 100 days after allogeneic SCT. Similar to that observed in our studies in mice, the bone loss was long term, persisting at 12 months post‐SCT. Furthermore, patients who received chemotherapy less than 100 days before SCT had significantly more bone loss at the hip. In these patients, serum levels of MCP1, but not routine biomarkers of bone turnover, including C‐terminal cross‐linking telopeptide of type‐1 collagen (β‐CTx), positively correlated with their bone loss. Hence, myelosuppressive therapies increase inflammation and directly contribute to bone loss. Administration of an osteoclast inhibitor before the initiation of cancer therapy is likely to have the best outcome in preventing bone loss in patients with cancer.

IL1RAP expression as a measure of leukemic stem cell burden at diagnosis of chronic myeloid leukemia predicts therapy outcome

IL1RAP expression as a measure of leukemic stem cell burden at diagnosis of chronic myeloid leukemia predicts therapy outcome

Low-dose busulphan conditioning and neonatal stem cell transplantation preserves vision and restores hematopoiesis in severe murine osteopetrosis

OBJECTIVE Infantile malignant osteopetrosis is a fatal disease caused by lack of functional osteoclasts. In most of patients, TCIRG1, encoding a subunit of a proton pump essential for bone resorption, is mutated. Osteopetrosis leads to bone marrow failure and blindness due to optic nerve compression. Oc/oc mice have a deletion in Tcirg1 and die around 3 to 4 weeks, but can be rescued by neonatal stem cell transplantation (SCT) after irradiation conditioning. However, as irradiation of neonatal mice results in retinal degeneration, we wanted to investigate whether conditioning with busulphan prior to SCT can lead to preservation of vision and reversal of osteopetrosis in the oc/oc mouse model. MATERIALS AND METHODS Pregnant dams were conditioned with busulphan and their litters transplanted with 1 x 10(6) normal lineage-depleted bone marrow cells intravenously or intraperitoneally. Mice were followed in terms of survival and engraftment level, as well as with peripheral blood lineage analysis, bone and eye histopathology and a visual-tracking drum test to assess vision. RESULTS Busulphan at 15 mg/kg was toxic to oc/oc mice. However, six of seven oc/oc mice conditioned with busulphan 7.5 mg/kg survived past the normal lifespan with 10% engraftment, correction of the skeletal phenotype, and normalization of peripheral blood lineages. Busulphan, in contrast to irradiation, did not have adverse effects on the retina as determined by histopathology, and 8 weeks after transplantation control and oc/oc mice retained their vision. CONCLUSION Low-dose busulphan conditioning and neonatal SCT leads to prolonged survival of oc/oc mice, reverses osteopetrosis and prevents blindness even at low engraftment levels.

Lentiviral gene transfer of TCIRG1 into peripheral blood CD34(+) cells restores osteoclast function in infantile malignant osteopetrosis.

Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by non-functional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to restore the resorptive function of IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA. CD34(+) cells from peripheral blood of five IMO patients and from normal cord blood were transduced with lentiviral vectors expressing TCIRG1 and GFP under a SFFV promoter, expanded in culture and differentiated on bone slices to mature osteoclasts. qPCR analysis and western blot revealed increased mRNA and protein levels of TCIRG1, comparable to controls. Vector corrected IMO osteoclasts generated increased release of Ca(2+) and bone degradation product CTX-I into the media as well as increased formation of resorption pits in the bone slices, while non-corrected IMO osteoclasts failed to resorb bone. Resorption was approximately 70-80% of that of osteoclasts generated from cord blood. Furthermore, transduced CD34(+) cells successfully engrafted in NSG-mice. In conclusion we provide the first evidence of lentiviral-mediated correction of a human genetic disease affecting the osteoclastic lineage.