Siddhartha Mukherjee

Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia

Mesenchymal cells contribute to the ‘stroma’ of most normal and malignant tissues, with specific mesenchymal cells participating in the regulatory niches of stem cells. By examining how mesenchymal osteolineage cells modulate haematopoiesis, here we show that deletion of Dicer1 specifically in mouse osteoprogenitors, but not in mature osteoblasts, disrupts the integrity of haematopoiesis. Myelodysplasia resulted and acute myelogenous leukaemia emerged that had acquired several genetic abnormalities while having intact Dicer1. Examining gene expression altered in osteoprogenitors as a result of Dicer1 deletion showed reduced expression of Sbds, the gene mutated in Schwachman–Bodian–Diamond syndrome—a human bone marrow failure and leukaemia pre-disposition condition. Deletion of Sbds in mouse osteoprogenitors induced bone marrow dysfunction with myelodysplasia. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced oncogenesis.

Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts

Cells of the osteoblast lineage affect the homing and the number of long-term repopulating haematopoietic stem cells, haematopoietic stem cell mobilization and lineage determination and B cell lymphopoiesis. Osteoblasts were recently implicated in pre-leukaemic conditions in mice. However, a single genetic change in osteoblasts that can induce leukaemogenesis has not been shown. Here we show that an activating mutation of β-catenin in mouse osteoblasts alters the differentiation potential of myeloid and lymphoid progenitors leading to development of acute myeloid leukaemia with common chromosomal aberrations and cell autonomous progression. Activated β-catenin stimulates expression of the Notch ligand jagged 1 in osteoblasts. Subsequent activation of Notch signalling in haematopoietic stem cell progenitors induces the malignant changes. Genetic or pharmacological inhibition of Notch signalling ameliorates acute myeloid leukaemia and demonstrates the pathogenic role of the Notch pathway. In 38% of patients with myelodysplastic syndromes or acute myeloid leukaemia, increased β-catenin signalling and nuclear accumulation was identified in osteoblasts and these patients showed increased Notch signalling in haematopoietic cells. These findings demonstrate that genetic alterations in osteoblasts can induce acute myeloid leukaemia, identify molecular signals leading to this transformation and suggest a potential novel pharmacotherapeutic approach to acute myeloid leukaemia.

Gremlin 1 Identifies a Skeletal Stem Cell with Bone, Cartilage, and Reticular Stromal Potential

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Pharmacologic targeting of a stem/progenitor population in vivo is associated with enhanced bone regeneration in mice.

Drug targeting of adult stem cells has been proposed as a strategy for regenerative medicine, but very few drugs are known to target stem cell populations in vivo. Mesenchymal stem/progenitor cells (MSCs) are a multipotent population of cells that can differentiate into muscle, bone, fat, and other cell types in context-specific manners. Bortezomib (Bzb) is a clinically available proteasome inhibitor used in the treatment of multiple myeloma. Here, we show that Bzb induces MSCs to preferentially undergo osteoblastic differentiation, in part by modulation of the bone-specifying transcription factor runt-related transcription factor 2 (Runx-2) in mice. Mice implanted with MSCs showed increased ectopic ossicle and bone formation when recipients received low doses of Bzb. Furthermore, this treatment increased bone formation and rescued bone loss in a mouse model of osteoporosis. Thus, we show that a tissue-resident adult stem cell population in vivo can be pharmacologically modified to promote a regenerative function in adult animals.

Activin A promotes multiple myeloma-induced osteolysis and is a promising target for myeloma bone disease

Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-β family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated JNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distal-less homeobox–5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials.

Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage.

Mutation of the RB-1 and p53 tumor suppressors is associated with the development of human osteosarcoma. With the goal of generating a mouse model of this disease, we used conditional and transgenic mouse strains to inactivate Rb and/or p53 specifically in osteoblast precursors. The resulting Rb;p53 double mutant (DKO) animals are viable but develop early onset osteosarcomas with complete penetrance. These tumors display many of the characteristics of human osteosarcomas, including being highly metastatic. We established cell lines from the DKO osteosarcomas to further investigate their properties. These immortalized cell lines are highly proliferative and they retain their tumorigenic potential, as judged by their ability to form metastatic tumors in immunocompromised mice. Moreover, they can be induced to differentiate and, depending on the inductive signal, will adopt either the osteogenic or adipogenic fate. Consistent with this multipotency, a significant portion of these tumor cells express Sca-1, a marker that is typically associated with stem cells/uncommitted progenitors. By assaying sorted cells in transplant assays, we demonstrate that the tumorigenicity of the osteosarcoma cell lines correlates with the presence of the Sca-1 marker. Finally, we show that loss of Rb and p53 in Sca-1-positive mesenchymal stem/progenitor cells is sufficient to yield transformed cells that can initiate osteosarcoma formation in vivo.

Dual inhibition of akt/mammalian target of rapamycin pathway by nanoparticle albumin-bound-rapamycin and perifosine induces antitumor activity in multiple myeloma.

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway mediates multiple myeloma (MM) cell proliferation, survival, and development of drug resistance, underscoring the role of mTOR inhibitors, such as rapamycin, with potential anti-MM activity. However, recent data show a positive feedback loop from mTOR/S6K1 to Akt, whereby Akt activation confers resistance to mTOR inhibitors. We confirmed that suppression of mTOR signaling in MM cells by rapamycin was associated with upregulation of Akt phosphorylation. We hypothesized that inhibiting this positive feedback by a potent Akt inhibitor perifosine would augment rapamycin-induced cytotoxicity in MM cells. Perifosine inhibited rapamycin-induced phosphorylated Akt, resulting in enhanced cytotoxicity in MM.1S cells even in the presence of interleukin-6, insulin-like growth factor-I, or bone marrow stromal cells. Moreover, rapamycin-induced autophagy in MM.1S MM cells, as evidenced by electron microscopy and immunocytochemistry, was augmented by perifosine. Combination therapy increased apoptosis detected by Annexin V/propidium iodide analysis and caspase/poly(ADP-ribose) polymerase cleavage. Importantly, in vivo antitumor activity and prolongation of survival in a MM mouse xenograft model after treatment was enhanced with combination of nanoparticle albumin-bound–rapamycin and perifosine. Utilizing the in silico predictive analysis, we confirmed our experimental findings of this drug combination on PI3K, Akt, mTOR kinases, and the caspases. Our data suggest that mutual suppression of the PI3K/Akt/mTOR pathway by rapamycin and perifosine combination induces synergistic MM cell cytotoxicity, providing the rationale for clinical trials in patients with relapsed/refractory MM. Mol Cancer Ther; 9(4); 963–75. ©2010 AACR.

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

Significance Mutations in genes encoding proteins that function in splicing of mRNA precursors occur frequently in myelodysplastic syndromes (MDS) and certain leukemias. However, the mechanism by which the mutated splicing factors function has begun to be elucidated only recently. Here we use genome-editing techniques to introduce a common MDS mutation in the gene Serine/arginine-rich splicing factor 2 (SRSF2), which encodes an RNA-binding splicing regulator, in cultured blood cells. We show that splicing of several hundred transcripts, including some with possible relevance to disease, is altered. We further show that mutant SRSF2 is sufficient to induce these changes and does so by binding to RNA sequence elements in the misregulated mRNAs with altered specificity. Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein–protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

High-Dose Zoledronic Acid Impacts Bone Remodeling with Effects on Osteoblastic Lineage and Bone Mechanical Properties

Purpose: The increasing incidence of osteonecrosis of the jaw and its possible association with high cumulative doses of bisphosphonate led us to study the effects of high doses of zoledronic acid (ZA) on bone remodeling. Experimental Design: Five-week-old C57BL6 mice were treated with saline or ZA weekly for 3 weeks at increasing doses (0.05-1 mg/Kg). Effects of ZA on bone remodeling were studied using standard assays. Results: We observed an increase in bone mineral density and content in treated animals at doses of 0.05 mg/Kg, which was not further enhanced at higher doses of ZA. Trabecular bone volume at the proximal tibia and the distal femur assessed by histomorphometry and microCT, respectively, increased significantly in ZA-treated groups. There was however no difference between 0.5 and 1 mg/kg, suggesting a ceiling effect for ZA. ZA led to decreased numbers of osteoclasts and osteoblasts per bone perimeter that paralleled a significant reduction of serum levels of TRAC5b and osteocalcin in vivo. Effects on osteoblasts were confirmed in in vitro assays. Mechanical testing of the femur showed increased brittleness in ZA-treated mice. Conclusions: High doses of ZA inhibit both osteoclast and osteoblasts function and bone remodeling in vivo interfering with bone mechanical properties. No dose response was noted beyond 0.5 mg/kg suggesting that lower doses of ZA may be adequate in inhibiting bone resorption. Our data may help inform future studies of ZA use with respect to alternate and lower doses in the treatment of patients with cancer bone disease. (Clin Cancer Res 2009;15(18):5829–39)