Angela Pennisi

The proteasome inhibitor, bortezomib suppresses primary myeloma and stimulates bone formation in myelomatous and nonmyelomatous bones in vivo

Multiple myeloma (MM), a hematologic malignancy of terminally differentiated plasma cells is closely associated with induction of osteolytic bone disease, induced by stimulation of osteoclastogenesis and suppression of osteoblastogenesis. The ubiquitin‐proteasome pathway regulates differentiation of bone cells and MM cell growth. The proteasome inhibitor, bortezomib, is a clinical potent antimyeloma agent. The main goal of this study was to investigate the effect of bortezomib on myeloma‐induced bone resorption and tumor growth in SCID‐rab mice engrafted with MM cells from 16 patients. Antimyeloma response of bortezomib, which was evident in >50% of 16 experiments and resembled clinical response, was associated with significant increased bone mineral density (BMD) and osteoblast numbers, and reduced osteoclast numbers in myelomatous bones. This bone anabolic effect, which was also visualized on X‐ray radiographs and confirmed by static and dynamic histomorphometric analyses, was unique to bortezomib and was not observed in hosts responding to melphalan, a chemotherapeutic drug widely used to treat MM. Bortezomib also increased BMD and osteoblasts number and reduced osteoclasts number in nonmyelomatous implanted bones. In vitro bortezomib directly suppressed human osteoclast formation and promoted maturation of osteoblasts. We conclude that bortezomib promotes bone formation in myelomatous and nonmyelomatous bones by simultaneously inhibiting osteoclastogenesis and stimulating osteoblastogenesis. As clinical and experimental studies indicate that bone disease is both a consequence and necessity of MM progression our results suggest and that bortezomibs effects on bone remodeling contribute to the antimyeloma efficacy of this drug. Am. J. Hematol., 2009.

Atacicept (TACI-Ig) inhibits growth of TACI(high) primary myeloma cells in SCID-hu mice and in coculture with osteoclasts.

APRIL (a proliferation-inducing Ligand) and BLyS/BAFF (B-lymphocyte stimulator/B-cell-activating factor of the TNF (tumor necrosis factor) family have been shown to be the survival factors for certain myeloma cells in vitro. BAFF binds to the TNF-related receptors such as B-cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) and BAFFR, whereas APRIL binds to TACI and BCMA and to heparan sulfate proteoglycans (HSPG) such as syndecan-1. TACI gene expression in myeloma reportedly can distinguish tumors with a signature of microenvironment dependence (TACIhigh) versus a plasmablastic signature (TACIlow). We tested the effect of atacicept (formerly TACI-Ig, which blocks APRIL and BAFF) and BAFFR-Ig (which blocks BAFF only) on primary myeloma growth in the SCID-hu model and in coculture with osteoclasts. With only few exceptions, atacicept and to a lesser extent BAFFR-Ig, inhibited growth of TACIhigh but not TACIlow myeloma samples in vivo and ex vivo, and the response rate was inversely correlated with TACI expression. Most TACIhigh myeloma cells were molecularly classified as being low risk with our recently described 70-gene model. APRIL and BAFF were highly expressed by osteoclasts and were upregulated in myeloma cells after coculture with osteoclasts. Our findings suggest that APRIL plays an essential role in the survival of TACIhigh bone marrow-dependent myeloma cells and TACI gene expression may be a useful predictive marker for patients who could benefit from atacicept treatment.

The ephrinB2/EphB4 axis is dysregulated in osteoprogenitors from myeloma patients and its activation affects myeloma bone disease and tumor growth

Myeloma bone disease is caused by uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Bidirectional signaling between the cell-surface ligand ephrinB2 and its receptor, EphB4, is involved in the coupling of osteoblastogenesis and osteoclastogenesis and in angiogenesis. EphrinB2 and EphB4 expression in mesenchymal stem cells (MSCs) from myeloma patients and in bone cells in myelomatous bones was lower than in healthy counterparts. Wnt3a induced up-regulation of EphB4 in patient MSCs. Myeloma cells reduced expression of these genes in MSCs, whereas in vivo myeloma cell-conditioned media reduced EphB4 expression in bone. In osteoclast precursors, EphB4-Fc induced ephrinB2 phosphorylation with subsequent inhibition of NFATc1 and differentiation. In MSCs, EphB4-Fc did not induce ephrinB2 phosphorylation, whereas ephrinB2-Fc induced EphB4 phosphorylation and osteogenic differentiation. EphB4-Fc treatment of myelomatous SCID-hu mice inhibited myeloma growth, osteoclastosis, and angiogenesis and stimulated osteoblastogenesis and bone formation, whereas ephrinB2-Fc stimulated angiogenesis, osteoblastogenesis, and bone formation but had no effect on osteoclastogenesis and myeloma growth. These chimeric proteins had similar effects on normal bone. Myeloma cells expressed low to undetectable ephrinB2 and EphB4 and did not respond to the chimeric proteins. The ephrinB2/EphB4 axis is dysregulated in MM, and its activation by EphB4-Fc inhibits myeloma growth and bone disease.

Bone marrow endothelial cells in multiple myeloma secrete CXC-chemokines that mediate interactions with plasma cells.

Bone marrow endothelial cells (EC) from patients with multiple myeloma (MM) were found to express and secrete higher amounts of the CXC‐chemokines CXCL8/interleukin (IL)‐8, CXCL11/interferon‐inducible T‐cell alpha chemoattractant (I‐TAC), CXCL12/stromal cell‐derived factor (SDF)‐1α, and CCL2/monocyte chemotactic protein(MCP)‐1 than EC from human umbilical vein (HUVEC), considered as a healthy counterpart. Paired plasma cells and several MM cell lines expressed cognate receptors of each chemokine to a variable extent. When cells were exposed to chemokines, CXCL8/IL‐8 and CXCL12/SDF‐1α stimulated their proliferation and all chemokines stimulated cell chemotaxis. It is suggested that angiogenesis also favours MM progression through the release of CXC‐chemokines.

Human placenta-derived adherent cells prevent bone loss, stimulate bone formation, and suppress growth of multiple myeloma in bone.

Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta‐derived adherent cells (PDACs) are mesenchymal‐like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)–rab model of medullary myeloma‐associated bone loss. Intrabone injection of PDACs into nonmyelomatous and myelomatous implanted bone in SCID‐rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose‐dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID‐rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis. STEM CELLS 2011;29:263–273

Role of Bruton's tyrosine kinase in myeloma cell migration and induction of bone disease

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Brutons tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B‐lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL)−6– or stroma–dependent cell lines and osteoclasts. SDF‐1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM‐A13, reduced their migration toward stromal cell‐derived factor‐1 (SDF‐1). Pretreatment with LFM‐A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID‐rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF‐1 but had no effect on short‐term growth. BTK expression was correlated with cell‐surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell‐surface CXCR4‐expressing myeloma cells. BTK was not upregulated by IL‐6 while its inhibition had no effect on IL‐6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell‐surface CXCR4 and migrated toward SDF‐1. LFM‐A13 suppressed migration and differentiation of osteoclast precursors as well as bone‐resorbing activity of mature osteoclasts. In primary myeloma‐bearing SCID‐rab mice, LFM‐A13 inhibited osteoclast activity, prevented myeloma‐induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell‐surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma‐induced bone disease. Am. J. Hematol. 88:463–471, 2013.

Consequences of Daily Administered Parathyroid Hormone on Myeloma Growth, Bone Disease, and Molecular Profiling of Whole Myelomatous Bone

Background Induction of osteolytic bone lesions in multiple myeloma is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Current management of myeloma bone disease is limited to the use of antiresorptive agents such as bisphosphonates. Methodology/Principal Findings We tested the effects of daily administered parathyroid hormone (PTH) on bone disease and myeloma growth, and we investigated molecular mechanisms by analyzing gene expression profiles of unique myeloma cell lines and primary myeloma cells engrafted in SCID-rab and SCID-hu mouse models. PTH resulted in increased bone mineral density of myelomatous bones and reduced tumor burden, which reflected the dependence of primary myeloma cells on the bone marrow microenvironment. Treatment with PTH also increased bone mineral density of uninvolved murine bones in myelomatous hosts and bone mineral density of implanted human bones in nonmyelomatous hosts. In myelomatous bone, PTH markedly increased the number of osteoblasts and bone-formation parameters, and the number of osteoclasts was unaffected or moderately reduced. Pretreatment with PTH before injecting myeloma cells increased bone mineral density of the implanted bone and delayed tumor progression. Human global gene expression profiling of myelomatous bones from SCID-hu mice treated with PTH or saline revealed activation of multiple distinct pathways involved in bone formation and coupling; involvement of Wnt signaling was prominent. Treatment with PTH also downregulated markers typically expressed by osteoclasts and myeloma cells, and altered expression of genes that control oxidative stress and inflammation. PTH receptors were not expressed by myeloma cells, and PTH had no effect on myeloma cell growth in vitro. Conclusions/Significance We conclude that PTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis and attenuated bone resorption and myeloma growth; mechanisms involve increased osteoblast production of anti-myeloma factors and minimized myeloma induction of inflammatory conditions.

Inhibitor of DASH proteases affects expression of adhesion molecules in osteoclasts and reduces myeloma growth and bone disease

Dipeptidyl peptidase (DPP) IV activity and/or structure homologues (DASH) are serine proteases implicated in tumourigenesis. We previously found that a DASH protease, fibroblast activation protein (FAP), was involved in osteoclast‐induced myeloma growth. Here we further demonstrated expression of various adhesion molecules in osteoclasts cultured alone or cocultured with myeloma cells, and tested the effects of DASH inhibitor, PT‐100, on myeloma cell growth, bone disease, osteoclast differentiation and activity, and expression of adhesion molecules in osteoclasts. PT‐100 had no direct effects on viability of myeloma cells or mature osteoclasts, but significantly reduced survival of myeloma cells cocultured with osteoclasts. Real‐time PCR array for 85 adhesion molecules revealed upregulation of 17 genes in osteoclasts after coculture with myeloma cells. Treatment of myeloma/osteoclast cocultures with PT‐100 significantly downregulated 18 of 85 tested genes in osteoclasts, some of which are known to play roles in tumourigenesis and osteoclastogenesis. PT‐100 also inhibited osteoclast differentiation and subsequent pit formation. Resorption activity of mature osteoclasts and differentiation of osteoblasts were not affected by PT‐100. In primary myelomatous severe combined immunodeficient (SCID)‐hu mice PT‐100 reduced osteoclast activity, bone resorption and tumour burden. These data demonstrated that DASH proteases are involved in myeloma bone disease and tumour growth.

Establishment and exploitation of hyperdiploid and non-hyperdiploid human myeloma cell lines

The establishment of clinically relevant human myeloma cell lines is central for our understanding of myeloma pathogenesis and development of novel therapies for the disease. Unfortunately, most available lines were generated from extramedullary sites, harbored multiple genetic abnormalities and categorized as non‐hyperdiploid. In contrast, hyperdiploid myeloma cell lines, which represent more than 50% of patients, are rare. We established procedures for establishment of stroma‐dependent myeloma lines by passaging primary myeloma cells, in severe combined immunodeficient‐human (SCID‐hu) or SCID‐rab mice followed by maintenance in co‐culture with stromal cells. We described the establishment and characterization of two hyperdiploid (LD and CF) and two non‐hyperdiploid (JB and BN) cell lines. Using our animal models, we also established bortezomib‐sensitive and ‐resistant BN lines. These cell lines were cellularly, phenotypically and molecularly characterized using flow cytometry immunophenotyping, DNA content, G‐band and multicolor spectral karyotyping (SKY) and global gene expression profiling. All four cell lines were infected with lentiviral‐expressing luciferase for detection of tumour cells at high sensitivity level and for monitoring myeloma growth in co‐cultures and in vivo by live animal imaging. These myeloma cell lines and the procedures used for their establishment provide essential tools for studying myeloma biology and therapy.

Relevance of Pathological Complete Response after Neoadjuvant Therapy for Breast Cancer.

Breast cancer is a heterogeneous disease, and the different biological subtypes have different prognostic impacts. Neoadjuvant trials have recently become popular as they offer several advantages compared to traditional adjuvant trials. Studies have shown that patients who achieve pathological complete response (pCR) after neoadjuvant treatment have a better long-term outcome. Consequently, increasing the rate of pCR became the end point of neoadjuvant trials with the expectation of translation into improved survival. However, the definition of pCR has lacked uniformity, and the prognostic impact of achievement of pCR on survival in different breast cancer subtypes is uncertain. In this review, we present the controversies associated with the use of pCR as an end point in neoadjuvant trials.