Targeting Notch inhibitors to the myeloma bone marrow niche decreases tumor growth and bone destruction without gut toxicity

Background:Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by bone destruction[1]. Chemokine signaling by skeletal cells or by other cells of the bone marrow niche regulates bone formation and resorption[2]. Recent studies have found that CXCL7 enhanced the osteoclast formation in mouse bone marrow cells[3, 4]. Whether CXCL7 plays a role in human osteoclastogenesis especially in RA patients remains unclear.Objectives:To examine the functional role of CXCL7 in the induction of osteoclastogenesis in RA.Methods:The level of CXCL7 in CD14+monocyte supernatant was assessed via enzyme-linked immunosorbent assay. Osteoclastogenesis of CD14+monocyte from RA patients and healthy donors were evaluated by TRAP staining and F-actin ring immunofluorescence. Bone resorption pit was observed by scanning electron microscopy. We performed quantitative reverse transcription polymerase chain reaction (RT-PCR) to detect changes in osteoclast markers. RAW264.7 macrophages were also used to investigate specific signaling pathway by which CXCL7 stimulated during osteoclast formation.Results:CXCL7 level in CD14+monocyte supernatant from RA patients (5690 ±627.05 pg/ml) was significantly higher than that in healthy controls (2301 ±535.52 pg/ml) (n=5, P<0.001). In the presence of M-CSF and RANKL, CXCL7 promoted osteoclast formation(Figure 1A and B) and increased bone resorption area(Figure 1C) of CD14+monocyte from healthy donors in the low concentration (10ng/ml) group (n=3, p < 0.05). While in high concentration of CXCL7 (50ng/ml) group, there were no significant changes in the number of osteoclasts. Transcription level of the osteoclast markers RANK, cathepsin K, and MMP-9 was significantly increased in the CXCL7 (10 ng/mL) group after 3 days in the presence of M-CSF and sRANKL (n=3, p < 0.05). When using CD14+ monocyte from RA patients, the optimal concentration of CXCL7 was 50ng/ml, which significantly increased the number of osteoclasts (Figure 2A and B)and bone resorption area (Figure 2C) (n=3, p < 0.01). Flow cytometry analysis revealed that a higher proportion of CD14+monocytes expressed CXCR2 from healthy donors than those from RA patients (n=6, p < 0.01). Consistent with the results obtained in CD14+monocytes, the effects of exogenous CXCL7 on osteoclast formation were also observed in RAW264.7 cells (p < 0.01). The addition of CXCL7 dramatically promoted phosphorylation ERK1/2 in RAW264.7 cells, but it did not affect the phosphorylation of P65.Conclusion:CXCL7 level in CD14+monocyte supernatant was higher in RA patients than that of healthy donors. CXCL7 promoted osteoclastogenesis in CD14+monocyte both from RA patients and healthy donors. CXCL7 could be a potential therapeutic target for bone destruction in RA.References:[1] McInnes, I.B. and G. Schett, The pathogenesis of rheumatoid arthritis. N Engl J Med, 2011. 365(23): p. 2205-19.[2] Brylka, L.J. and T. Schinke, Chemokines in Physiological and Pathological Bone Remodeling. Front Immunol, 2019. 10: p. 2182.[3] Nakao, K., et al., IGF2 modulates the microenvironment for osteoclastogenesis. Biochem Biophys Res Commun, 2009. 378(3): p. 462-6.[4] Goto, Y., et al., CXCR4(+) CD45(-) Cells are Niche Forming for Osteoclastogenesis via the SDF-1, CXCL7, and CX3CL1 Signaling Pathways in Bone Marrow. Stem Cells, 2016. 34(11): p. 2733-2743.Acknowledgments :We gratefully thank the Medical Research Center of Peking University Third Hospital for providing experimental equipment and technical support.Disclosure of Interests:None declared

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Pim Inhibition Suppresses Osteoclastogenesis and Tumor Growth in Myeloma

Blood ◽

10.1182/blood.v126.23.4203.4203 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4203-4203 ◽

Cited By ~ 1

Author(s):

Jumpei Teramachi ◽

Masahiro Hiasa ◽

Asuka Oda ◽

Hirofumi Tenshin ◽

Ryota Amachi ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Growth ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Research Funding ◽

Bone Destruction ◽

Cathepsin K ◽

Marrow Stromal Cells ◽

Bone Lesions ◽

Rank Ligand

Abstract Devastating bone destruction in multiple myeloma (MM) still remains a significant clinical problem. In pursuing factors responsible for MM tumor expansion and bone destruction, we found that the serine/threonine kinase Pim-2 is constitutively over-expressed as an anti-apoptotic mediator, and further up-regulated in MM cells when cocultured with bone marrow stromal cells or osteoclasts (OCs) (Leukemia, 2011). We also demonstrated that Pim inhibition is able to induce bone formation while suppressing MM tumor growth (Leukemia, 2015). However, the impact of Pim inhibition on MM-induced bone resorption remains unknown. Therefore, the present study was undertaken to clarify the role of Pim-2 in osteoclastogenesis enhanced in MM and the therapeutic effects of Pim inhibition on mutual interaction between MM cells and OCs. Pim-2 was highly expressed almost exclusively in cathepsin K-positive mature OCs on the surface of bone but not in other bone marrow cells in normal mouse bone tissues. RANK ligand and TNF-α induced the expression of Pim-2 in monocytes and RAW264.7 preosteoclastic cells at mRNA and protein levels. Inhibitors of the classical NF-κB pathway, SN50 or IMG2001, abolished Pim-2 up-regulation in RAW264.7 cells by RANK ligand or TNF-α, while Pim inhibition marginally affected the nuclear translocation of NF-κB subunits, p50 and p65, as well as the promoter activity of NF-κB, suggesting Pim-2 up-regulation downstream of the NF-κB pathway. Pim-2 appeared to be up-regulated along with c-fos, NFATc1 and cathepsin K during osteoclastogenesis. The Pim inhibitor SMI-16a potently suppressed the RANK ligand-induced expression of c-fos, NFATc1 and cathepsin K in RAW264.7 cells, and abolished osteoclastogenesis and bone resorption enhanced by MM cell conditioned media on hydroxyapatite-coated dishes. Furthermore, the Pim inhibition was found to suppress [Ca2+ ]i oscillation and thereby nuclear translocation of NFATc1, a critical transcription factor for osteoclastogenesis. MM cells and acid-producing OCs are mutually interacted in bone lesions to enhance MM tumor growth and bone destruction while creating an acidic milieu, thereby forming a progressive vicious cycle. Pim-2 was also up-regulated in MM cells when cocultured with OCs as well as bone marrow stromal cells, and to lesser extent merely by acidic conditions. Interestingly, acidic conditions rather preferentially enhanced the cytotoxic effects of the Pim inhibitor SMI-16a on MM cells even in cocultures with OCs or bone marrow stromal cells. Finally, treatment with SMI-16a reduced OC numbers in bone lesions together with tumor reduction and the restoration of bone formation in mouse MM models with intra-tibial injection of murine 5TGM1 MM cells. These results collectively demonstrated that Pim-2 play a critical role in osteoclastogenesis and tumor growth in acidic bone lesions in MM, and further corroborated that Pim-2 is a pivotal therapeutic target for MM bone disease and tumor progression. Disclosures Abe: Novartis Pharma K.K.: Speakers Bureau; Takeda Pharmaceutical Company Limited: Research Funding; Kyowa Hakko Kirin Company, Limited: Research Funding; Astellas Pharma Inc.: Research Funding; Ono Pharmaceutical Co.,Ltd.: Research Funding; GlaxoSmithKline plc: Research Funding.

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Abstract 103: Disruption of Notch Signaling targeted to the myeloma bone marrow microenvironment simultaneously inhibits tumor growth and prevents bone loss without inducing gut toxicity

10.1158/1538-7445.sabcs18-103 ◽

2019 ◽

Author(s):

Adam J. Ferrari ◽

Kevin McAndrews ◽

Jessica H. Nelson ◽

James T. Bell ◽

Venkatesan Srinivasan ◽

...

Keyword(s):

Bone Marrow ◽

Bone Loss ◽

Tumor Growth ◽

Notch Signaling ◽

Bone Marrow Microenvironment ◽

Gut Toxicity

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Bone disease as the first manifestation of systemic AL-amyloidosis

Terapevticheskii arkhiv ◽

10.26442/00403660.2020.07.000775 ◽

2020 ◽

Vol 92 (7) ◽

pp. 85-89

Author(s):

L. P. Mendeleeva ◽

I. G. Rekhtina ◽

A. M. Kovrigina ◽

I. E. Kostina ◽

V. A. Khyshova ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Disease ◽

Plasma Cells ◽

Interventricular Septum ◽

Bone Destruction ◽

Amyloid Deposition ◽

Bone Lesions ◽

Al Amyloidosis ◽

Linear Type

Our case demonstrates severe bone disease in primary AL-amyloidosis without concomitant multiple myeloma. A 30-year-old man had spontaneous vertebral fracture Th8. A computed tomography scan suggested multiple foci of lesions in all the bones. In bone marrow and resected rib werent detected any tumor cells. After 15 years from the beginning of the disease, nephrotic syndrome developed. Based on the kidney biopsy, AL-amyloidosis was confirmed. Amyloid was also detected in the bowel and bone marrow. On the indirect signs (thickening of the interventricular septum 16 mm and increased NT-proBNP 2200 pg/ml), a cardial involvement was confirmed. In the bone marrow (from three sites) was found 2.85% clonal plasma cells with immunophenotype СD138+, СD38dim, СD19-, СD117+, СD81-, СD27-, СD56-. FISH method revealed polysomy 5,9,15 in 3% of the nuclei. Serum free light chain Kappa 575 mg/l (/44.9) was detected. Multiple foci of destruction with increased metabolic activity (SUVmax 3.6) were visualized on PET-CT, and an surgical intervention biopsy was performed from two foci. The number of plasma cells from the destruction foci was 2.5%, and massive amyloid deposition was detected. On CT scan foci of lesions differed from bone lesions at multiple myeloma. Bone fragments of point and linear type (button sequestration) were visualized in most of the destruction foci. The content of the lesion was low density. There was no extraossal spread from large zones of destruction. There was also spontaneous scarring of the some lesions (without therapy). Thus, the diagnosis of multiple myeloma was excluded on the basis based on x-ray signs, of the duration of osteodestructive syndrome (15 years), the absence of plasma infiltration in the bone marrow, including from foci of bone destruction by open biopsy. This observation proves the possibility of damage to the skeleton due to amyloid deposition and justifies the need to include AL-amyloidosis in the spectrum of differential diagnosis of diseases that occur with osteodestructive syndrome.

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