Pim Inhibition Suppresses Osteoclastogenesis and Tumor Growth in Myeloma

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.

Download Full-text

Myeloma–Bone Interaction: A Vicious Cycle via TAK1–PIM2 Signaling

Cancers ◽

10.3390/cancers13174441 ◽

2021 ◽

Vol 13 (17) ◽

pp. 4441

Author(s):

Takeshi Harada ◽

Masahiro Hiasa ◽

Jumpei Teramachi ◽

Masahiro Abe

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Critical Role ◽

Bone Destruction ◽

Marrow Stromal Cells ◽

Bone Lesions ◽

Vicious Cycle ◽

Growth And Survival ◽

Wnt Inhibitors

Multiple myeloma (MM) has a propensity to develop preferentially in bone and form bone-destructive lesions. MM cells enhance osteoclastogenesis and bone resorption through activation of the RANKL–NF-κB signaling pathway while suppressing bone formation by inhibiting osteoblastogenesis from bone marrow stromal cells (BMSCs) by factors elaborated in the bone marrow and bone in MM, including the soluble Wnt inhibitors DKK-1 and sclerostin, activin A, and TGF-β, resulting in systemic bone destruction with loss of bone. Osteocytes have been drawn attention as multifunctional regulators in bone metabolism. MM cells induce apoptosis in osteocytes to trigger the production of factors, including RANKL, sclerostin, and DKK-1, to further exacerbate bone destruction. Bone lesions developed in MM, in turn, provide microenvironments suited for MM cell growth/survival, including niches to foster MM cells and their precursors. Thus, MM cells alter the microenvironments through bone destruction in the bone where they reside, which in turn potentiates tumor growth and survival, thereby generating a vicious loop between tumor progression and bone destruction. The serine/threonine kinases PIM2 and TAK1, an upstream mediator of PIM2, are overexpressed in bone marrow stromal cells and osteoclasts as well in MM cells in bone lesions. Upregulation of the TAK1–PIM2 pathway plays a critical role in tumor expansion and bone destruction, posing the TAK1–PIM2 pathway as a pivotal therapeutic target in MM.

Download Full-text

Vector-averaged gravity regulates gene expression of receptor activator of NF-κB (RANK) ligand and osteoprotegerin in bone marrow stromal cells via cyclic AMP/protein kinase A pathway

Bone ◽

10.1016/s8756-3282(02)00680-4 ◽

2002 ◽

Vol 30 (4) ◽

pp. 553-558 ◽

Cited By ~ 26

Author(s):

M Kanematsu ◽

K Yoshimura ◽

M Takaoki ◽

A Sato

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Protein Kinase ◽

Cyclic Amp ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Rank Ligand ◽

Receptor Activator ◽

Kinase A

Download Full-text

Targeting MEK1/2 Signaling Cascade by AS703026, a Novel Selective MEK1/2 Inhibitor, Induces Pleiotropic Anti-Myeloma Activity in Vitro and In Vivo.

Blood ◽

10.1182/blood.v114.22.3848.3848 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3848-3848 ◽

Cited By ~ 2

Author(s):

Yu-Tzu Tai ◽

Kihyun Kim ◽

Xian-Feng Li ◽

Mariateresa Fulciniti ◽

Weihua Song ◽

...

Keyword(s):

Bone Marrow ◽

Cell Growth ◽

Tumor Growth ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Time Dependent ◽

Dependent Manner

Abstract Abstract 3848 Poster Board III-784 The mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in the pathogenesis of human multiple myeloma (MM) by promoting interactions of MM cells with bone marrow stromal cells (BMSCs) that secrete cytokines and growth factors for MM cell growth, survival, and resistance to chemotherapeutic drugs. Accumulating studies have supported targeting this signaling pathway in MM. Here we investigate cytotoxicity of AS703026, a novel selective MEK1/2 inhibitor with highly oral bioavailability, in MM cell lines and patient MM cells and define its mechanisms of action. AS703026, more potently (∼9-10 fold) than AZD6244, inhibits growth and survival of MM cells and cytokine-induced osteoclast differentiation. It specifically blocks baseline and adhesion-induced pERK1/2, but not pSTAT3. Selective MEK1/2 inhibition by AS703026 led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest, as shown by increased subG0 cells, and concurrently abolished S phase cells. AS703026 also reduced expression of c-maf oncogene in a time-dependent manner, suggesting a MEK1/2-dependent regulation of c-maf that may contribute MM cell growth inhibition. AS703026 further induced apoptosis in MM cells, as manifested by caspase 3 and PARP cleavages in a time-dependent manner. It blocked osteoclastogenesis in vitro, as measured by number of TRAP-positive multinuclear cells following culturing PBMCs with RANKL and M-CSF. Importantly, AS703026 sensitized drug-resistant MM cells to a broad spectrum of conventional (dexamethasone, melphalan), as well as novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. Synergistic or additive cytotoxicity (combination index < 1) induced by these combinations was further validated by annexin-V/PI staining and flow cytometric analysis. Combining these agents led to a significantly increased apoptosis and cell death than AS703026 alone, confirming enhanced cytotoxicity against MM cells. In vivo studies demonstrate that treatment of MM cell line H929-bearing mice with AS703026 (n=4 at 30 mg/kg; n=6 at 15 mg/kg), but not vehicle alone (n=6), blocked MM tumor growth in a dose-dependent manner (p<0.008 at 30 mg/kg; p<0.02 at 15 mg/kg). Immunoblotting and immunohistochemistrical staining showed that AS703026-reduced tumor growth was associated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nM) triggered significant cytotoxicity against the majority of patients with relapsed and refractory MM (>84%, n=18), regardless mutation status of 3 RAS and BRAF genes. Bone marrow stromal cells-induced viability of MM patient cells is similarly blocked within the same dose range. Our results therefore strongly support clinical protocols evaluating AS703026, alone or with other anti-MM agents, to improve patient outcome in MM. Disclosures: Chauhan: Progenra, Inc: Consultancy. Richardson:Keryx Biopharmaceuticals: Honoraria. Clark:EMD Serono: Employment. Ogden:EMD Serono: Employment. Andreas:EMD Serono: Employment. Rastelli:EMD Serono: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Download Full-text

Daratumumab Inhibits AML Metabolic Capacity and Tumor Growth through Inhibition of CD38 Mediated Mitochondrial Transfer from Bone Marrow Stromal Cells to Blasts in the Leukemic Microenvironment

Blood ◽

10.1182/blood-2019-128592 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1385-1385 ◽

Cited By ~ 3

Author(s):

Jayna J Mistry ◽

Charlotte Hellmich ◽

Jamie A Moore ◽

Christopher R Marlein ◽

Genevra Pillinger ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Research Funding ◽

Marrow Stromal Cells ◽

Post Transplantation ◽

Mitochondrial Transfer ◽

Nsg Mouse

Background Acute myeloid leukemia (AML) is dependent on the bone marrow microenvironment, where bone marrow stromal cells (BMSCs) are an important tumor supporting cell type. We have previously demonstrated that, contrary to the Warburg hypothesis, AML blasts rely on oxidative phosphorylation for survival and are dependent on increased mitochondrial levels compared to non-malignant CD34+ progenitor cells. Moreover, we found that AML blasts meet their high metabolic demands by transferring in mitochondria from surrounding BMSC. We have also recently described how mitochondria are transferred from BMSC to myeloma cells in a pro-tumoral, CD38 dependent, mechanism. As the leukemia-initiating cells in AML may reside within the CD34+/CD38+ compartment, we examined the mitochondrial transfer and the resultant metabolic and functional consequences of inhibiting CD38 using daratumumab in the setting of the AML microenvironment. Methods Primary AML blasts and primary AML BMSC were isolated from patients bone marrow in accordance with the Declaration of Helsinki. BMSC were separated by adherence and then characterised using flow cytometry for expression of CD90+, CD73+, CD105+ and CD45-. Mitochondrial transfer was assessed in vitro using qPCR and MitoTracker staining based methods. In vivo experiments using an NSG AML xenograft model were carried out with darartumumab (or control) treatment given on days 9 and 16 post AML transplant. Tumor engraftment and growth were monitored weekly by live animal in vivo imaging. Post transplantation, AML mitochondrial content and transfer were assessed by evaluation of murine mitochondrial DNA in human AML blasts by species specific PCR analysis. Post transplantation mitochondrial function was measured by TMRM and Seahorse analysis. Results In-vitro experiments using MitoTracker Green demonstrate that daratumumab inhibits the transfer of mitochondria from BMSC to AML. In-vivo, daratumumab treatment significantly reduced tumor growth in human xenograft mouse model. Furthermore, we found that two doses of daratumumab resulted in reduced mitochondrial potential and oxygen consumption rate in the AML cells derived from the BM microenvironment of the AML engrafted NSG mice. Finally, examination of human AML cells sorted from NSG mouse bone marrow confirmed that mouse mitochondrial DNA content in the human AML blasts was reduced from animals treated with daratumumab compared to animals with AML treated with vehicle control. Conclusion Daratumumab treatment inhibits mitochondrial transfer from BMSC to AML in the BM microenvironment, resulting in a reduction of pro-tumoral oxidative phosphorylation in the blasts and subsequent reduced leukemia growth, which is associated with improved animal survival. While it is likely that daratumumab functions through a number of mechanisms of action, here we show in the NSG mouse model (which lacks functional B cells, T cells and NK cells and where macrophages and dendritic cells are defective) that inhibition of mitochondrial transfer in AML can be added to the list of mechanisms of action for daratumumab. These data support the further investigation of daratumumab as a therapeutic approach for the treatment of this mitochondrial dependent tumor. Disclosures Bowles: Janssen: Research Funding; Abbvie: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

Download Full-text