Drug Resistant Myeloma Cells Cause More Severe Bone Destruction in Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 839-839
Author(s):  
Mu Hao ◽  
Zhimin Gu ◽  
Reinaldo Franqui Machin ◽  
Hongwei Xu ◽  
Gregory S. Thomas ◽  
...  
Keyword(s):  
Bone Disease ◽  
Osteoclast Differentiation ◽  
Bone Destruction ◽  
Conditioned Media ◽  
In Vivo Studies ◽  
Drug Resistant ◽  
Newly Diagnosed ◽  
Lytic Lesions ◽  
Trap Staining

Abstract Background: Multiple myeloma (MM) is the second most common hematologic malignancy in the US and is characterized by osteolytic disease. More than 80% of MM patients have osteolysis, leading to in fractures, severe bone pain, spinal cord compression, and hypercalcemia, greatly compromising patient quality of life and resulting in mortality. While bone exists in a dynamic state between resorption and deposition, the molecular mechanisms underlying bone destruction in MM remain elusive. One contributing factor is heparanase (HPSE). In our work, we have previously shown that the expression of NEK2 and HPSE are closely correlated. We have also shown that high expression of chromosomal instability-related genes (e.g. NEK2) induces drug resistance and poor prognosis in MM. Though tumor burden is dramatically decreased in remission, bone destruction is poorly repaired, suggesting drug resistant MM cells may play a critical role in inducing bone destruction or in preventing the repair of bone disease in remission. Materials and Methods: Gene expression profiling (GEP) data and bone lytic lesions detected by MRI were collected from 333 newly diagnosed MM patients from the Total Therapy 2 (TT2) clinical trial. MM cell lines with stable overexpression or knockdown of NEK2 and/or HPSE were created using lentiviral delivery. Protein and mRNA expression was assessed by immunoblotting and real-time PCR, respectively. TRAP staining was used to assess osteoclast differentiation. In vivo studies were performed in the KaLwRij 5TGM1 MM mouse model in 3 different groups: empty vector (EV), NEK2 overexpression (NEK2 OE), or NEK2 OE with HPSE shRNA. Bone density and imaging assessment was performed via X-ray and microCT scanning. HPSE, NFκB, and USP7 contributions were examined using the specific small molecule inhibitors SST0001, BSM-345541, and P5091, respectively. Results: In correlating NEK2 expression with osteolytic lesion from 333 newly diagnosed MMs, we found the expression of NEK2 was significantly higher in MM patients with bone lytic lesion than those without lesions. NEK2 expression was also higher in patients with severe bone disease (7 or more lesions) than those fewer bone lytic lesions (< 7). To determine if NEK2 causes bone lytic lesions in MM, we performed in vivo studies injecting NEK2 OE 5TGM1 cells into KaLwRij mice. MicroCT showed clearly that bone volume and thickness of trabecular bone were decreased significantly in mice injected with NEK2 OE cells compared to EV cells. Strikingly, treatment with the HPSE inhibitor SST0001 dramatically rescued bone mass loss induced by NEK2 overexpression. Histological TRAP staining of decalcified bone sections revealed a significant increase in the number of osteoclasts in the bone surface area in mice injected with NEK2 OE cells compared to EV-treated mice, while addition of the SST0001 significantly reduced the number of osteoclast. Because osteoclast causes bone resorption, we examined the effect of applying conditioned media from MM cells with or without NEK2 overexpression to pre-osteoclast cells derived from MM patients, C57BL6 mice, and the pre-osteoclast RAW264.7 cell line. We found significantly increased mature osteoclasts (>3 nuclei) after culture with media collected from NEK2 MM cells compared to conditioned media collected from EV cells. Consistently, osteoclast differentiation-associated genes (i.e. CTSK, NFATc1, RANK, and TRAP) were upregulated in pre-osteoclasts cultured with NEK2 OE conditioned media. SST0001 suppressed osteoclast differentiation induced by NEK2, strongly suggesting that HPSE mediates NEK2 induced osteoclastogensis and bone destruction in MM. Mechanistic studies revealed that NEK2 directly binds to and activates USP7 resulting in deubiquitination of NFκB. Inhibition of NFκB decreased NEK2-induced HPSE expression induced by NEK2 at both the mRNA and protein level. Conclusions: Our findings suggest that high NEK2 expression may be a biomarker for bone disease in MM. We demonstrate that a subpopulation of drug-resistant, NEK2-overexpressing MM cells leads to increased production and activity of HPSE, and increased osteoclast differentiation contributing to severe bone disease. Mechanistically, we show that NEK2 interacts with USP7, stabilizing NFκB and upregulating HPSE. Our results demonstrate drug resistant MM cells may not only promote tumor cell survival but also contribute to bone destruction in MM. Disclosures No relevant conflicts of interest to declare.

Brain-Derived Neurotrophic Factor Enhances Osteoclastogenesis in Multiple Myeloma via Upregulation of RANKL/OPG Ratio Both in Vitro and in a Murine Model of Myeloma Bone Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 568-568
Author(s):  
Li-Sha Ai ◽  
Chun-Yan Sun ◽  
Tao Guo ◽  
Ya-Dan Wang ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Disease ◽  
Neurotrophic Factor ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Myeloma Bone Disease ◽  
Trap Staining

Abstract Abstract 568 Osteolytic bone disease is a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption uncoupled with osteoblastic bone formation. Myeloma-induced osteoclastogenesis is largely depending on the increase of receptor activator of NF-κB ligand (RANKL) and decrease of osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain-derived neurotrophic factor (BDNF) was identified as an MM-derived factor correlated with increased RANKL level and contributed to myeloma bone destruction. On the other hand, tyrosine receptor kinase B (TrkB), the receptor of BDNF, was found to be abundantly expressed by osteoblasts (OBs). Since OBs are the main source of RANKL and OPG in bone, here we sought to evaluate the involvement of BDNF/TrkB in the crosstalk between myeloma cells and OBs, as well as the effects of BDNF on RANKL/OPG ratio and myeloma bone disease. Co-cultures of OBs with pre-osteoclasts were performed in a non-contacted transwell system and treated with various concentration of BDNF. Osteoclast formation was detected with a tartrate-resistant acid phosphatase (TRAP) staining kit. Then, RANKL and OPG levels were measured when OBs cultures were exposed to BDNF or co-cultured with three human myeloma cell lines (RPMI8226, ARH-77 and U266). K252a (an inhibitor of TrkB) was present or absent in these systems to assess the effects of BDNF on RANKL/OPG expression in OBs. The involvement of downstream signaling molecules activated by BDNF in OBs was also investigated in this study, with the use of U0126 and a specific small interfering RNA (siRNA) for TrkB. For in vivo study, ARH-77 cells were stably transfected with an antisense short-hairpin RNA construct to BDNF (AS-ARH) or empty vector (EV-ARH). These cells were then intravenously injected to severe combined immunodeficiency (SCID) mice, to test their capacity to induce MM bone disease. Radiographs of mice tibiae and vertebrae were taken weekly by X ray. Changes in total body bone mineral density (BMD) of mice skeleton were recorded. At the end of the experiment, bone sections were stained with hematoxylin and eosin staining or TRAP staining. Secretion levels of RANKL and OPG in mice bone marrow were measured by ELISA. We showed that BDNF increased RANKL and decreased OPG production in OBs in a time- and dose-dependent manner, thus contributing to osteoclast formation in vitro. In addition, these effects were completely abolished by K252a and TrkB-siRNA (P < 0.05). BDNF regulates RANKL/OPG expression in OBs through the TrkB/ERK signaling pathway. Our in vivo results indicated that mice injected with AS-ARH cells, which expressed low levels of endogenous BDNF, were preserved and exhibited no radiologically identifiable osteolytic lesions. In addition, mice in AS-ARH group also had a lower incidence of vertebral compression deformities and paralysis in comparison with mice in EV-ARH group (P < 0.05). Further more, bones harboring AS-ARH cells showed marked reduction of RANKL/OPG ratio and osteoclast density when compared to the controls harboring EV-ARH cells (P < 0.05). Our results demonstrate that BDNF is an important contributor to osteoclastogenesis in MM. Antisense inhibition of BDNF in MM cells remarkably inhibited osteolytic bone destruction in SCID-ARH mice model. BDNF-induced bone destruction is partially mediated by MM-OB interactions via upregulation of RANKL/OPG ratio in the bone marrow milieu. These findings suggest targeting BDNF may become a new therapeutic strategy to improve patient outcome in MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bacterial infections exacerbate myeloma bone disease

2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Rui Liu ◽  
Yuping Zhong ◽  
Rui Chen ◽  
Shiyi Chen ◽  
Yazhu Huang ◽  
...  
Keyword(s):  
Complete Remission ◽  
Bone Disease ◽  
Bacterial Infections ◽  
Osteoclast Differentiation ◽  
Bone Lesion ◽  
In Vivo Studies ◽  
Binding Ability ◽  
E Coli ◽  
Myeloma Bone Disease

AbstractMultiple myeloma is characterized by osteolytic lesions caused by reduced bone formation and activated bone resorption. An important feature of myeloma is a failure of bone healing after successful treatment. In this work, clinical studies indicated a highly positive correlation between bone marrow bacteria abundance and bone lesion numbers of myeloma patients in complete remission. Coculture experiments demonstrated that marrow Escherichia coli (E. coli) promotes osteoclast differentiation and inhibits osteoblast differentiation. Mechanism studies showed that E. coli lipopolysaccharides (LPS) activated NF-κB p65 signaling and reduced phosphorylated smad1/5/9 binding ability with RUNX2 promoter, leading to decreased RUNX2 expression in osteoblast progenitors. Additionally, LPS enhanced phosphorylated NF-κB p65 binding ability with NFATc1 promoter, leading to increased NFATc1 expression in osteoclast progenitors. In vivo studies revealed E. coli contributes to osteolytic bone lesion, and elimination of E. coli infection assists healing of bone lesion in mouse model of myeloma in complete remission. These findings establish a heretofore unrecognized effect for E. coli in the genesis of myeloma bone disease and suggest a new treatment strategy.

scholarly journals Dental Implant Site Drilling and Induced Morphological Changes Correlated with Temperature in Pig’s Rib Used as the Human Jaw Model

2021 ◽  
Vol 11 (6) ◽  
pp. 2493
Author(s):  
Karol Kirstein ◽  
Michalina Horochowska ◽  
Jacek Jagiełło ◽  
Joanna Bubak ◽  
Aleksander Chrószcz ◽  
...  
Keyword(s):  
Bone Tissue ◽  
Morphological Changes ◽  
Bone Destruction ◽  
Microscopic Investigation ◽  
In Vivo Studies ◽  
Drilling Speed ◽  
Tissue Destruction ◽  
Destruction Zone ◽  
Jaw Model

The bone tissue destruction during drilling is still one of the crucial problems in implantology. In this study, the influence of drilling speed, coolant presence, and its temperature on bone tissue was tested using swine rib as a biological model of human jaws. The same method of drilling (with or without coolant) was used in all tested samples. The microscopic investigation estimated the size of the destruction zone and morphology of bone tissue surrounding the drilling canal. The achieved results were statistically elaborated. The study proved that the optimal drilling speed was ca. 1200 rpm, but the temperature of the used coolant had no significant influence on provoked bone destruction. Simultaneously, the drilling system without coolant compared to this with coolant has statistical importance on drilling results. Further in vivo studies will verify the obtained results.

Activity profile of two 5-nitroindazole derivatives over the moderately drug-resistant Trypanosoma cruzi Y strain (DTU TcII): in vitro and in vivo studies

Parasitology ◽  
2020 ◽  
Vol 147 (11) ◽  
pp. 1216-1228
Author(s):  
Cristina Fonseca-Berzal ◽  
Cristiane França da Silva ◽  
Denise da Gama Jaen Batista ◽  
Gabriel Melo de Oliveira ◽  
José Cumella ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Positive Impact ◽  
Cardiac Cells ◽  
In Vivo Studies ◽  
Drug Resistant ◽  
Activity Profile ◽  
Reference Drug ◽  
Novel Drugs

AbstractIn previous studies, we have identified several families of 5-nitroindazole derivatives as promising antichagasic prototypes. Among them, 1-(2-aminoethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one, (hydrochloride) and 1-(2-acetoxyethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one (compounds 16 and 24, respectively) have recently shown outstanding activity in vitro over the drug-sensitive Trypanosoma cruzi CL strain (DTU TcVI). Here, we explored the activity of these derivatives against the moderately drug-resistant Y strain (DTU TcII), in vitro and in vivo. The outcomes confirmed their activity over replicative forms, showing IC50 values of 0.49 (16) and 5.75 μm (24) towards epimastigotes, 0.41 (16) and 1.17 μm (24) against intracellular amastigotes. These results, supported by the lack of toxicity on cardiac cells, led to better selectivities than benznidazole (BZ). Otherwise, they were not as active as BZ in vitro against the non-replicative form of the parasite, i.e. bloodstream trypomastigotes. In vivo, acute toxicity assays revealed the absence of toxic events when administered to mice. Moreover, different therapeutic schemes pointed to their capability for decreasing the parasitaemia of T. cruzi Y acute infected mice, reaching up to 60% of reduction at the peak day as monotherapy (16), 79.24 and 91.11% when 16 and 24 were co-administered with BZ. These combined therapies had also a positive impact over the mortality, yielding survivals of 83.33 and 66.67%, respectively, while untreated animals reached a cumulative mortality of 100%. These findings confirm the 5-nitroindazole scaffold as a putative prototype for developing novel drugs potentially applicable to the treatment of Chagas disease and introduce their suitability to act in combination with the reference drug.

scholarly journals Host−guest complexation-mediated codelivery of anticancer drug and photosensitizer for cancer photochemotherapy

2019 ◽  
Vol 116 (14) ◽  
pp. 6618-6623 ◽  
Author(s):  
Guocan Yu ◽  
Benyue Zhu ◽  
Li Shao ◽  
Jiong Zhou ◽  
Manik Lal Saha ◽  
...  
Keyword(s):  
Side Effects ◽  
Click Reaction ◽  
Tumor Model ◽  
Therapeutic Outcome ◽  
In Vivo Studies ◽  
Drug Resistant ◽  
Hydrophobic Core ◽  
Anticancer Efficacy ◽  
Tumor Accumulation

Although platinum-based anticancer drugs prevail in cancer treatment, their clinical applications are limited by the severe side effects as well as their ineffectiveness against drug resistant cancers. A precise combination of photodynamic therapy (PDT) and chemotherapy can synergistically improve the therapeutic outcome and thereby may overcome drug resistance through a multipronged assault. Herein, we employ the well-defined cavity of a discrete organoplatinum(II) metallacage (M) to encapsulate octaethylporphine (OEP), a photosensitizer, forming a dual-functionalized system M⊃OEP that is wrapped into the hydrophobic core of the nanoparticles (MNPs) self-assembled from an amphiphilic diblock copolymer. Using a copper-free click reaction, a targeting ligand is conjugated on the surface of the MNPs, aiming to specifically deliver a chemotherapeutic drug and a photosensitizer to cancer cells. Benefiting from the enhanced permeability and retention effect and active targeting capability, high tumor accumulation of MNPs is achieved, leading to an improved therapeutic outcome and reduced side effects. In vivo studies demonstrate that the combination of chemotherapy and PDT exhibits a superior antitumor performance against a drug-resistant tumor model attributed to their synergistic anticancer efficacy.

Arsenic Trioxide Shows Synergistic Anti-Myeloma Effects When Combined with Bortezomib and Melphalan In Vitro and Helps Overcome Resistance of Multiple Myeloma Cells to These Treatments in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2467-2467
Author(s):  
Richard A. Campbell ◽  
Haiming Chen ◽  
Daocheng Zhu ◽  
Janice C. Santos ◽  
Benjamin Bonavida ◽  
...  
Keyword(s):  
Arsenic Trioxide ◽  
Cell Lines ◽  
Combined Therapy ◽  
In Vivo Studies ◽  
Low Doses ◽  
Drug Resistant ◽  
Early Results ◽  
Post Implantation

Abstract Arsenic trioxide (ATO) induces apoptosis of plasma cells through a number of mechanisms including inhibiting DNA binding by NF-κB. These results suggest that this agent may be synergistic when combined with other active anti-myeloma drugs. To evaluate this we examined the effect of ATO alone and in combination with anti-myeloma treatments evaluated in vitro with MTT assays and using our severe combined immunodeficient (SCID)-hu murine myeloma models. First, we determined the effects of combining ATO with bortezomib or melphalan on the myeloma cell lines RPMI8226 and U266. Cell proliferation assays demonstrated marked synergistic anti-proliferative effects of ATO at concentrations ranging from 5x10−5M – 5x10−9M and melphalan concentrations ranging from 3x10−5M – 3x10−9M. Similar effects were observed when these cell lines were treated with bortezomib and varying concentrations of ATO (5x10−5 M – 5x10−10 M). We also investigated the potential of ATO to increase the efficacy of anti-myeloma therapies in our SCID-hu murine model LAGλ–1 (Yang H et al. Blood 2002). Each SCID mouse was implanted with a 0.5 cm3 LAGλ–1 tumor fragment into the left hind limb muscle. Mice were treated with ATO alone at 6.0 mg/kg, 1.25 mg/kg, 0.25 mg/kg, and 0.05 mg/kg intraperitoneally (IP) daily x5/week starting 19 days post-implantation. Mice receiving the highest dose of ATO (6.0 mg/kg) showed marked inhibition of tumor growth and reduction of paraprotein levels while there was no effect observed in all other treatment groups. Next, 27 days following implantation of our LAGλ–1 intramuscular (IM) tumor, LAGλ–1 mice were treated with ATO (1.25 mg/kg) IP, bortezomib (0.25 mg/kg), or the combination of both drugs at these doses in the schedules outlined above. ATO or bortezomib treatment alone had no anti-myeloma effects at these low doses consistent with our previous results whereas there was a marked decrease in both tumor volume (57%) and paraprotein levels (53%) in mice receiving the combined therapy. The combination of melphalan and ATO was also evaluated in this model. LAGλ–1 bearing mice received therapy with melphalan IP x1/weekly at 12.0 mg/kg, 6.0 mg/kg, 0.6 mg/kg, and 0.06 mg/kg starting 22 days post-implantation and showed no anti-myeloma effects. Twenty-eight days following implantation of LAGλ–1 tumor, mice received ATO (1.25 mg/kg) or melphalan (0.6 mg/kg) alone at doses without anti-myeloma effects, or the combination of these agents at these doses. The animals treated with these drugs alone showed a similar growth and increase in paraprotein levels to control mice whereas the combination of ATO and melphalan at these low doses markedly suppressed the growth of the tumor by &gt;50% and significantly reduced serum paraprotein levels. These in vitro and in vivo studies suggest that the addition of ATO to other anti-myeloma agents is likely to result in improved outcomes for patients with drug resistant myeloma. Based on these results, these combinations are now in clinical trials with promising early results for patients with drug resistant myeloma.

Expression of Soluble Syndecan-1 or Heparanase by Myeloma Cells Enhances Levels of Angiogenic Growth Factors Present in Endothelial Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3448-3448
Author(s):  
Yang Yang ◽  
Ashley Elizabeth Frith ◽  
Allison Theus ◽  
Veronica Macleod ◽  
Ralph D. Sanderson
Keyword(s):  
Endothelial Cells ◽  
Growth Factors ◽  
Growth Factor ◽  
Fold Increase ◽  
Conditioned Media ◽  
High Rate ◽  
In Vivo Studies ◽  
Angiogenic Growth Factors ◽  
Myeloma Cells

Abstract Multiple myeloma is a devastating cancer with a high rate of morbidity and mortality. Our previous in vivo studies demonstrate that both shed syndecan-1 and heparanase can promote myeloma tumor growth, metastasis and angiogenesis. To examine the mechanism underlying this enhanced angiogenesis, human umbilical vein endothelial cells (HUVEC) were cocultured with cells of the CAG myeloma cell line (vector-only controls, CAGcontrol) or CAG cells engineered to express high levels of either soluble syndecan-1 ectodomain (CAGssyn1 ) or heparanase ( CAGHPSE ). After coculture for 48 hours, levels of angiogenic growth factors present in the endothelial cells were examined. The goal was to determine if expression of either soluble syndecan-1 or heparanase by CAG myeloma cells altered growth factor levels relative to those present when control CAG cells were used. Co-culture with CAGssyn1 or CAGHPSE cells did not enhance endothelial levels of FGF-2, while levels of hepatoma-derived growth factor (HDGF) and hepatocyte growth factor (HGF) were elevated in endothelia growing in the presence of CAGssyn1 cells but not CAGHPSE cells or CAGcontrol cells. However, VEGF levels present in endothelial cells were substantially enhanced by the presence of CAGssyn1 (1.9-fold increase) or CAGHPSE cells (1.6-fold increase). Surprisingly, levels of VEGF in conditioned media of cocultures containing either CAGssyn1 or CAGHPSE cells was low. In contrast, when cultured in the absence of HUVECs, VEGF levels were elevated in conditioned media of both CAGssyn1and CAGHPSE cells. Addition of this conditioned media containing high levels of VEGF to HUVECs growing in the absence of CAG cells did not result in an elevation of VEGF levels in the endothelial cells. Together, these experiments suggest that VEGF expression is upregulated in CAG cells expressing high levels of shed syndecan-1 or heparanase and that VEGF becomes associated with the endothelial cells only when they are cultured in the presence of the myeloma cells. This cross-talk between myeloma and endothelial cells may lead to the enhanced angiogenesis that occurs in vivo in tumors formed by myeloma cells producing high levels of shed syndecan-1 and/or heparanase.

Immunoexpression of Macrophage Inflammatory Protein-1 Alpha on Bone Marrow Trephine Biopsies Correlates with the Extent of Bone Disease in Newly Diagnosed Patients with Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5119-5119
Author(s):  
Maria Roussou ◽  
Anna Tasidou ◽  
Efstathios Kastritis ◽  
Magdalini Migkou ◽  
Maria Gavriatopoulou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Disease ◽  
Newly Diagnosed ◽  
Santa Cruz ◽  
Myeloma Bone Disease ◽  
Inflammatory Protein ◽  
Lytic Lesions ◽  
Intermediate Expression ◽  
Macrophage Inflammatory Protein

Abstract Macrophage inflammatory protein-1 alpha (MIP-1alpha) is a low molecular weight chemokine that belongs to the RANTES family and is a potent osteoclast stimulator. Previous studies have shown that malignant plasma cells (PCs) from myeloma cell lines produce MIP-1alpha, while MIP-1alpha levels are elevated in the bone marrow plasma and the serum of patients with multiple myeloma (MM) and correlate with the extent of bone disease. The purpose of our study was to evaluate the role of MIP-1alpha immunoexpression on bone marrow trephine biopsies in myeloma bone disease and examine possible correlations between MIP-1alpha expression with survival and prognostic factors of MM. We evaluated formalin fixed paraffin-embedded bone marrow sections of 130 patients with newly diagnosed MM (66M/64F, median age: 68 years). Bone marrow sections were subjected to immunohistochemistry study using the anti-MIP- 1alpha monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The immunoreactivity of MIP-1alpha was examined on the basis of positive PCs with the following cut off values: &lt;20% positive PCs (negative expression group, group I), 20–50% positive PCs (intermediate expression group, group II) and &gt;50% positive PCs (high expression group, group III). Moreover MIP-1alpha was measured using ELISA methodology (R&D Systems, Minneapolis, MN, USA) in the serum of the patients. The extent of bone disease was assessed radiographically, and grading was performed as follows: group A included 43 patients (33%) who had no lytic lesions and/or osteoporosis only, group B included 24 patients (18%) who had lytic lesions in 1–3 areas, whereas group C included 63 patients (48%) with lytic lesions in &gt;3 areas and/or a bone fracture. Thirty-seven (28%) patients had negative MIP-1alpha expression, 17 (13%) intermediate expression and 79 (59%) high expression of MIP-1alpha. MIP-1alpha expression of ≥20% PCs in the trephine biopsies significantly correlated with the extent of bone disease (Figure 1, ANOVA p&lt;0.0001). Furthermore, 91 patients (70%) had values of serum MIP-1alpha &gt;14 pg/ml, which was the median value observed in 20 controls of similar age and gender (p&lt;0.001). MIP-1alpha serum levels also correlated with the extent of bone disease (ANOVA p&lt;0.0001). In terms of overall survival, no significant association was observed in relation to MIP-1alpha expression. Increased immunoexpression of MIP-1alpha was associated with high plasma cell infiltration in the bone marrow (r=0.348, p&lt;0.0001), low platelet count (r=0.282; p=0.0012), hypercalcemia (r=0.246; p=0.022), elevated serum creatinine (r=0.258, p=0.027), and advanced disease stage (ISS, p=0.034). Our findings underline, for the first time, the increased incidence of high immunoexpression of MIP-1alpha in the malignant PCs of myeloma patients and the correlation between the expression of MIP-1alpha by myeloma PCs in the trephine biopsies and the extent of lytic bone disease. These results confirm the significant role of MIP-1alpha in the pathogenesis of myeloma bone disease and suggest that MIP-1alpha may represent a rational therapeutic target for the management of bone destruction in myeloma. Figure Figure

Next Generation XPO1 Inhibitor KPT-8602 for the Treatment of Drug-Resistant Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1818-1818 ◽  
Author(s):  
Joel G Turner ◽  
Jana L Dawson ◽  
Christopher L Cubitt ◽  
Erkan Baluglo ◽  
Steven Grant ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Ex Vivo ◽  
Single Agent ◽  
Resistant Cell ◽  
Drug Resistant ◽  
Newly Diagnosed ◽  
Induced Apoptosis ◽  
Drug Resistant Cell

Abstract Purpose Human multiple myeloma (MM) remains an incurable disease despite relatively effective treatments, including proteasome inhibitors, immunomodulator-based therapies, and high-dose chemotherapy with autologous stem cell rescue. New agents are needed to further improve treatment outcomes. In previous studies, we have shown that inhibitors of the nuclear export receptor XPO1, in combination with bortezomib, carfilzomib, doxorubicin, or melphalan, synergistically induced apoptosis in MM cells in vitro, in vivo and ex vivo without affecting non-myeloma cells. In early clinical trials, the oral, brain penetrating XPO1 inhibitor selinexor showed clear anti-myeloma activity however adverse events have been recorded, including nausea and anorexia. Our purpose was to investigate the use of oral KPT-8602, a novel small-molecule inhibitor of XPO1 with minimal brain penetration, which has been shown to have reduced toxicities in rodents and primates while maintaining potent anti-tumor effects. Experimental Procedures To test the efficacy of KPT-8602, we treated human MM cell lines (both parental and drug-resistant) with KPT-8602 ± currently used MM drugs, including bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, and VP-16. Human MM cell lines assayed included RPMI-8226 (8226), NCI-H929 (H929), U266, and MM1.S, PI-resistant 8226-B25 and U266-PSR, doxorubicin-resistant 8226-Dox6 and 8226-Dox40, and melphalan-resistant 8226-LR5 and U266-LR6 cell lines. MM cells (2-4x106 cells/mL) were treated for 24 hours with KPT-8602 (300 nM), followed by treatment with one of the listed anti-MM agents for an additional 24 hours. MM cells were then assayed for cell viability (CellTiter-Blue, Promega). In addition, cells were treated with KPT-8602 ± anti-MM agents concurrently for 20 hours and assayed for apoptosis by flow cytometry. In vivo testing was done in NOD/SCID-g mice by intradermal injection of U266 MM cells. Treatment started 2 weeks after tumor challenge with KPT-8602 (10 mg/kg) ± melphalan (1 or 3 mg/kg) 2X/week (Tuesday, Friday) or with KPT-8602 alone 5X weekly (10 mg/kg) (Monday-Friday). A parallel experiment was run using the clinical XPO1 inhibitor KPT-330 (selinexor). Ex vivo testing was performed on MM cells from newly diagnosed/relapsed patient bone marrow aspirates with KPT-8602 ± bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, or VP16. CD138+/light-chain+ cells were assayed for apoptosis by flow cytometry. Results Viability assay showed that KPT-8602 had low IC50values (~140 nM) as a single agent and functioned synergistically with bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16. (CI values < 1.0). This synergistic effect was less pronounced in myeloma cells when KPT-8602 was used in combination with dexamethasone or lenalidomide. KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 combination therapy also induced apoptosis in all MM cell lines tested, including drug-resistant cell lines, as shown by caspase 3 cleavage and flow cytometric analyses. NOD/SCID-gamma mouse tumor growth was reduced and survival increased in KPT-8602/melphalan-treated mice when compared to single-agent controls. In addition, mice treated with KPT-8602 5X weekly had significantly reduced tumor growth and increased survival when compared to 2X weekly drug administration. No toxicity was observed in KPT-8602-treated mice as determined by weight loss in both the 2X and 5X groups. In patient bone marrow biopsies, the combination of KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 was more effective than single agents at inducing apoptosis in CD138+/LC+ MM cells in both newly diagnosed and relapsed/refractory patient samples. Conclusions We found that the novel XPO1 inhibitor KPT-8602 sensitizes MM cells to bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 as shown by apoptosis in parental and drug-resistant cell lines and by cell viability assays. Sensitization was found to be synergistic. In addition, KPT-8602 was effective in treatment of human MM tumors in mice as a single agent or in combination with melphalan and was effective when combined with several MM drugs in MM cell lines and MM patient bone marrow aspirates. KPT-8602 may be a potential candidate for future clinical trials. Disclosures Shacham: Karyopharm: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties.

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