bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Role for Thrombin in Hematopoietic Stem/Progenitor Cell Homing.
Authors: Shirvaikar Neeta , Jalili Ali , Ratajczak Mariusz Z. , Janowska-Wieczorek Anna
Published: October 2019

Thrombin, an important serine protease, not only plays a pivotal role in platelet aggregation and coagulation, but also through activation of its receptor, seven transmembrane, G-protein-coupled receptor PAR-1, elicits numerous cellular responses in platelets and endothelial cells such as induction of adhesion molecules, production of chemokines, activation of matrix metalloproteinase (MMP)-2, cytoskeletal reorganization and migration. Thrombin is also one of the inflammatory molecules elevated during G-CSF mobilization of hematopoietic stem/progenitor cells (HSPC) and their collection by leukapheresis. We recently reported that components of leukapheresis products including thrombin enhance in vitro chemotaxis of CD34+ cells towards an SDF-1 gradient and in vivo homing to bone marrow (BM) niches in a murine model (Blood2005; 105:40). In this study we investigated whether thrombin enhances the homing-related responses of human HSPC (CD34+ cells) through MMPs, especially membrane-type (MT)1-MMP which is known to be localized on the leading edge of migrating cells and both activates latent proMMPs (MMP-2, -9) and itself has strong pericellular proteolytic activity. We found that stimulation of CD34+ cells with thrombin upregulates mRNA for MT1-MMP and MMP-9 as well as MT1-MMP protein expression (Western blot, flow cytometry) and proMMP-2 and proMMP-9 secretion (zymography). Thrombin was also found to (i) prime trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP, and (ii) activate MMP-2 in of co-cultures of CD34+ cells with stromal cells (BM fibroblasts and HUVEC) which secrete proMMP-2. We also found that SDF-1 upregulates mRNA and protein expression of MT1-MMP. Moreover, using confocal microscopy we demonstrate for the first time that in CD34+ cells, PAR-1, like CXCR4, is localized in the GM1 fraction of lipid rafts and stimulation of these cells with thrombin as well as SDF-1 increases incorporation of MT1-MMP into membrane lipid rafts. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibits MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards SDF-1. Thus we conclude that thrombin, through PAR-1 signalling and the SDF-1-CXCR4 axis, upregulates the incorporation of MT1-MMP into membrane lipid rafts and the interaction of these axes enhances the homing-related responses of HSPC towards SDF-1.
Kinetics of Autologous Stem Cell Mobilization Failure: Comparison of AMD3100/G-CSF, G-CSF, GM-/G-CSF, and Chemotherapy/G-CSF on Remobilization Success.
Authors: DiPersio John F. , Smith Angela , Sempek Dianne , Baker Albert , Jiang Steven , Vij Ravi , Cashen Amanda , Westervelt Peter
Published: October 2019

Background: No standard approach for the mobilization of peripheral hematologic stem and progenitor cells (HSPCs) has been established. High levels of circulating CD34+ cells, a surrogate marker for mobilization efficiency, are associated with less apheresis days. A higher dose of CD34+ cell transfused after high-dose chemotherapy decreases time to hematologic recovery. Consequently, a better understanding of variables associated with mobilization kinetics may further optimize stem cell collection and reduce complications associated with autologous stem cell transplants.

Methods: The Washington University (St. Louis, MO) transplantation database includes clinical parameters from 407 multiple myeloma (MM), 567 non-Hodgkin’s Lymphoma (NHL), and 164 Hodgkin’s disease (HD) pts who received an ASCT between 1995 and 2006. A retrospective analysis of this large pt population was conducted to determine factors associated with the mobilization kinetics of CD34+ cells.

Results: Figure 1 summarizes the mobilization kinetics as defined by number of days to reach a target of 2 × 10^6 CD34+ cells/kg. Overall, the median number of aphereses to reach the target were 1, 2, and 2 in MM, NHL, and HD, respectively. Daily median CD34+ yields in MM pts were 3.8, 1.2, and 0.5 × 10^6 on day 1–3, respectively. In NHL pts, yields were 1.4, 0.8, and 0.4 × 10^6 on day 1–3. In HD pts, yields were 1.8, 0.8, and 0.3 × 10^6 on day 1–3, respectively. The addition of chemotherapy increased the % of pts requiring only a single apheresis to reach the mobilization target. Figure 2 summarizes the mobilization kinetics for each re-mobilization regimen. In general, a limited number of cells was collected with each aphereses; >70% of pts failed to mobilize 2 × 10^6 CD34+ cells/kg. In contrast, remobilization with AMD3100 allowed the collection of sufficient CD34+ cells in 67% of pts; median number of apheresis to reach the target was 3.

Conclusions: Factors associated with mobilization kinetics of CD34+ cells include disease state and mobilization regimen. Re-mobilization is associated with high failure rates, re-mobilization regimens including AMD3100 are more successful.

Figure 1: Mobilization kinetics by disease state Figure 1:. Mobilization kinetics by disease state Figure 2: Mobilization kinetics by re-mobilization regimen Figure 2:. Mobilization kinetics by re-mobilization regimen
Single Agent AMD3100 Mobilization of Peripheral Blood Progenitor Cells for Autologous Transplantation in Patients with Multiple Myeloma (MM).
Authors: Flomenberg Neal , Comenzo Raymond , Badel Karin , Calandra Gary
Published: October 2019

Background: The addition of AMD3100 to a G-CSF mobilization regimen increases the number of hematopoietic stem and progenitor cells (HSPCs) that can be collected by apheresis, allowing the successful (re)mobilization and transplantation of MM patients. This single-arm, open-label, study was therefore initiated to determine if AMD3100 alone could successfully mobilize HSPCs in MM patients for autologous stem cell transplantation (ASCT). This is the first clinical trial of HSPCs mobilized by AMD3100 alone in the autologous transplant setting.

Methods: Patients received 240 μg/kg AMD3100 followed by apheresis 6 hours later. Dosing and apheresis could be repeated for up to 4 consecutive days or until ge;4 × 106 CD34+ cells/kg were collected. Post-transplant follow-up included daily WBC measurements until leukocyte engraftment (≥0.5 × 109/l for 3 consecutive days or ≥1 × 109/l for 1 day), and platelet engraftment (≥20 × 109/l for 7 days).

Results: Nine patients have been enrolled. The median number of CD34+ cells collected was 2.7 × 106/kg (Range:1.9– 4.6), completed in 4 apheresis days by 8 patients and 3 days by 1 patient. Six patients (67 %) mobilized ≥2 × 106 CD34+ cells/kg and 2 pts (22 %) mobilized ≥4 × 106 CD34+ cells/kg. All patients were transplanted; 1 patient received tandem transplants with study cells; 1 patient received tandem transplants, the first transplant with study cells followed by a second transplant with G-CSF mobilized cells. Median time to PMN engraftment was 10 days (Range: 10–11 days); median time to PLT engraftment was 21 days (Range: 15–33 days, except for 1 patient with platelet recovery some time between day 23 and 82). One patient with Plasma Cell Leukemia died of disease relapse at 6 months. Engraftment was durable with a follow-up of over 19 months in the earliest transplanted patient. AMD3100 was safe and generally well tolerated with the most common adverse events being injection site reactions, GI upset, and paresthesias. There were no drug-related severe adverse events.

Conclusions: AMD3100 alone mobilizes autologous HSPCs that provide prompt and durable engraftment, indicating that AMD3100 mobilizes functional HSPCs independent of the presence of G-CSF. These results are consistent with and reinforced by observations made using AMD3100 to mobilize allogeneic HSPCs [Devine et al., ASH 2005, #299]. While the number of cells mobilized by AMD3100 alone is relatively modest, AMD3100 plus G-CSF mobilizes a more primitive subset of CD34+ cells than G-CSF alone which could potentially translate into fewer CD34+ cells being required for transplantation when AMD3100 is part of the mobilization regimen [Seeger et al., ASH 2005, #1969]. Therefore, further evaluation of dose and schedule, as well as the role of CD34+ phenotype, is required to optimize HSPC mobilization with single-agent AMD3100.
An Update on the Role of Thalidomide (THAL) in Total Therapy 2 (TT2) for Newly Diagnosed Patients with Multiple Myeloma (MM): Analysis of Subgroups Defined by Standard Prognostic Factors (SPF) and Gene Expression Profiling (GEP)-Derived Subgroups.
Authors: Shaughnessy John D. , Haessler Jeffrey , Zeldis Jerry , Huang Yongsheng , Zhan Fenghuang , Sawyer Jeffrey , Anaissie Elias , van Rhee Frits , Tricot Guido , Epstein Joshua , Zangari Maurizio , Pineda-Roman Mauricio , Hollmig Klaus , Crowley John , Barlogie Bart
Published: October 2019

Background: THAL, whose activity in MM was discovered in the setting of advanced and refractory disease in the late 1990’s (Singhal, NEJM, 2000), has become the standard front-line therapy in combination with dexamethasone (DEX). In a randomized phase III tandem transplant trial, TT2, a higher complete response (CR) rate and longer event-free survival (EFS) had been observed on the THAL arm (Barlogie, NEJM, 2006). The similar overall survival (OS) on THAL and control arms had been attributed to the routine use of THAL as salvage therapy for the patients randomized to the No-THAL arm and the shorter post-relapse OS among patients randomized to the THAL arm.

Patients and Methods: With a median follow-up on TT2 of 53mo, 107 patients have relapsed and 219 died. Subset analyses were performed to determine whether THAL confers an OS advantage in any subgroup of patients.

Results: 6-yr EFS and OS rates are 48%/63% on THAL and 38%/58% on control arm (p=0.01/0.67). Post-relapse OS is now similar with median durations of 5.3mo/4.3mo among control/THAL arms (p=0.11). According to multivariate analyses of 11 standard prognostic factors, EFS was shorter among patients treated without THAL, in the presence of cytogenetic abnormalities (CA), B2M and LDH elevations and low albumin, whereas CR was favorable; OS was inferior with CA, high LDH, low albumin and in patients not receiving 2nd transplant or not achieving CR. Randomization to THAL was beneficial only in the >2 risk factor group: 6-yr OS was 47% in 31 patients on THAL and 12% in 31 control patients (Figure 1, p=0.01). When examined in the context of GEP (70 gene model-based high versus low risk groups) and inter-phase FISH data (amp1q21), available in 260 patients, the 57 with GEP low risk and absence of amp1q21 receiving THAL had 5-yr OS of 90% compared to 74% among 73 controls (p=0.13).

Conclusion: With longer follow-up of 53mo on TT2, EFS remains superior among patients randomized to THAL; post-relapse survival is no longer inferior among those randomized to THAL; THAL benefited a high-risk subgroup with >2 standard risk factors, whereas no significant `difference has yet emerged among genetically defined subgroups.

Figure Figure
Amplification of 1q21 Is Associated with Poor Outcome after Treatment with Bortezomib in Relapsed/Refractory Multiple Myeloma.
Authors: Drach Johannes , Sagaster Verena , Odelga Victoria , Kaufmann Hannes , Ackermann Jutta , Galhuber Markus , Zojer Niklas , Küenburg Elisabeth , Zielinski Christoph , Wieser Rotraud , Ludwig Heinz
Published: October 2019

Bortezomib is an active agent for treatment of multiple myeloma (MM) and may even be effective in patients (pts) with adverse prognostic factors including unfavorable cytogenetic abnormalities. However, it is unknown whether or not bortezomib may overcome the negative prognostic impact of a CKS1B gene amplification at chromosome 1q21 (amp1q21), which has recently been reported as a negative prognostic factor even in the setting of a total therapy approach. We therefore evaluated chromosome 1q21 among other abnormalities in 46 pts with relapsed/refractory MM who were treated with single-agent bortezomib (1.3 mg/m2 on days 1, 4, 8, and 11 every 3 weeks). Median age of pts was 63 years (range, 40 – 82 ) and median time to bortezomib therapy was 40 months (median number of prior therapies: 3; 96% of pts had high-dose pulsed dexamethasone, 61% thalidomide, 85% alkylating agents, and 41% high-dose melphalan). A response to bortezomib was noted in 45% of pts, with 17% of pts achieving a CR/near CR. Amp1q21 as determined by interphase FISH was observed in 20 of the 46 pts (43.5%). Treatment outcome after bortezomib was negatively affected by presence of amp1q21: The overall response rate was 30% (versus 58% in pts with normal 1q21; P = .06) and the CR/near-CR rate was 10% (versus 23%). Moreover, amp1q21 was associated with shortened time to treatment failure (median, 2.4 versus 6.6 months; P = .043) and overall survival (OS) (median, 4.4 versus 19.8 months; P = .003) compared to pts with normal 1q21. Beta-2-microglobulin and 14q32 translocations were unrelated to treatment outcome with bortezomib. In contrast, median OS was short in the presence of low serum albumin (4.8 versus 17.8 months; P = .036) and deletion of 13q14 (6.7 months versus median not yet reached; P = .06). Using amp1q21, low serum albumin, and deletion of 13q14 as risk factors, patients with significantly different median OS after bortezomib treatment were discriminated: Pts with ≥ 2 risk factors had a median OS of only 4.9 months as opposed to pts with 1 risk factor (median OS 16.5 months) or without any risk factor (median OS not yet reached) (P = .004). In conclusion, FISH-defined amp1q21 is a strong adverse prognostic factor for pts with relapsed/refractory MM treated with single-agent bortezomib. We are currently investigating whether or not bortezomib combinations may be more effective in MM pts with amp1q21.
A Novel Multiplex Drug Screening Assay for Identification of Potential Compounds with Anti-Myeloma Activity from a Library of 1120 Drugs.
Authors: Feng Rentian , Lokshin Anna , Gorelik Elieser , Lentzsch Suzanne
Published: October 2019

The majority of drug screening assays are aimed at selection of compounds that affect proliferation or survival of myeloma cells. However, this approach might fail to identify compounds with a potent therapeutic activity that are unable to directly inhibit tumor cell proliferation in vitro but might have potent anti-tumor activity in vivo by targeting the microenvironment of the myeloma cell. For this purpose we used a Multiplex drug-screening assay (MDSA) to identify compounds with potential anti-myeloma activity from a library of 1120 compounds provided by the Multiple Myeloma Research Foundation (MMRF). MDSA is based on use of the Luminex technology (LabMAP Multianalyte Profiling), and testing various myeloma producing factors (MPFs), such as cytokines, chemokines and growth factors that are important for myeloma cell proliferation and survival. The multiple myeloma cell lines MM1.S, RPMI-8226, and IM9 were tested for their capacity to secrete the full set of 31 cytokines, chemokines and growth factors. RPMI-8226 was selected for MDSA due to its high capacity to secrete MPFs (IL-8, VEGF, MCP-1, MIP-1α, MIP-1β, IP10, RANTES and SIL-6R). RPMI-8226 cells were treated with 10 10−6M of each compound (first screening phase) and 1 10−6M (secondary screening), and supernatants from 72-hour cultures were analyzed. The criterion of effective drugs for each cytokine was set up as the ability to inhibit or stimulate MPFs (exceed +/− 1.5 mean value of non-treated control). The resulting data on the drugs were graded by the degree to which they caused inhibition or stimulation of all MPFs (greater than 50% and greater than 90%).

A total of 205 of the 1,120 candidates were picked out from the first screening at 10 10−6M. Results from the second analysis (at 1 10−6M) indicated that 14 compounds achieved inhibition of all MPFs and dequalinium dichloride manifested the strongest inhibition of all MPFs. Forty drugs were able to selectively inhibit certain MPFs at levels that exceeded 50% and 14 drugs inhibited MPFs by 90%. With respect to stimulation of cytokine secretion, a total of 39 compounds demonstrated selective stimulation of some MPFs and three drugs (amethopterin (R, S), etoposide, and lasalocid sodium salt) induced stimulation at the level of 90% or greater.

Overall, MDSA is a powerful high throughput screening assay to analyze compounds with inhibitory or stimulatory effects on cytokines, chemokines and growth factors that are involved in the pathogenesis of multiple myeloma. Potent compounds identified in this study warrant further investigation for their anti-myeloma effects in vitro and in vivo.
Variation in DNA Repair Genes XRCC3, XRCC4, and XRCC5 and Risk of Myeloma.
Authors: Hayden Patrick , Tewari Prerna , Staines Anthony , Morris Derek , Crowley Dominique , Nieters Alexandra , Becker Nicholas , de Sanjose Silvia , Foretova L. , Maynardie Mark , Coco Pier Luigi , Boefetta Paolo , Brennan Paul , Browne Paul V. , Lawler Mark
Published: October 2019

Aberrant class switch recombination (CSR), the physiological process that regulates maturation of the antibody response, is believed to be an early event in the pathogenesis of myeloma. The genetic basis of CSR, from initiation of the DNA double-strand break through to detection and repair, has been elucidated. We hypothesised that germline polymorphisms in the genes implicated in DNA double strand break repair may contribute to susceptibility to myeloma. We therefore assessed 32 SNPs in 3 genes central to the DNA repair pathway in patients with myeloma and controls from the EpiLymph Study, a European study of the epidemiology of lymphoid neoplasms, and from an Irish hospital registry (306 cases, 263 controls). The genes examined were XRCC3, XRCC4, and XRCC5. XRCC3 is a member of the RecA/Rad51-related protein family that participates in homologous recombination. The XRCC4 protein forms a complex with DNA ligase IV and DNA-dependent protein kinase in the repair of DNA double-strand breaks by non-homologous end joining. XRCC5 encodes the 80-kilodalton subunit of the Ku heterodimer protein, the DNA-binding component of the DNA-dependent protein kinase. SNPs from the chosen genes were identified from HapMap CEU Phase I and II genotype data (Public Release #20; 2006-01-24). Haplotype-tagging SNPs (htSNPs) were chosen based on Tagger analysis (as implemented in Haploview Version 3.2). A SNP in GSTP1 was also genotyped to allow for comparison with allele frequencies previously generated from a myeloma cohort. Genotyping was performed using TaqMan®-based assays on the 7900 ABI HT platform. The drop-out rate was consistently less than 3% in all assays. For quality control (QC) purposes, duplicates of 10% of the samples were interspersed throughout the plates. The concordance rates for QC samples were greater than 98%. GSTP1 SNP results were comparable to previously published findings. For the htSNP rs963248 in XRCC4, Allele A was significantly more frequent in cases than in controls (86.4% vs.80.8%) (p=0.0105), as was the AA genotype (74% vs. 65%) (p=0.026). Haplotype analysis was performed using Cocaphase for rs963248 in combination with additional SNPs in XRCC4. The strongest evidence of association came from the A - T haplotype from rs963248-rs2891980 (80.9% vs. 74.5%; p=0.008). For XRCC5, the genotype GG from rs1051685 was detected in 10 cases from different national populations but in only 1 control (p=0.015). Interestingly, this SNP is located in the 3′ UTR of XRCC5. Overall, these data provide support for the hypothesis that common variation in the genes encoding DNA repair proteins contributes to susceptibility to myeloma.
Expression of Wnt-3a in Myeloma Cells Inhibits the Osteolytic Phenotype In-Vivo.
Authors: Qiang Ya-Wei , Yaccoby Shmuel , Shaughnessy John D.
Published: October 2019

Wnt signaling is a highly conserved signal transduction pathway involved in embryonic development. Inappropriate canonical Wnt signaling resulting in beta-catenin stabilization, is associated with several types of human cancers. Multiple myeloma plasma cells express Wnt receptors, Wnt ligands and soluble Wnt inhibitors. Wnt signaling is central to osteoblast and osteoclasts development and secretion of Wnt signaling inhibitors by myeloma cells is thought to contribute to the osteolytic phenotype seen in this disease and prostate cancer. While it is now clear that MM cells can signal through both canonical and non-canonical mechanisms, there are conflicting data as to the direct role of Wnt signaling in myeloma cell biology. Others have shown that Wnts cause proliferation of myeloma cells; while we have shown that canonical Wnts cause morphological changes and migration, but not cell proliferation. To further elucidate the role of canonical Wnt signaling in myeloma and myeloma bone disease we used limiting dilutions in the presence of G418 to create two independent stable clones of the myeloma cell line NCI-H929 expressing Wnt-3A (H929/W3A), which is not expressed in myeloma, and an empty vector (H929/EV). Because Wnt antibodies are not available we cloned Wnt-3A as a fusion protein with hemagglutinin (HA). Western blots against HA revealed a positive band of the expected size only in the H929/W3A clones. GST-E-cadherin binding assay and Western blot analysis revealed elevated levels of total and free beta-catenin in H929/W3A relative to H929/EV, however, there this was not associated with increased growth or proliferation by MTT assay. To determine the in-vivo growth characteristics and effects on bone resorption of Wnt-3A producing cells, we transplanted the lines into a human bone implanted the flank of SCID mice. Tumor growth rate as determined by increased production of human immunoglobulin in mice serum was significantly slower in the Wnt-3A transfected cells relative to controls (P < .05). Loss of bone mineral density (BMD) of the implanted bones engrafted with H929/W3A cells was lower than in bones engrafted with H929/EV cells (P < .05). Reduced tumor burden and BMD loss was also visualized on x-ray radiographs. Taken together these data indicate that all factors promoting bone resorption produced by or elicited by the myeloma cell line H929 are subordinate to canonical Wnt signaling and that prevention of bone destruction may help control myeloma progression.
Histone Deacetylase-6 (HDAC6) Modulates Akt and STAT3 Activity Via Heat Shock Protein (Hsp) 90 in Human Multiple Myeloma (MM) Cells.
Authors: Hideshima Teru , Bradner James E. , Yasui Hiroshi , Raje Noopur , Chauhan Dharminder , Podar Klaus , Tai Yu-Tzu , Mitsiades Constantine , Richardson Paul G. , Mazitschek Ralph , Schreiber Stuart L. , Anderson Kenneth C.
Published: October 2019

Histone deacetylase 6 (HDAC6) has an essential role to recruit ubiquitinated proteins to transport to aggresomes, which ultimately induces lysosomal protein degradation. We have shown that inhibition of proteasomes with bortezomib and of aggresomes with HDAC6 inhibitor Tubacin demonstrated significant cytotoxicity in MM cell lines and MM patient tumor cells in vitro (Hideshima T et al., PNAS2005, 102: 8597–8572). In this study, we further examined the biologic significance of HDAC6 inhibition by Tubacin in MM cells. We found that HDAC6 is constitutively associated with heat shock protein (Hsp) 90 in MM cell lines which is enhanced by Tubacin, as evidenced by co-immunoprecipitation. Since Akt and STAT3 have been shown to play important role in proliferation, anti-apoptosis, and drug resistance in MM cells; and all are client proteins of Hsp90, we next further examined whether inhibition of HDAC6 could modulate activities of these proteins via Hsp90. Importantly, Tubacin enhanced phosphorylation of Akt, associated with augmentation of Hsp90 acetylation. Hsp90 inhibitor 17-AAG downregulated Akt phosphorylation associated with enhanced interaction of Hsp90 with Akt, which was partially blocked by Tubacin. On the other hand, 17-AAG did not enhance acetylation of α-tubulin or ubiquitination of proteins, suggesting that Hsp90 does not affect HDAC6 function. Furthermore, we found that STAT3 is also constitutively associated with Hsp90. Importantly, both Tubacin and 17-AAG inhibit phosphorylation of STAT3 in a dose- and time-dependent fashion in MM cells. Taken together, our data indicate that HDAC6 has an important role not only in aggresomal protein degradation, but also in MM cell pathogenesis by modulating Akt and STAT3 signaling cascades via Hsp90 acetylation in MM cells.
Azacitidine Down-Regulates Both IL-6 Signalling and NFkB Activity in Human Myeloma Cells.
Authors: Khong Tiffany , Spencer Andrew
Published: October 2019

We have previously demonstrated that Azacitidine (AZA) at clinically achievable concentrations (<10μM) induces apoptosis in a range of genetically heterogeneous human myeloma cell lines (HMCL) and prolongs survival in the murine 5T33 model of systemic multiple myeloma (MM). HMCL U266 demonstrates autocrine IL-6 production, constitutive Stat3 phosphorylation and is resistant to conventional chemotherapy and glucocorticoids. Using U266 we have evaluated further the effect of AZA on human MM cells. AZA treatment (5mM daily) of U266 induced apoptosis at 24, 48 and 72 hours of 28%, 43% and 52%, respectively. Immunoblot analyses showed rapid falls in IL-6R and p-Stat3 (both within 4 hours) prior to evidence of proteolysis (PARP cleavage). ELISA of AZA-treated U266 conditioned media demonstrated a reduction in IL-6 and an immediate and sustained reduction in soluble IL-6R levels, consistent with reduced IL-6 synthesis and IL-6 receptor shedding, respectively. RT-PCR confirmed that the rapid falls in IL-6 and IL6-R were not secondary to reduced transcription with relative increases of 1.7 and 2.4-fold, respectively, by 8 hours post-AZA. Flow cytometry showed an initial rise and subsequent modest fall in both membrane and cytosolic gp130 while RT-PCR and immunoblot analyses showed a rapid fall in both SOCS3 transcriptional activity and protein levels, respectively. Time-course experiments with AZA 5μM and the addition of either physiological (100pg/ml) or supra-physiological (3ng/ml) concentrations of exogenous IL-6 over a 96-hour period demonstrated that 3ng/ml but not 100pg/ml IL-6 was capable of partially restoring Stat3 phosphorylation but that neither concentration of exogenous IL-6 prevented AZA-induced cell death. NFkB activity was evaluated with a luciferase reporter enzyme system following treatment for 24 hours with AZA 1μM, 5μM and 10μM. Marked suppression of NFkB activity was seen at both the 5μM and 10μM dose levels. Flow cytometric evaluation of phospho-IkB and IkB showed a modest decline in the pIkB:IkB ratio over the same period. Finally, combined treatment of U266 with AZA and dexamethasone demonstrated synergistic killing (synergism quotient = 1.2) when AZA was administered prior to dexamethasone but not in the reverse order. We conclude that AZA induces down-regulation of both IL-6 signalling and NFkB activity. Furthermore, our data suggest that the former effect is most likely mediated by rapid AZA-induced inhibition of protein synthesis. The mechanism of NFkB inhibition requires further clarification. AZA represents a potentially novel therapeutic approach to MM.
Stromal Cell-Derived Factor-1 Stimulates the Growth of Myeloma Cells in Concert with IL-6 in Both Autocrine and Paracrine Manners.
Authors: Kim Seong-Woo , Lee Hyo-Jin , Yun Hwan-Jung , Kim Samyong , Jo Deog-Yeon
Published: October 2019

Stromal cell-derived factor-1 (SDF-1), which is constitutively expressed and produced in bone marrow (BM) stromal cells (BMSCs), functions via its receptor CXCR4 in the homing of myeloma cells to the bone marrow. In addition, SDF-1 inhibits dexamethasone-induced cell apoptosis. However, it has not been clarified whether SDF-1 stimulates myeloma cell growth. In this study, we explored the possibility that SDF-1 functions as an autocrine growth factor of myeloma cells. RPMI-8226 myeloma cells and primary CD138+ cells obtained from the BM of patients with multiple myeloma expressed SDF-1 mRNA, as revealed by RT-PCR analysis. SDF-1 was detected by ELISA in the supernatants of cultures of these cells grown for three days in serum-free medium, which indicates that myeloma cells are able to produce and secrete SDF-1. SDF-1 (added at a concentration of up to 100 ng/mL) alone had no discernible effects on the proliferation of RPMI-8226 cells and primary BM CD138+ cells in a serum-free condition. Interleukin-6 (IL-6) stimulated a moderate level of proliferation of these cells, while the combination of SDF-1 and IL-6 markedly enhanced the proliferation of these cells. IL-6 did not influence CXCR4 expression on myeloma cells. However, SDF-1 up-regulated significantly the expression of IL-6 receptor in these cells, as determined by Western blot analysis. SDF-1 and IL-6 enhanced in an additive fashion the phosphorylation of Akt and ERK1/2, but did not increase the phosphorylation of STAT3. Pertussis toxin (PTX, 200 ng/mL) and AMD3100 (100 nM), which is a specific antagonist of CXCR4, inhibited the proliferation of RPMI-8226 cells by 35% and 37%, respectively, in cultures grown for four days under serum-free conditions. To determine the effects of blocking the SDF-1/CXCR4 axis on the growth of myeloma cells in the presence of BMSCs, RPMI-8226 cells were co-cultured with MS-5 murine BMSCs. The addition of PTX and AMD3100 (100 nM) twenty-four hours after the beginning of the co-culture markedly decreased the number and size of cobblestone-like areas underneath the stromal cells. These results indicate that myeloma cells can produce SDF-1, and that SDF-1 in concert with IL-6 enhances the proliferation of myeloma cells in both a paracrine and autocrine manner.
SDX-308, a Structural Analog of Etodolac, Inhibits NF-κB Activity Resulting in Significant Inhibition of Osteoclast Formation/Activity and Multiple Myeloma Cell Growth.
Authors: Feng Rentian , Anderson Gulsum , Xiao Guozhi , Elliott Gary , Leoni Lorenzo M. , Mapara Markus Y. , Roodman David , Lentzsch Suzanne
Published: October 2019

Multiple myeloma (MM) is characterized by increased osteoclast activity resulting in bone destruction and the development of lytic bone lesions. Current treatment modalities have resulted in an increased overall survival in MM patients and new drugs are required that specifically inhibit bone destruction. Etodolac is a non-steroidal anti-inflammatory drug that is approved for treatment of degenerative joint disease and rheumatoid arthritis. SDX-101, an R-enantiomer of Etodolac, was recently demonstrated to induce cytotoxicity, overcome drug resistance, and enhance the activity of dexamethasone in MM. SDX-308 is a novel and more potent etodolac structural analog with a more favorable safety profile than the racemic etodolac due to a lack of significant COX-inhibitory activity.

In this study, we focused on effects of SDX-308 on osteoclastogenesis and MM cells. SDX-308 required a 10-fold lower concentration (5x10−6M) than SDX-101 (50x10−6M) to induce potent inhibition (60–80%) of osteoclast formation using mononuclear bone marrow cells from MM patients and healthy donors. Depending on the MM cell line (MM.1S, RPMI-822, or OPM2), SDX-308 required 10- to 100-fold lower concentration (1–10x10−6M) to inhibit MM cell proliferation compared to SDX-101 (10–100x10−6M). In addition, SDX-308 (7.5x10−6M) completely inhibited bone resorption as determined by dentin-based bone resorption assays. We found that pre-treatment of RAW264.7 osteoclast-like cells with SDX-308 decreased constitutive and RANKL-stimulated NF-κB activation measured by luciferase activity. Further, SDX-308 inhibited phosphorylation of p65, IκBα and p65 nuclear translocation in RAW264.7 cells. In addition, SDX-308 effectively suppressed TNFα-induced IKK-γ and IkB-α phosphorylation and degradation and subsequent NF-κB activation in human MM cells. Even in higher concentrations, SDX-101 was less effective in inhibiting NF-κB signaling.

In conclusion, these results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity by specifically targeting the NF-κB signaling pathway. These results show that SDX-308 is a promising therapeutic candidate for inhibiting tumor cell growth and elevated osteoclast activity in MM.
Delays in the Cancer Pathway from Symptom Onset to Initiation of Treatment – A Retrospective Study in Patients with Non-Hodgkin’s Lymphoma.
Authors: Nassur Reem , Mangel Joy , Chin-Yee Ian
Published: October 2019

Background: Cancer patients may experience significant delays along the clinical pathway from the onset of symptoms to initiation of treatment. Few studies have examined the nature or the extent of these delays in patients with non-Hodgkin’s lymphoma (NHL).

Method: London Regional Cancer Program (LRCP) is a tertiary care centre with a referral base of 1.5 million people in Southwestern Ontario. In this study, the charts of all patients with newly diagnosed NHL that were seen at the LRCP between Jan 1 – Dec 31, 2004 were reviewed retrospectively in order to measure and document the amount of time spent at each stage of the cancer pathway. Key time intervals from symptom onset to initiation of treatment were recorded. Associations between the waiting times and clinical/sociodemographic factors were also explored.

Results: The charts of 116 patients were reviewed. The median overall waiting time from symptom onset to the initiation of treatment was 137 days. Median waiting time for each interval in the pathway were as follows: from symptom onset to first visit with primary care physician: 17 days; from first visit with physician to surgical consultation: 28 days; from surgical consult to the definitive biopsy: 13 days; from definitive biopsy to pathological diagnosis: 10 days; from pathological diagnosis to hematology referral: −5 days; from hematology referral to hematology consultation: 8 days; from hematology consult to completion of staging: 8 days; and from final staging to initiation of treatment: 5 days. The time from symptom onset to treatment initiation varied somewhat by clinical factors such as gender, age, and presence of B-symptoms. Patients with B-symptoms experienced shorter delays (119 d) than patients without B-symptoms (154 d). Male patients had shorter pathway times (113 d) compared to female patients (138 d). Patients at the extremes of age (age 20–29 & age >80) tended to have shorter delays than patients between the ages of 30–79 (ages 20–29: 60d, age >80: 85d, ages 30–79:137 d). Whether patients were referred from London vs. from another municipality did not appear to influence delays (137 d).

Conclusions: NHL patients are experiencing significant delays in the assessment and treatment of their cancer. Most prior studies have focused primarily on delays from the point of referral to a cancer specialist to initiation of treatment. The results from our study suggest that a significant proportion of the patient’s clinical path is being spent at the prediagnosis phase (from the time of first physician visit until pathological diagnosis, 57 days) as opposed to from the time of diagnosis until the initiation of treatment (18 days). Efforts to reduce delays at both the pre- and post-diagnosis phases of the patient path may reduce patient anxiety with subsequent improvements in quality of life. The clinical impact of these delays on patient outcome is uncertain and requires further study.
Targeting Aurora Kinases as Therapy in Multiple Myeloma.
Authors: Shi Yijiang , Reiman Tony , Li Weiqun , Maxwell Christopher , Pilarski Linda , Feldman Rick , Lichtenstein Alan
Published: October 2019

The aurora kinases A and B are critical for facilitating cell cycle transit from G2 through to cytokinesis. As important regulators of the mitotic event, they are being tested as potential targets in cancer therapy. Multiple myeloma (MM) is a B cell malignancy characterized by progressive genetic instability, suggesting a disruption of cell cycle checkpoints has occurred that normally arrest cells at G2M or within mitosis when injury to the mitotic machinery occurs. Since these deficient checkpoints would prevent cell cycle arrest and potential repair and may render MM cells susceptible to apoptotic death in mitosis, we tested the anti-myeloma effecy of two separate agents that inhibit aurora kinases. Both agents induced cytoreduction of MM cell lines and primary myeloma samples at nM concentrations while normal peripheral blood lymphocytes and CLL cells were not affected. MM cells were not protected by IL-6 or activating mutations of RAS. Anti-myeloma effects were characterized by induction of tetraploidy followed by apoptosis. The myeloma apoptotic effect correlated well with the inhibition of aurora kinase activity as shown by reduction of histone 3B phosphorylation (substrate of auroras). Furthermore, stable ectopic over-expression of aurora kinase A significantly protected MM cells against aurora inhibitors but had no effect on apoptosis induced by velcade. As expression of the centrosomal protein RHAMM in MM cells may contribute to genetic instability, we tested effects of RHAMM over-expression on the sensitivity to aurora inhibitors. Although RHAMM over-expression in transfected MM cells was very modest, it significantly enhanced sensitivity to apoptosis induced by aurora kinase inhibitors. These results suggest the potential for aurora kinase inhibitors in multiple myeloma especially in patients where RHAMM is over-expressed.
Oncolytic Virotherapy for Multiple Myeloma.
Authors: McCart Judith Andrea , Deng Hui , Tang Nan , Sleep Gillian , Mehta Navneet , Stief Amaalia , Yang Xiu-Zhi , Cottick Kimberly , McKerlie Colin , Trudel Suzanne , Stewart Keith
Published: October 2019

Oncolytic virotherapy for cancer employs viruses which specifically lyse tumour cells, leaving normal cells intact. Double-deleted vaccinia virus (vvDD) is tumour-specific due to mutations that restrict viral replication to dividing cells. This is the first use of vvDD to treat a hematologic malignancy. vvDD expressing enhanced green fluorescent protein (EGFP) was used to infect multiple myeloma (MM) cell lines. We used flow cytometry (FACS) analysis to quantify EGFP positive cells in parallel to colorimetric (MTT) and microscopic cell viability assays. FACS results indicated vvDD efficiently (27–73%) infected the MM cell lines RPMI8226, MY5, H929, U266 and LP1 by 72 hours post infection. MTT assays revealed a 7–8 fold decrease in cell viability of infected cells compared to controls. MM cell lines were grown as subcutaneous xenografts (My5) or systemically (RPMI8226) in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, and tumour size/paralysis was monitored after systemic delivery of vvDD. My5 tumours responded to ip vvDD, resulting in a significant delay of tumour growth compared to controls, while 80% of mice treated with vvDD 3 days after iv RPMI8226 tumour injection were cured. Analysis of mouse tissues for viral biodistribution and pathogenicity showed that the ovary was the second highest viral containing tissue though it was 4 logs lower than tumour. Ovaries showed some evidence of follicular necrosis in treated tissues. All other tissues were unaffected. Primary human MM (CD138+) cells obtained from bone marrow aspiration were readily (50–60%) infected with vvDD while normal mononuclear cells from the same samples were not infected. At the same time primary human CD138+ cells were infected with vvDD at an MOI of 1 or mock infected and then plated in matrigel. vvDD treated cells showed significantly reduced colony formation compared to mock-infected controls. These results suggest that vvDD is a safe and effective treatment for multiple myeloma and should be considered for phase I testing in these patients.
Host Immune Responses Against CT Antigens in Multiple Myeloma Patients.
Authors: Lendvai Nikoletta , Gnjatic Sacha , Ritter Erika , Chen Yao-Tseng , Coughlin Christina , Vonderheide Robert H. , Niesvizky Ruben , Bhardwaj Nina , Chen-Kiang Selina , Old Lloyd J. , Cho Hearn J.
Published: October 2019

The type I Melanoma Antigen GEne (MAGE) proteins belong to the Cancer-Testis (CT) family of tumor-associated antigens and are widely expressed in solid and hematologic malignancies. They are immunogenic and frequently elicit spontaneous immune responses in patients with CT antigen-expressing tumors, particularly in malignant melanoma. In melanoma patients, there is high concordance between humoral and cellular immunity. Based on these findings, CT antigens are widely investigated as potential antigenic targets for tumor-specific therapeutic vaccines. We previously showed that the type I MAGE proteins CT7 (MAGE-C1), CT10 (MAGE-C2) and MAGE-A3 were commonly detected in primary myeloma specimens, and expression of CT7 and MAGE-A3 was correlated with abnormally elevated plasma cell proliferation. These findings suggest that type I MAGE may be rational targets for vaccine therapy in multiple myeloma. Therefore, it is important to determine if type I MAGE elicit cellular or humoral immune responses in myeloma patients. To investigate this hypothesis, we assessed cellular immunity against CT7 and humoral immunity against a broad panel of CT antigens. To quantify CT7-specific cellular immunity, expanded, polyclonal pools of T cells from the bone marrow, the tumor microenvironment, were co-cultured in interferon gamma (IFNγ) ELISpot assays with autologous antigen-presenting cells (APC) transduced with in vitro transcribed mRNA coding for CT7 or control antigens. CT antigen-specific humoral immunity was examined by ELISA assay using patient serum or plasma and recombinant CT antigens. This analysis demonstrated that 2/9 patients exhibited specific T cell immunity against CT7 in their bone marrow lymphocytes as measured by IFNγ secretion. These same two patients had positive titers for other CT antigens; one for MAGE-A1 (another type I MAGE), the other for SSX-1 (a structurally distinct CT antigen). Interestingly, neither patient had positive serology for CT7. Serum from 16 other myeloma patients did not have detectible antibody titers for a broad panel of CT antigens. These results show that CT antigens are immunogenic in myeloma patients, with cellular responses against CT7 and humoral responses against MAGE-A1 and SSX-1. However, unlike other types of cancer, there appears to be discordance between humoral and cellular immunity against CT7 in multiple myeloma. This may be due in part to the significant derangements of humoral immunity in this disease. These results support further investigation of immunologic therapies targeting type I MAGE in myeloma, especially therapeutic vaccine strategies.
Clonotypic Bone Marrow-Derived Endothelial Progenitor Cells in Multiple Myeloma.
Authors: Braunstein Marc J. , Carrasco Daniel R. , Kahn David , Sukhdeo Kumar , Protopopov Alexei , Zhang Yunyu , Anderson Kenneth C. , Batuman Olcay
Published: October 2019

In multiple myeloma (MM), bone marrow-derived endothelial progenitor cells (EPCs) contribute to tumor neoangiogenesis and their levels covary with tumor mass and prognosis. Recent X-chromosome inactivation studies in female patients showed that, similar to tumor cells, EPCs are clonally restricted in MM. Genomic profiling of MM using high-resolution array comparative genomic hybridization (aCGH) has been previously utilized to mine the genome and find clinical correlates in MM patients. In this study, clonotypic aspects of bone marrow-derived EPCs and MM cells were investigated using aCGH and expression profiling analysis. Confluent EPCs were outgrown from bone marrow aspirates by adherence to laminin. EPCs were >98% vWF/CD133/KDR+ and <1% CD38+. The laminin-nonadherent bone marrow fraction enriched for tumor cells was >50% CD38+. For aCGH and for gene expression profiling, genomic DNA and total RNA from EPCs and MM cells were hybridized to human oligonucleotide arrays (Agilent Technologies) and human cDNA microarrays (Affymetrix), respectively. High resolution aCGH with segmentation analysis showed that EPCs and MM cells in one of ten cases share identical patterns of chromosomal gains and losses, while another 5 cases shared multiple focal copy number alterations (CNAs) including gains and losses. The genomes of EPCs and MM cells additionally displayed exclusive CNAs, but these were far fewer in EPCs than in MM cells. In 3 patients, EPCs harbored a common 0.6Mb deletion at 1q21 not shared by MM cells. Pertinent genes in this region that could affect proliferation and tumor suppression include N2N, NBPF10, and TXNIP. Validation studies of aCGH findings by other methods are ongoing. Gene expression profiling showed decreased expression of 1q21 region genes (e.g., calgranulin C and lamin A/C). A genome-wide comparison of patients’ MM cells and EPCs, which is focused on their shared genetic characteristics, will be presented.
2-Methoxyestradiol in Combination with Velcade® Results in an Augmentation of NFκB Inhibition, In Vitro Antiproliferative Activity and In Vivo Antitumor Efficacy in the RPMI-8226 Myeloma Model.
Authors: Burke Patricia , Swartz Glenn M. , Waters Cassandra L. , Chen Xiaoru , LaVallee Theresa M. , Treston Anthony M. , Fogler William E. , Sidor Carolyn F.
Published: October 2019

2-Methoxyestradiol (2ME2, Panzem®) is an endogenous metabolite of estradiol with antiproliferative, proapoptotic, and antiangiogenic activity. Some of these activities are mediated through the downregulation of HIF1-α. Panzem® capsules have been evaluated in several oncology clinical trials, including multiple myeloma where some patients have been on therapy for more than 4 years with stable disease. A new formulation of 2ME2 with enhanced absorption, Panzem® NCD, is currently in Phase 1 and 2 clinical studies in oncology. In anticipation of combination studies with Panzem® NCD, we evaluated the effectiveness of 2ME2 alone or with the proteosome inhibitor, Velcade® (bortezomib), in the RPMI-8226 myeloma xenograft tumor model. Preliminary in vitro studies indicated significantly increased antiproliferative activity when 2ME2 (1.5μM) was combined with Velcade® (5nM) compared to Velcade® alone (p<0.05 ANOVA). Additionally, 2ME2 in combination with Velcade® also resulted in decreased nuclear NFκB in response to TNFα, as compared to either agent alone. Subsequent to these studies, female CB17 SCID mice were injected subcutaneously with 1 x 106 RPMI-8226 tumor cells. Starting 10 days after tumor cell inoculation when mean tumor volume was approximately 100–200 mm3, cohorts of mice (n=8/group) began treatment with (a) vehicle alone ad libitum in the drinking water; (b) 2ME2 300 mg/kg ad libitum in the drinking water; (c) Velcade® 1.5 mg/kg i.p., biw; or (d) 2ME2 (300 mg/kg) in combination with Velcade® (1.5 mg/kg). On study day 27, mean tumor volume in vehicle-treated, control mice was approximately 3400 mm3. Under these experimental conditions, treatment with either 2ME2 alone or Velcade® alone generated a treatment/control (T/C) value of 0.66 and 0.64, respectively. In comparison, 2ME2 in combination with Velcade® resulted in a T/C value of 0.35. Velcade® is a well recognized and commonly used antineoplastic agent in multiple myeloma. The degradation of HIF1-α is accomplished through the proteosome. These studies demonstrate that a combination strategy using Velcade® and 2ME2 can enhance the antitumor effectiveness of either agent alone in the treatment of a xenograft model of human multiple myeloma.