bluuebunny/crossref_metadata_2025 huggingface dataset:
Xanthohumol Induces Apoptosis in B-CLL In Vitro by Sustained Endoplasmic Reticulum Stress and Inhibition of NFκ-B.
Authors: Lust Sofie , Vanhoecke Barbara , Van Gele Mireille , Kaileh Mary , Boelens Jerina , Poelman Martine , VandenBerghe Wim , Philippé Jan , Offner Fritz
Published: April 2020

Introduction

Correct folding of new proteins is supervised in the endoplasic reticulum (ER) unfolded protein response (UPR). Misfolded proteins recruit the chaperone Grp78 that is thereby released from the transcription factors ATF6, IRE-1 leading to compensatory increase in Grp78, and PERK, leading to phosphorylation of eIF2α and block of further protein translation. UPR overload leads to ER stress and cell death. Targeting the endoplasmic reticulum (ER) is a new strategy explored in B-CLL. The hop-derived chalcone Xanthohumol (X) has been characterized as a ‘broad-spectrum’ cancer chemopreventive agent. Recently, we demonstrated that X induces dose- and time-dependent cell death of MCF7/6 breast cancer cells accompanied by ER stress. X induces apoptosis and cleavage of poly(ADP)-ribose-polymerase (PARP) in B-CLL in vitro. The present study investigates the branches of the UPR in relation to X induced apoptosis of B-CLL cells.

Materials and methods. Lymphocytes were isolated by Lymphoprep from 15 patients with B-CLL after informed consent. CD19 positive cells were selected by EasySep positive selection kit. Apoptosis was assessed by flow-cytometry (AnnexinV-PI). Western Blotting was used for Grp78, ATF6, XBP1, phospho-eIF2a, eIF2a, ATF4, CHOP, phospho-IKK, IKK, PARP, caspase-9, -8, -7, -4, cleaved caspase-3, mcl-1, bcl-xL, bax, bak, and bid. NF-kB activity was assessed by EMSA. Quantitative RT-PCR was performed to analyze Grp78 mRNA levels. Bcl-2 protein level was detected by flow cytometry and reactive oxygen species (ROS) by fluorescence microscopy.

Results and conclusion

X induced an upregulation of Grp78 mRNA levels which was not translated in an increase in protein. X treatment stimulated a rapid and sustained phosphorylation of eIF2a, suggesting the involvement of PERK. In contrast, the ER-stress transducers ATF6 and IRE1 were not activated. X-induced ER stress was associated with strong induction of the pro-apoptotic protein CHOP and inhibition of the NF-kB pathway. Furthermore, the pro-apoptotic effect of X was accompanied by an accumulation of ROS, a downregulation of the anti-apoptotic proteins mcl-1, bcl-xL, bcl-2 and processing of caspase-3, -7 and -9.In conclusion, the chalcone X is capable of inducing cell death with down-regulation of bcl-2, mcl-1, bcl-xL, and activation of the caspase cascade. This is accompanied by ER-stress as evidenced by the upregulation of Grp78 mRNA levels, induction of a rapid and sustained phosphorylation of eIF2a, upregulation of CHOP, and inhibition of the NF-kB signaling.
Intense Reversal of Bone Marrow Angiogenesis after Consolidation Therapy with Alemtuzumab in Advanced Chronic Lymphocytic Leukemia.
Authors: Molica Stefano , Montillo Marco , Ribatti Domenico , Mirabelli Rosanna , Tedeschi Alessandra , Ricci Francesca , Veronese Silvio , Vacca Angelo , Morra Enrica
Published: April 2020

We evaluated microvessel area of bone marrow (BM) samples from 20 patients with advanced CLL (i.e., symptomatic Binet stage A, B or C) who received at least 8 weeks, after the end of treatment with fludarabine, subcutaneous alemtuzumab, three times weekly for 6 weeks, at escalating dose up to 10 mg. The patient sample included 14 males and 6 females with a median age of 51 years (range 44–60). After a median number of 6 cycles of fludarabine (range, 4–13), 11 (55%) patients could be classified in complete remission (CR) and 9 (45%) in partial remission (PR) (7 nodular-PR and 2 PR). Interestingly, the rate of CR increased to 90% (18 CR; P=0.03; Fisher’s exact test) after treatment with alemtuzumab. In keeping with hematological responses, significant changes in BM microvessel area were observed The decrease of BM angiogenesis, observed virtually in all patients, was a continuous process characterizing the sequential use of fludarabine and alemtuzumab (P = 0.002; Kruskal-Wallis test). This conspicuous feature was easily demonstrable in either ZAP-70-positive (P = 0.02) or ZAP-70-negative (P = 0.001) patients.

Residual disease was assessed using a consensus polymerase chain reaction (PCR) methodology to detect the clonality of IgH sequences. Thirteen out of 20 (65%) patients changed from a monoclonal to a polyclonal pattern of IgH after alemtuzumab consolidation. We wondered whether molecular response could translate into a change in the BM microvessel area. A separate evaluation carried out in patients with a persistent monoclonal pattern and in those who changed to a polyclonal pattern of IgH after alemtuzumab therapy showed a significant reduction of BM microvessel area only in the latter (P = 0.0002).

A new information provided by the present study concerns the impact of the cumulative dose of alemtuzumab on either molecular response or reduction in BM angiogenesis. Analysis carried out after setting as cut-off the median cumulative dose of alemtuzumab (i.e., 184 mg; range, 134–187) revealed a dose-response relationship. Only two (15.3%) out of 13 patients who received a cumulative dose of alemtuzumab higher than 184 mg had persistent monoclonal IgH in comparison to 5 (71.4%) out 7 patients who had received a cumulative dose of alemtuzumab lower than median (P = 0.007). Similarly, a significant decrease in the extent of microvessel area was observed among patients whose cumulative dose was equal or higher than 184 mg (P = 0.0001) while the same did not apply for a cumulative dose of alemtuzumab lower than 184 mg (P = 0.09).

Overall, these data demonstrate a decrease in BM vascularity after treatment with alemtuzumab, which reflects both molecular response and cumulative dose of this drug, and suggest a potential use of alemtuzumab as anti-angiogenic agent in the treatment of advanced CLL.
Epigenetic Modulation by Vidaza™ induces Apoptosis and Overcomes In Vitro Drug Resistance in Human Multiple Myeloma Cells.
Authors: Kiziltepe Tanyel , Catley Laurence , Hideshima Teru , Raje Noopur , Yasui Hiroshi , Vallet Sonia , Ikeda Hiroshi , Okawa Yutaka , Anderson Kenneth C.
Published: April 2020

Methylation of DNA is an epigenetic modification that plays an important role in the regulation of gene expression in mammalian cells. Although DNA methylation is required for normal cell development and function, aberrant methylation and the resulting aberrant expression of genes, such as tumor suppressor genes and oncogenes, contribute to the development of malignancies. Interestingly, aberrant DNA methylation has recently emerged as one of the most frequent molecular alterations in hematologic malignancies, providing a powerful rationale to use inhibitors of DNA methylation as a novel means of targeting hematologic malignancies. Vidaza™ [Pharmion Corporation] (5-azacytidine), an FDA approved drug for the treatment of myelodysplastic syndromes, is an inhibitor of DNA methylation. Multiple myeloma (MM) is currently an incurable hematological malignancy despite all the conventional and novel therapies, and we here examined the biological effects using Vidaza™ on human MM cells. We demonstrate here that Vidaza™ has significant cytotoxicity in both conventional therapy sensitive (MM1S, RPMI-8226) and resistant (MM1R, RPMI-Dox40, RPMI-LR5) MM cell lines, as well as freshly isolated patient MM tumor cells, with an IC50 of 1.25–4 mM at 72 hours in vitro. Importantly, no cytotoxic effects of Vidaza™ were detected in peripheral blood mononuclear cells (PBMNC) obtained from healthy volunteers at ≤20 mM, suggesting a therapeutic index. Moreover, Vidaza™ overcame the survival and growth advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells (BMSC). Vidaza™ induced apoptosis in MM cells, as determined by flow cytometric analysis using PI and Annexin V staining. Vidaza™-induced apoptosis was associated with PARP, caspase 8 and caspase 9 cleavage. Importantly, pan-caspase inhibitor zVAD-fmk, significantly, but only partially, inhibited apoptosis induced by Vidaza™, suggesting the involvement of both caspase dependent and independent pathways. Taken together, our studies therefore demonstrate that Vidaza™ induces apoptosis and overcomes in vitro drug resistance in MM cells. Ongoing studies are delineating the mechanism of action of Vidaza™ against MM cells to further provide the preclinical rationale for clinical evaluation of Vidaza™, alone or in combination with other agents, to improve patient outcome in MM.
Elimination of the Need for Urine Studies during Diagnostic Studies of Monoclonal Gammopathies by the Combined Use of Serum Immunofixation and Serum Free Light Chain Assays.
Authors: Katzmann Jerry A. , Dispenzieri Angela , Kyle Robert , Snyder Melissa R. , Plevak Mathew F. , Larson Dirk R. , Abraham Roshini S. , Lust John A. , Melton L. Joseph , Rajkumar S. Vincent
Published: April 2020

Due to the diagnostic sensitivity of serum free light chain quantitation for monoclonal light chain diseases, it has been suggested that urine assays no longer need be performed as part of the diagnostic algorithm for monoclonal proteins. We reviewed our experience to determine the relative diagnostic contribution of urine assays.

Methods: Patients with a monoclonal gammopathy and monoclonal urinary protein at initial diagnosis who also had a serum immunofixation and serum free light chain quantitation within 30 days of diagnosis were identified (n = 428). The laboratory results for serum protein electrophoresis, serum immunofixation, serum free light chain, urine protein electrophoresis, and urine immunofixation were reviewed.

Results: The patients in this cohort had diagnoses of multiple myeloma, primary amyloid, monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, solitary plasmacytomas, and other less frequently detected monoclonal gammopathies. By definition of the cohort, all 428 had a monoclonal urine protein. 86% had an abnormal serum free light chain K/L ratio, 81% had an abnormal serum protein electrophoresis, and 94% had an abnormal serum immunofixation. In only 2 patients, however, were all 3 serum assays normal. Both of these were patients with monoclonal gammopathy of undetermined significance (idiopathic Bence Jones proteinuria).

Conclusion: Discontinuation of urine studies and reliance on a diagnostic algorithm using solely serum studies (protein electrophoresis, immunofixation, and free light chain quantitation), missed 2 of the 428 monoclonal gammopathies (0.5 %) with urinary monoclonal proteins, and these 2 cases required no medical intervention.
BAX UTR G (-248) A Polymorphism Has No Impact on Survival in Patients with Multiple Myeloma.
Authors: Paneesha Shankaranarayana , Hawkins K. , Davies F. , Hooper L. , Starczynski J. , Pepper C. , Morgan Gareth , Fegan C. , Pratt Guy
Published: April 2020

Multiple myeloma is a malignant tumour of plasma cells that remains incurable for the vast majority of patients with a median survival of 2–3 years. The bax protein is a member of the Bcl-2 family and is an important pro-apoptotic protein. Low BAX expression has been shown to be associated with prolonged survival in patients with myeloma(1). A novel polymorphism, G(-248)A in the BAX promoter in patients with Chronic lymphocytic leukaemia (CLL) has shown to be associated with reduced Bax protein expression, progressive disease and treatment failure(2, 3). We report here the impact of this polymorphism on survival in 304 (M: 168; F: 136) patients with myeloma

Materials and Methods

This study included 158 (M: 89; F: 69) patients from MRC Myeloma VII trial and 146 (M: 79; F: 67) under our care from January 1992 and February 2006. DNA was extracted from myeloma patients and healthy volunteer’s peripheral blood using a standard salting out method. Analysis of the 5-UTR polymorphism was undertaken using method already published by our centre(3). Patients received either intensive or non intensive antimyeloma therapy as per standard practice. Statistical analysis was carried out using SAS software.

Results

The frequency of GG and GA/AA genotypes was 81.6% and 18.4% in myeloma patients as compared to 85.2% and 14.8% in normal controls. Median age at diagnosis was 58 yr (range 34–87 yrs). (GA/AA Group: 58 yrs (42–85); GG Group: 58 yr (34–87)). There were no significant differences in the sex distribution, β2 microglobulin, haemoglobin, bone lesions, calcium and age between the GA/AA & GG groups with p values 0.72, 0.46, 0.95, 0.61, 0.49 and 0.46 respectively. Abnormal creatinine levels were significantly few in GA/AA group (p value: 0.002). Median survival for the AA/GA group was 48 months (95% CI 36 – 61 months) compared with 54 months in the GG group (95% CI 46 – 63 months)[p=0.5 log-rank test] with a median follow up of 62 months (range 1 to 141).

Conclusions

Our results show that the GA/AA polymorphism has no impact on the overall survival in patients with myeloma. Further work is ongoing in order to assess the impact of this polymorphism on response in various treatment groups.
Interruption of Bisphosphonates (BP) Therapy in Multiple Myeloma (MM) Patients and Osteonecrosis of the Jaw (ONJ).
Authors: Badros Ashraf , Goloubeva Olga , Weikel Dianna , Rapoport Aaron P. , Akpek Gorgun , Yanovich Saul , Meiller Timothy , Sausville Edward
Published: April 2020

ONJ is a serious complication in MM patients; incidence is time-dependent with highest risk seen after long-term use of BP in older MM patients, often after dental surgical procedure (Badros, et al JCO 2006). Therapy has focused on debridement of necrotic bone, infection management, pain control and discontinuation of BP. The association between BP and ONJ remains provisional; theoretically and anecdotally, however there is evidence that BP withdrawal may allow for recovery of normal osteoclast function and reversal of antiangiogenic effects of the drug on the periosteum allowing for bone healing. We have observed an increased risk of skeletal complications in MM patients after long interruptions of BP therapy (6–12+months), even though patients had been on BP therapy for a median of 3 yrs (range: 2–10+yrs). Table below summarizes the complications that occurred off BP therapy in MM patients with ONJ (n=28) and without ONJ who stopped BP ahead of dental procedures (n=20). Skeletal complications included avascular necrosis of the hip (AVN), fractures, new lytic lesions on skeletal surveys and bone pain; all occurred in relapsed patients with active MM. These events highlighted the importance of BP cornerstone for treatment of skeletal complications of MM; of the 28 ONJ patients, 8 died from progressive MM and 3 patients had non-healing oral lesions and did not resume BP. Twelve of 17 ONJ patients were restarted on BP after complete healing of the oral lesions; all of whom had relapsed MM and bone disease. They received pamidronate every 3 months. So far 3 of 12 patients (25%) had recurrent ONJ and none had skeletal complications. Of the 20 patients who stopped BP before dental procedures, 3 (15%) went on to develop ONJ. Fifteen had restarted BP 2–3 months after the procedure site was well-healed; one patient developed ONJ at the healed extraction site 6 months after restarting BP therapy. This patient was in remission on maintenance thalidomide but with extensive myeloma bone disease. In conclusion, interruption of BP in MM patients with ONJ as well as before and after dental procedures had inconsistent results with regards to both healing and prevention of ONJ. On the other hand, the damage of holding BP therapy is evident with increased frequency of skeletal complications. Until further studies define the optimal frequency and duration of BP therapy, we propose continuous administration of BP therapy probably less frequently after 2 years of monthly infusions with careful assessment of the risk status of each individual patient.

Skeletal complications off bisphosphonate therapy FracturesΔ AVN New lytic lesions Bone pain Δ Fractures affecting long bones or ribs. λ Two had bone disease as first site of relapse. π One patient had a femoral Infarction and severe pain. ONJ; n=28 5 5 2 4 Dental Procedure; n=20 3 1 3λ 1π
Neutropenia-Associated Mutations in PRDM5, a Novel Epigenetic Regulator of Hematopoiesis.
Authors: Duan Zhijun , Huang Shi , Person Richard E. , Badolato Raffaele , Donadieu Jean , Horwitz Marshall S.
Published: April 2020

The growth factor independent 1 (Gfi1) oncoprotein is an intrinsic regulator of hematopoiesis, and its mutations are associated with congenital neutropenia. We performed a yeast two-hybrid screen with Gfi1, in order to begin dissecting the gene network through which it acts and also toward identifying other genes potentially responsible for cases of hereditary neutropenia lacking mutations in Gfi1 or ELA2 (encoding neutrophil elastase, the most common cause of hereditary neutropenia). From a limited number of proteins found to interact with Gfi1 in the yeast two-hybrid screen, we evaluated PRDM5, a previously uncharacterized zinc finger protein belonging to the PR domain (PRDI-BF1 and RIZ homology) -containing family of tumor suppressor genes. We screened for mutations in the gene encoding PRDM5 among 230 unrelated patients with otherwise genetically unexplained congenital neutropenia and discovered two with heterozygous amino acid sequence substitutions that were not detected in twice the number of controls. Consequently, we systematically characterized PRDM5, and found that it functions as a novel epigenetic regulator of hematopoiesis: First, PRDM5 is a sequence-specific DNA-binding transcription factor as revealed by random sequence selection. Second, by using chromatin immunoprecipitation (ChIP)-based cloning along with “ChIP-on-chip” assays, we identified approximately two hundred protein-coding genes (including hematopoietic transcription factors) and thirty-four microRNA genes as putative PRDM5 targets, a subset of which we confirmed by additionally performing gel retardation and transfection reporter assays. Significantly, one of the targets of PRDM5—just as is true of Gfi1—is ELA2. Third, depletion of PRDM5 by siRNA in both HeLa and HEK293 cells changed the cell cycle program, and, as revealed by microarray and real-time RT-PCR assays, PRDM5 knockdown in HEK293 cells altered the expression of ninety of the protein-coding targets and eight of the microRNA targets. Fourth, transcriptional profiling revealed that the expression of about five thousand genes, including many involved in the regulation of cell proliferation and differentiation, changed more than five-fold following PRDM5 knockdown in HEK293 cells. Fifth, we found that PRDM5 regulates transcription by associating with the histone deacetylase HDAC1 and the histone methyltransferase G9a and recruits them to its target genes. Treatment of cells with the histone deacetylase inhibitors trichostatin A (TSA) or 4-phenylbutyric acid (PBA) deregulates PRDM5 target genes. Finally, the two variants observed in patients with congenital neutropenia alter the DNA-binding activity of PRDM5 and interfere with its function as a transcriptional repressor in reporter assays, suggesting that, just as with Gfi1, rare mutations in a global regulator of hematopoiesis may contribute to the genetic mechanisms causing neutropenia.
Phenyhexyl Isothiocyanate, a Novel Inhibitor of Histone Deacetylation and Methylation, Induce Growth Arrest and Apoptosis of Myeloma Cells.
Authors: Lu Quanyi , Feng Jane , Seiter Karen , Ahmed Tauseef , Chiao Dicky J.W. , Liu Delong
Published: April 2020

Histone deacetylase (HDAC) inhibitors are emerging as a promising family of new agents in the therapy of hematologic malignancies. We have shown that phenyhexyl isothiocyanate(PHI) is a novel histone deacetylase inhibitor and can also modulate histone methylation in leukemia cells. In this study we investigated the effect of PHI on human myeloma cell line RPMI8226 in vitro.

We have observed that cell proliferation was inhibited by PHI in a dose and time dependent manner. Cell cycle analysis indicated an arrest in G0/G1 phase, and significant apoptosis was detected in PHI treated RPMI8226 cells. The accumulation of P21 and reduced Levels of PCNA were detected. We also examined the effect of PHI on vascular endothelial growth factor (VEGF) production by the myeloma cells. Treatment with 20μM PHI for 24hrs led to a decrease of VEGF concentration by 50% of that secreted by the control cells. VEGF production further decreased to 25% when the cells were exposed to PHI for 48hrs. These data suggest that myeloma cells are sensitive to the novel HDAC inhibitor, and PHI may become a novel agent in multiple myeloma therapy.

PHI Inhibit VEGF production by Myeloma Cells PHI Inhibit VEGF production by Myeloma Cells
Monokine Induced by Interferon-γ (MIG) Serum Levels Are a Marker of Disease Load and Correlate with Prognosis in Multiple Myeloma.
Authors: Schreder Martin , Koch Gudrun , Heintel Daniel , Strasser-Weippl Kathrin , Frischmuth Katrin , Jaeger Ulrich , Ludwig Heinz , Zojer Niklas
Published: April 2020

Background and Aims: Monokine-induced by interferon-γ (MIG) is a chemokine that is produced by monocytes and macrophages in response to interferon-γ and acts as a chemoattractant mainly to T-lymphocytes in inflammatory processes. MIG has also been suggested to act in an autocrine loop to stimulate tumour cells through its receptor CXCR3, which is known to be expressed in myeloma cells. However, it is presently unclear if MIG is of biologic significance in myeloma in vivo. We have recently shown that multiple myeloma oncogene 1 (MUM1) expression in myeloma cells correlates with prognosis in this disease (Heintel D et al., ASH 2005), and MUM1 was found to upregulate MIG gene expression in B cell malignancies (Uranishi M et al., Leukemia 2005). This led us to evaluate the potential prognostic significance of MIG serum levels in a series of well characterized myeloma patients.

Methods: 105 newly diagnosed multiple myeloma patients (median age 69.3 years, range 39.4–90.5) were enrolled. 18 patients presented with Durie/Salmon stage I disease, 9 had stage II and 78 had stage III. MIG serum levels were determined by a commercially available ELISA (R&D Systems). Serum samples from 17 MGUS patients and 37 age-matched healthy volunteers were used as controls.

Results: MIG serum levels were elevated in multiple myeloma patients (median 161.3 pg/ml, range 9.37–1966.0) compared to MGUS patients (median 92.7 pg/ml, range 6.29–1303.1) and healthy controls (median 106.2 pg/ml, range 51.0–390.6). For analysis of myeloma cases, a cut-off level for MIG of 200pg/ml (=95th percentile of MIG in controls) was chosen to identify low and high MIG expressers. Using the cut-off as defined above, 63 patients with low MIG serum levels and 42 high MIG expressers were identified in a population of 105 myeloma patients. MIG serum levels in myeloma patients showed strong correlations with several markers of tumour load including low albumin and high β2-microglobulin. Interestingly, no correlation was found with C-reactive protein levels, indicating that MIG is not associated with an inflammatory response in myeloma. Median survival was significantly shorter in patients with high MIG serum levels compared to patients with low MIG expression (median not reached vs. 17.0 months, p<0.001; see figure).

Conclusions: MIG serum levels correlate with markers of disease burden in myeloma and high MIG levels are associated with a poor outcome in this disease.

Overall Survival: low vs. high MIG serum levels Overall Survival: low vs. high MIG serum levels
Significance of Serum Free Light Chain Estimation with Detectable Serum Monoclonal Protein on Immunofixation Electrophoresis.
Authors: Mehta Jayesh , Stein Regina , Vickrey Eric , Resseguie William , Singhal Seema
Published: April 2020

The serum free light chain (SFLC) assay is useful in detecting monoclonal protein when there no detectable M protein on immunofixation electrophoresis (IFE). There are limited data on its value when IFE is positive. In a small series of 5 patients achieving CR, normalization of SFKLR was found to precede IFE negativity by a few weeks (Moesbauer et al. ASH 2005). Results on 231 serum samples from myeloma patients (most on therapy) where serum IFE showed IgA κ (n=33), IgA λ (n=13), IgG κ (n=153), or IgG λ (n=32), and where simultaneous SFLC and immunoglobulin (Ig) estimation had been performed were analyzed. Samples with >1 monoclonal band or multiple oligoclonal bands were excluded. The serum free κ:λ ratio (SFKLR; normal 0.26–1.65) was abnormal in 113 (49%) and normal in 118 (51%). IgG and IgA levels were compared in the context of normal versus abnormal SFKLR within each of the 4 isotypes (IgA κ, IgAλ, IgG κ, IgG λ). The table below shows that involved Ig levels were higher with abnormal than with normal SFKLR. However, uninvolved Ig levels were higher with normal than with abnormal SFKLR suggesting that normalization of SFKLR may mark a response to therapy - improved uninvolved Ig levels being evidence of response.

Monoclonal protein Immunoglobulin Abnormal SFKLR Normal SFKLR P IgA kappa IgA 1640 (190–4000) 515 (102–2230) 0.048 IgA kappa IgG 419 (118–1120) 404 (197–1740) 0.39 IgA lambda IgA 408 (159–696) 704 (180–779) 0.17 IgA lambda IgG 619 (495–1510) 1530 (533–1700) 0.025 IgG kappa IgA 42 (7–225) 94 (7–642) 0.0009 IgG kappa IgG 1490 (585–5560) 1260 (327–2690) 0.004 IgG lambda IgA 32 (7–121) 96 (19–562) 0.047 IgG lambda IgG 2060 (555–12300) 1050 (432–2830) 0.018

However, does normalization of SFKLR universally herald IFE negativity? This is an important unanswered question because SFKLR is normal in a high proportion of samples which still show monoclonal protein on IFE. The figures below show scatter plots of IgG and IgA for each of the 4 isotypes for normal vs abnormal SFKLR. Within each plot, there is no obvious pattern distinguishing normal (x) from abnormal (o) SFKLR. However, there are a number of normal SFKLR points with high involved and low uninvolved Ig levels where a normal SFKLR is difficult to explain.

Figure Figure Figure Figure

We conclude that the SFLC assay often reveals normal SFKLR even when there is a detectable monoclonal protein in the serum. Whether this always predicts eventual paraprotein clearance and achievement of IFE negativity in patients on therapy is unknown, and needs to be studied prospectively.
Characterization of Bortezomib Resistant Human Multiple Myeloma Cell Line (HMCL): A Clinically Relevant Model for Novel Drug Development in Multiple Myeloma (MM).
Authors: Chanan-Khan Asher Alban , Ersing Noreen , Pera Paula , Bernaki Ralph , Coignet Lionel , Goodrich David W. , Haung Raya , Porter Carl W.
Published: April 2020

Introduction: Bortezomib (B) is the first in class proteasome inhibitor that received FDA approval for the treatment of MM patients with relapsed or refractory disease. Despite impressive clinical activity all patients develop resistance to B. The underlying mechanism of resistance to B remains undetermined. Mechanisms to overcome B resistance or development of new therapeutic agent(s) with activity in context of B resistance are limited due to unavailability of established preclinical B resistant MM models. To overcome this challenge we developed a B resistant HMCL.

Methods: we chronically exposed the OPM-2 HMCL to sub-lethal doses of B. Surviving cells were harvested, re-cultured and dose of B incrementally increased over prolong period of time. The resulting resistant cells were further characterized using array Comparative Genomic Hybridization (aCGH), spectral karyotyping (SKY) and gene expression profiling.

Results: After several passages we were able to induce B resistance in this HMCL. Final clone (OPM-2BR) demonstrate resistance to 2 × IC50 dose of B. While SKY and aCGH analysis demonstrated significant differences when compared with parent OPM-2WT (wild type) HMCL, gene expression profiling of the resistant and parental lines demonstrated significant upregulation in the expression of a number of ATP-binding cassette transporters. For example, the breast cancer resistance protein (ABCG2) is upregulated 4-fold in the resistant cell line compared to the parental cell line. This data suggests that drug efflux mediated by drug transporters represents one potential mechanism of resistance to B.

Conclusion: Chronic in vitro B exposure results in induced resistance in HMCL-OPM-2BR. Resistant cell line demonstrates cytogenetic variability when compared to the parent cell line. Induction of resistance is stable and provides an important preclinical tool to investigate mechanism(s) of B resistance as well as new drug development for B resistant myeloma patients. Detailed analysis of this cell lines including therapeutic interventions investigating resistance reversal will be presented at the meeting.
Cyclooxygenase-2 (COX-2) in Multiple Myeloma: Prognostic Marker or Therapeutic Target?.
Authors: Owen Roger G. , Fan Im , O’Connor Sheila J.M. , Davies Faith E. , Rollett Rebecca A. , Child J. Anthony , Rawstron Andy C.
Published: April 2020

Cyclooxygenase-2 (COX-2) is the key enzyme involved in prostaglandin synthesis. It appears to support the growth of a number of solid tumours including colon, breast, ovary, lung and uterine cervix and may be an important therapeutic target in at least some of these tumours. COX-2 expression has recently been evaluated (by immunohistochemistry using polyclonal anti-COX-2 antibodies) in multiple myeloma (MM) where expression was documented in 33–57% of patients. COX-2 expression in these studies was strongly associated with an adverse outcome. In addition there is some emerging data to suggest that the use of aspirin in MM may improve survival rates. In order to further evaluate this we have used a monoclonal antibody (Clone SP21, Labvision, Fremont, Ca) to assess COX-2 expression in both normal and neoplastic plasma cells. 52 specimens were assessed using standard streptavidin-biotin immunoperoxidase techniques using a known COX-2+ colon cancer as a positive control. Strong uniform COX-2 expression was seen in 32/33 (97%) of myeloma patients assessed and was also documented in all patients with MGUS (n=10). COX-2 expression was also documented in reactive plasmacytic lesions (oral mucosa, skin and lymph node, n=6) as well as normal bone marrow plasma cells (n=6). Megakaryocytes stained positively in all bone marrow biopsies examined and provided a useful positive internal control while erythroid, myeloid and lymphoid cells were consistently negative. We would conclude that COX-2 is strongly expressed by both normal and neoplastic plasma cells suggesting that COX-2 is a potential therapeutic target in MM. The apparent increase in the proportion of myeloma patients expressing COX-2 in the present study reflects the use of a monoclonal antibody in our immunohistology studies. The fact that polyclonal antibodies identify a lower proportion of patients who appear to have an inferior outcome suggests that the level of expression is of prognostic significance rather than its presence or absence. This is worthy of further study using more appropriate techniques such as RQ-PCR.
Detection of FGFR3 by Immunohistochemistry in Multiple Myeloma Is Highly Predictive of t(4;14) by FISH and Is Associated with CD56 (NCAM1) Expression.
Authors: Bahlis Nizar J. , Roland Birgitte , Maggliocco Toni , Auer Ivona , Stewart Douglas A. , Mansoor Adnan
Published: April 2020

Recent studies have demonstrated that neural cell adhesion molecule CD56 (NCAM1) is involved in multiple adhesive interactions with several different classes of ligands on the cell surface and in the extracellular matrix. One of these ligands is fibroblast growth factor receptor (FGFR) family. While a direct interaction between CD56 (NCAM1) and FGFR3 is yet to be reported we have speculated that such interaction might exist in myeloma cells with aberrant FGFR3 expression. In order to test this hypothesis we have retrospectively examined the presence of t(4;14) in 127 multiple myeloma samples and its correlation with the presence of deletion 13 by FISH; FGFR3 and cyclin D1 expression by immunohistochemistry and CD56 expression as determined by flow cytometry. FISH detected t(4;14) in 32 (25.1%) of 127 MM patient specimens. 71.4% of t(4;14) positive samples were found to have deletion of chromosome 13, in contrast to only 34% in t(4;14) negative samples. Aberrant expression of FGFR3 was detected by immunohistochemistry in 14/21 or 66.7% of t(4;14) positive samples. No expression of FGFR3 was detected in 10 t(4;14) negative samples. As expected low Cyclin D1 expression by IHC was detected in t(4;14) positive samples (5/21 or 23.8%). More importantly all samples positive for FGFR3 by IHC were CD56 positive (14/14 or 100%) and in 21/22 (95.5%) of t(4;14) positive in contrast to 62% in t(4;14) samples. Further work is currently being conducted in order to examine the downstream signaling pathways that might be activated as a result of such possible interaction.
Antiproliferative and Proapoptotic Effect of Vitamin D in Bortezomib (B) Resistant Human Myeloma Cell Line (HMCL).
Authors: Chanan-Khan Asher Alban , Romano Michelle , Pera Paula , Ersing Noreen , Muindi Josephia , Trump Donald L. , Johnson Candace
Published: April 2020

-Introduction: B is a proteasome inhibitor approved for treatment of multiple myeloma (MM) patients. Despite initial clinical efficacy all patients eventually become resistant to bortezomib and die of progressive disease. Development of newer agents with novel antitumor mechanisms is thus warranted. In vitro antiproliferative and proapoptotic effects of vitamin D are reported in various tumor models including MM. This is mediated through vitamin D receptor (VDR) which is also expressed on MM cells. Effect of calcitriol (D3) or its analog paricalcitol (D2) in HMCL resistant to B have not been previously examined. We investigated the antitumor potential of D3 and D2 in HMCL sensitive or resistant to B.

Method & Results: HMCL sensitive (OPM-2) or resistant to B (OPM-2/BR) were treated with various concentration of D2 or D3. Significant growth inhibition and induction of apoptosis was noted with D2 and D3 that was maximal at 3 days (p<0.01). This antitumor response was noted despite resistance to B. Treatment of MM cells with vitamin D (D2 or D3) was associated with a loss of full-length PARP, decrease in p21 and induction of p27. Levels of the pro-survival molecules P-Akt and P-Erk were also reduced while total Akt and Erk remain unchanged.

Conclusion: We demonstrate that the antiproliferative and proapoptotitc effect of vitamin D in HMCL are mediated through interruption of the Akt pathway and were independent of resistance to B. We have initiated a phase I clinical trial to investigate the clinical utility of paricalcitol in relapsed or refractory MM patients.
OSU03012, a Novel Celecoxib Derivative, Induces Apoptosis in Multiple Myeloma Cells Independently of Caspase Activation.
Authors: Zhang Shuhong , White Valerie L. , Johnson Amy , Chen Ching-Shih , Farag Sherif S.
Published: April 2020

Multiple myeloma (MM) is a clonal disorder affecting terminally differentiated B cells, with the accumulation of plasma cells in the bone marrow. Previous studies showed that OSU03012 is a novel celecoxib derivative lacking cyclooxygenase-2 inhibitory activity that induces apoptosis in various types of cancer cells and is being developed as an anti-cancer therapy in the NCI Rapid Access to Intervention Therapy (RAID). Here, we examined the in vitro effect of OSU03012 in MM cell lines (U266, ARH-77, IM-9 and RPMI8226). Cytotoxicity data indicated that mean LC50 (lethal concentration 50%) of OSU03012 was 6.25±0.86 μM at 24 hours and 4.23±0.87 μM at 72 hours in these four cell lines. Using annexin V/PI (propidium iodide) flow cytometry assay, OSU03012 was shown to induce apoptosis in MM cells. OSU03012 activated caspases-8, -9, and -3, induced PARP (POLY ADP-RIBOSE Polymerase) cleavage, and reduced survivin and XIAP expression after 6 and 24 hour exposure. Although the caspase inhibitor Q-VD-OPH treatment strongly blocked OSU03012-induced PARP cleavage, it did not inhibit OSU03012-induced apoptosis of MM cells. The pan-caspase inhibitor z-VAD-fmk did not prevent OSU03012 mediated cell death. Cell death with OSU03012 treatment was associated with significant down-regulation of phospho-Akt. Several substrates of AKT, including phospho-GSK-3 beta (Ser9), phospho-FoxO1a (Ser256) and phospho-MDM2 (Ser166) were also down-regulated by OSU03012 drug. OSU03012 triggered both early (6h) and late (24h) down-regulation of cyclin D1 expression, but cyclin A and B1 expression was down-regulated only at 24h. There was no induction of p21 or p27 protein levels by OSU03012. After 24-hour exposure, low concentration (1–5 μM) OSU03012 arrested MM cell lines in the G1 phase of the cell cycle while high concentration (10 μM) OSU03012 induced G2 phase arrested. OSU03012 decreased both phospho-Stat3 (Ser727) and Stat3 expression. OSU03012 has on effect on phosphorylated MAP kinase kinase1/2 (pMEK1/2) but it decreased MEK1/2 expression at 24h. The expression levels of Bcl-2 family proteins, Bcl-2, Mcl-1, BAX, and BIM did not alter with OSU03012 treatment suggesting that Bcl-2 members may not play direct or significant roles in inducing cell death. Taken together, we conclude that OSU03012 is potently active against MM cells by predominantly caspase-independent mechanisms, and may involve downstream pathways consequent to phopho-Akt down-regulation. These studies provide preclinical rationale for investigating OSU03012 in the treatment of MM.
OSU-HDAC42, a Novel Histone Deacetylase Inhibitor, Induces Apoptosis in a Caspase-Dependent Manner and Induces p21WAF1/CIP1 and p16 Expression in Multiple Myeloma Cell Lines.
Authors: White Valerie L. , Zhang Shuhong , Lucas David , Chen Ching-Shih , Farag Sherif S.
Published: April 2020

Multiple myeloma (MM) is a neoplastic disorder characterized by accumulation of slowly-proliferating clonal plasma cells. OSU-HDAC42 [a.k.a. (S)-HDAC-42] is a novel histone deacetylase inhibitor that induces apoptosis in various types of cancer cells and is being developed as an anti-cancer therapy in the NCI Rapid Access to Intervention Therapy (RAID) program. In this study, we tested the in vitro activity of OSU-HDAC42 against human MM cells.

OSU-HDAC42 induced myeloma cell death, with an LC50 of less than 1.6μM after 48 hours in the four cell lines tested - U266, IM-9, RPMI 8226 and ARH-77 using the MTT assay. OSU-HDAC42 induced cleavage of caspases 3, 8 and 9, as well as polyADP-ribose polymerase (PARP). Addition of the pan-caspase inhibitor Q-VD-OPH before exposure to the drug prevented apoptosis at 48 hours, as determined by Annexin V/propidium iodide staining. These results indicate that OSU-HDAC42 induced apoptosis by a mainly caspase-dependent manner. Bax expression was up-regulated at 24 and 48 hours, while Bcl-2 remains relatively constant. Mcl-1 showed increasing cleavage at increasing doses of OSU-HDAC42. These findings support a mitochondrial pathway of apoptosis. Cell cycle suppressor proteins p21WAF1/CIP1 and p16 were also significantly induced after treatment with the drug, suggesting that OSU-HDAC42 may also acts on pathways to halt cell cycle progression.

In addition, the gp130 (signal-transducing) subunit of the IL-6 receptor was down-regulated by OSU-HDAC42 exposure. The tyrosine-phosphorylated form of STAT3, which is phosphorylated by dimerized gp130, was also dramatically reduced following incubation with OSU-HDAC42, supporting the finding that gp130 expression is diminished. As IL-6 is an important growth and survival factor for MM cells, down-regulation of gp130 may be an important mechanism for the activity of OSU-HDAC42 against MM cells. TRAIL, FasL, XIAP, and p53 expression were not affected by OSU-HDAC42. While other HDAC inhibitors have been shown to activate the death receptor pathway or down-regulate XIAP, this was not observed with OSU-HDAC42 in myeloma cells.

In conclusion, OSU-HDAC42 has in vitro activity against myeloma cells and acts via activation of caspases, inducing the cell cycle suppressors p21WAF1/CIP1 and p16, as well as interfering with the IL-6 signal transduction pathway.
Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.
Authors: Paneesha Shankaranarayana , Adya Raghu , Khanji Hemali , Leung Ed , Vijayasekar C. , Pratt Guy , Borg Anton , Rose Peter , Morris Alan
Published: April 2020

Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection.

Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE).

Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.
PAD Is a Highly Effective Regimen for the Treatment of Patients with Relapsed Refractory Multiple Myeloma.
Authors: Delforge Michel , Janssens Ann , Dierickx Daan , Maertens Johan , Theunissen Koen , Vandenberghe Peter , Verhoef Gregor
Published: April 2020

Background and aim of the study:

Bortezomib (Velcade®) is a first-in-class potent and reversible proteasome inhibitor that has been approved for the treatment of relapsed and/or refractory multiple myeloma (MM). With single agent bortezomib, complete (CR) and partial (PR) response rates have been reported in 27% of patients with relapsed refractory (Richardson 2003), and in 38% of patients with relapsed MM (Richardson 2005). Addition of dexamethasone (dex) to bortezomib can increase the response rates (Jagannath, 2005), and a synergistic anti-myeloma effect has been described when bortezomib is combined with cytotoxic drugs like anthracyclins and melphalan (Ma, 2003). With the combination of PAD (bortezomib, adriamycin and dex) a 95% CR+PR rate in untreated myeloma patients has been reported (Oakervee, 2005), but data on the effectiveness of PAD in the relapsed, refractory setting are currently lacking.

Patients and Treatment:

We have studied the anti-myeloma effect of PAD in 8 unselected relapsed refractory MM patients (M/F:6/2; mean age 61 y, range:50–75). The mean time since diagnosis was 6 years (range:2–11). Previous treatment consisted of a median of 6 treatment lines including autologous (n = 6) and allogeneic (n = 2) transplantation, various forms of combination chemotherapy (n = 8) including anthracyclins (n = 7), pulse dose dex (n = 7), thalidomide −/+ dex (n = 7), bortezomib (n = 8) without (n = 3) or with dex (n = 5). PAD was administered as previously published (Oakervee, 2005), but with 12 days of dex administration also during during the odd cycles.

Results:

A median of 4 cycles of PAD were administered (range: 2–6). The CR+ PR rate was 88% according to the EBMT criteria: one patient achieved a CR (IF+), one a VGPR, and 5 patients a PR. One patient had a limited amount of paraprotein secretion but multiple extramedullary plasmacytomas. After 2 cycles of PAD she had > 50% reduction in tumormass on PET-CT imaging. On average, the best response occurred already after 2 cycles of PAD. 4/8 patients relapsed after a mean interval of 5 months and 3 patients are still in remission at + 5 months. Two patients developed septicaemia with one toxic death. Hematological grade III/IV toxicities included: neutropenia (4/8), and thrombocytopenia (4/8). Neuropathy pre-existed in 4/8 patients and was not aggravated with appropriated dose reductions of bortezomib.

Conclusion:

Using a combination of bortezomib, adriamycin and dexamethasone (PAD), we could obtain at least a PR in 8/8 unselected highly refractory myeloma patients who had, amongst many other agents, previously been exposed to bortezomib and/or adriamycin and/or dexamethasone. Major toxicities of PAD were hematological with one toxic death because of septicaemia. We believe that our observation warrants further studies of the PAD regimen in relapsed MM.