Validation of Potential Chemosensitivity Genes in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1818-1818
Author(s):  
Lin Yang ◽  
Xingding Zhang ◽  
Hua Wang ◽  
Robert Z. Orlowski ◽  
Xingxu Huang
Keyword(s):  
Multiple Myeloma ◽  
Patient Outcomes ◽  
High Throughput ◽  
Conflicts Of Interest ◽  
Tyrosine Phosphatase ◽  
Expression System ◽  
Myeloma Cells ◽  
Bortezomib Treatment ◽  
Junction Protein ◽  
Chemotherapy Agents

Abstract Abstract 1818 Chemotherapy agents are extremely important in the treatment of liquid malignancies, such as multiple myeloma (MM). Unfortunately, chemotherapy resistance in MM therapy is the most significant cause of treatment failure. The ability to predict, treat, or circumvent resistance is extremely likely to improve clinical outcomes. Thus, identification of novel genes that play a crucial role in MM progression and chemosensitivity is necessary to understand this disease better at the molecular level. Moreover, these genes and their products may serve as new therapeutic targets for MM, whose expression could improve patient outcomes or served as a predictor for chemotherapy outcome. To identify potential chemosensitivity genes, establishing a high-throughput method for validation of targets becomes urgently needed. Toward this purpose, we have successfully developed a high-throughput siRNA based functional target validation approach and identified 34 potential chemosensitivity genes. Our preliminary studies focusing on one of the candidate gene, TJP1 (tight junction protein 1), suggested that targeting TJP1 led to tumor cell resistant to several chemotherapy agents, including doxorubicin (Dox), cisplatin (Cis), methotrexate (MTX), and bortezomib. Further analysis with 264 bortezomib treated MM patients indicated that expression level of TJP1 correlated with patient response to bortezomib. Two clones and pooled RPMI 8226 MM cell line, which were developed against bortezomib treatment in our lab, showed loss of TJP1 expression, suggesting a role of TJP1 may play in bortezomib resistance development. More importantly, TJP1 targeting in myeloma cells resulted in cell resistance to bortezomib treatment. Protein tyrosine phosphatase, receptor type, O1 (PTRPO) was identified in our screening as well. We first examined the expression of PTPRO in a panel of myeloma cell lines, and found that PTPRO expression was dramatically decreased in several drug resistant lines compared to their parental sensitive cells. Moreover, using a Lentiviral cDNA expression system to overexpress PTPRO, we observed that PTPRO significantly inhibited cell growth and sensitized drug sensitivity of several drug lines by triggering apoptosis. Finally, evaluation of the publicly available gene expression profiling data gathered from primary plasma cells on the Multiple Myeloma Genomics Portal, and the linked clinical annotation describing patient outcomes, has shown that over-expression of PTPRO is associated with a superior overall survival compared to patients whose myeloma show low levels of PTRPO expression, suggesting this may be a prognostic marker as well. Together, we have successfully identified a group of potential chenmosensitivity genes, in which two of the genes (TJP1 and PTPRO) have been further validated to sensitize the drug treatment in multiple myeloma cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Decursin and Doxorubicin Are in Synergy for the Induction of Apoptosis via STAT3 and/or mTOR Pathways in Human Multiple Myeloma Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Jinsil Jang ◽  
Soo-Jin Jeong ◽  
Hee-Young Kwon ◽  
Ji Hoon Jung ◽  
Eun Jung Sohn ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Combination Treatment ◽  
Tyrosine Phosphatase ◽  
Combined Treatment ◽  
Parp Cleavage ◽  
Myeloma Cells ◽  
Stat3 Signaling Pathway ◽  
Human Multiple Myeloma ◽  
Induction Of Apoptosis ◽  
Apoptotic Activity

Background. Combination cancer therapy is one of the attractive approaches to overcome drug resistance of cancer cells. In the present study, we investigated the synergistic effect of decursin fromAngelica gigasand doxorubicin on the induction of apoptosis in three human multiple myeloma cells.Methodology/Principal Findings. Combined treatment of decursin and doxorubicin significantly exerted significant cytotoxicity compared to doxorubicin or decursin in U266, RPMI8226, and MM.1S cells. Furthermore, the combination treatment enhanced the activation of caspase-9 and -3, the cleavage of PARP, and the sub G1 population compared to either drug alone in three multiple myeloma cells. In addition, the combined treatment downregulated the phosphorylation of mTOR and its downstream S6K1 and activated the phosphorylation of ERK in three multiple myeloma cells. Furthermore, the combined treatment reduced mitochondrial membrane potential, suppressed the phosphorylation of JAK2, STAT3, and Src, activated SHP-2, and attenuated the expression of cyclind-D1 and survivin in U266 cells. Conversely, tyrosine phosphatase inhibitor pervanadate reversed STAT3 inactivation and also PARP cleavage and caspase-3 activation induced by combined treatment of doxorubicin and decursin in U266 cells.Conclusions/Significance. Overall, the combination treatment of decursin and doxorubicin can enhance apoptotic activity via mTOR and/or STAT3 signaling pathway in multiple myeloma cells.

Expression of TRAIL Receptors on the Multiple Myeloma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4868-4868
Author(s):  
Juan Li ◽  
Junhe Li ◽  
Shaokai Luo ◽  
Yin Zhao
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Mononuclear Cells ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Killing Effect ◽  
Myeloma Cells ◽  
Decoy Receptors ◽  
Trail Receptors ◽  
Chemotherapy Agents

Abstract Objective To study the different expression of death receptors and decoy receptors on mononuclear cells from patients with multiple myeloma and myeloma cell line KM3 and compare the different expression of TRAIL receptors after chemotherapy or exposure to doxorubicin, to explore the mechanisms by which TRAIL selectively kills tumor cells. Methods Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry was used to investigate the expression of four receptors on mononuclear cells in 23 multiple myeloma patients and myeloma cell line KM3 and 15 controls, we furthermore compared the changes of expression mode after chemotherapy and incubation of KM3 cell with sub-clinical concentration of Doxorubicin. Results There finds only DR4 and DR5 on KM3 cell line without the expression of DcR1 and DcR2. Expression of DR4 and DR5 on mononuclear cells of MM patients is higher than that of controls (P<0.05), but DcR1 and DcR2 expression was lower than that of controls (P<0.05), after chemotherapy and exposure to Doxorubicin, the expression of DR5 on MM cells was up-regulated (P<0.05) Conclusions The expression of four receptors on myeloma cells and normal controls was significantly different, which might account for the selective killing effect of TRAIL on MM cells. DR5 was up-regulated on KM3 when incubating with Doxorubicin and after chemotherapy which suggests chemotherapy agents might enhance the apopotosis of MM cells through up-regulating of DR5 receptor.

Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1915-1915
Author(s):  
Unn-Merete Fagerli ◽  
Thorsten Stühmer ◽  
Toril Holien ◽  
Randi Utne Holt ◽  
Ove Bruland ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factors ◽  
Cell Lines ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Transcriptional Response ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

A Novel Small Molecule with Epigenetic Activity Prolongs Survival in a Mouse Model of Multiple Myeloma Refractory to Standard Drugs

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5121-5121
Author(s):  
Sergei Vatolin ◽  
Khan Nazeer Shahper ◽  
Yvonne Parker ◽  
Daniel Lindner ◽  
Frederic J. Reu
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Chromatin Condensation ◽  
Xenograft Model ◽  
Myeloma Cells ◽  
Tail Vein ◽  
Tail Vein Injection ◽  
Mouse Xenograft Model ◽  
Number Of Patients

Abstract Abstract 5121 Multiple myeloma refractory to bortezomib, IMiDs™, and conventional therapies represents an unmet medical need. An increasing number of patients progress to this stage since treatment related mortality has decreased. To test promising compounds for activity in this setting we established an NSG mouse xenograft model with serial transplantation by tail vein injection of myeloma cells from a patient with IgG kappa myeloma relapsed and refractory to all standard drugs. Eight days after tail vein injection monoclonal human IgG can be detected in serum. Bone marrow engraftment in young (6–12 weeks) NSG mice after sublethal radiation (275cGy) is close to 100% (n=32). Untreated mice die within less than 2 months, usually with liver and spleen metastasis (anti-human CD138 flow cytometry). In a drug screen that used a novel method developed in our lab, chromatin condensation PCR, we identified a non nucleoside compound (4I3) that potently (1mM) reactivated expression of epigenetically silenced genes and displayed cancer-specific growth and survival inhibition in myeloma cell lines but not normal cells. Normal bone marrow cells continued to divide at doses 10x higher than required to kill 80% of myeloma cells. 4I3 suppressed DNMT1 protein but rapid cell kill (within 1–2 days) suggested additional mechanisms which we currently investigate. Given IV to mice after documentation of engraftment by IgG serum immunoblots, it prolonged survival in an ongoing experiment. Updated results will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Exposure to Novel Agents Contribute to Extramedullary Spread of Multiple Myeloma Cells and Altered Expression of Adhesion Molecules: A Clinicopathologic Study of 91 Consecutive Autopsy Cases

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1832-1832
Author(s):  
Sohtaro Mine ◽  
Shotaro Hagiwara ◽  
Amane Tagashira ◽  
Toru Igari ◽  
Akiyoshi Miwa
Keyword(s):  
Multiple Myeloma ◽  
Adhesion Molecules ◽  
Conflicts Of Interest ◽  
Remission Rate ◽  
Novel Agents ◽  
Altered Expression ◽  
Myeloma Cells ◽  
Factors Associated ◽  
Patient Profile ◽  
Duration Of Illness

Abstract Abstract 1832 Background Recent development of novel agents such as bortezomib, lenalidomide, and thalidomide improved the remission rate and survival of multiple myeloma. However, the end stage of myeloma is still uncontrollable. The extramedullary disease (EMD) progression of myeloma was frequently observed in heavily treated patients, whereas few data exist about its incidence and predictive factors. It is well known that adhesion molecules play a role in disease progression and drug resistance. We investigated factors associated with the EMD progression based on the autopsy cases. In addition, the effect of the exposure to novel agents on the expression of adhesion molecules on myeloma cells was analyzed. Methods We reviewed autopsy reports and medical records of 91 multiple myeloma cases between 1979 and 2012 at the National Medical Center for Global Health and Medicine in Tokyo, Japan. The sites of myeloma cell invasion, infection, hemorrhage and renal complications were studied gross and microscopically. Patient profile, duration of illness, type of monoclonal gammopathy, clinical stage and history of treatment were studied. Durie & Salmon's criteria was used for diagnosis and staging. Factors associated with EMD were statistically analyzed using Student's t-test and the chi-square test. NCAM, VCAM, ICAM, and LFA-1 immunostaining in the bone marrow was performed. Results In 91 autopsy cases, 62.6% was male. Mean age and the duration of illness was 63.0 (38–85) years old and 40.6 months (1–156). Eighteen patients (19.8%), 23 (25.3%), and 15 (16.4%) were treated with novel agents, SCT and both respectively. EMD progression of myeloma cells was observed in 65 patients (71.4%). Frequent sites of EMD were spleen (48.9%), liver (37.8%), kidney (31.1%), lymph nodes (28.6%), lung (25.6%), pancreas (20.0%), and gastro-intestinal tract (18.9%). The incidence of EMD was significantly higher in patients treated with novel agents than in patients without novel agents (94.4% vs. 65.8%, p=0.016). The risk factors of EMD were novel agents, longer duration of illness, and adverse cytogenetic abnormalities. In the cases with novel agents, the expression of NCAM was significantly low (11.1% vs. 57.4%, p=0.049) compared to the cases without novel agents. However, the expression of VCAM was significantly higher in the cases with novel agents than the cases without novel agents (40.0% vs. 0%, p=0.01). Conclusion Multi-organ involvement of myeloma is not rare in autopsy cases of the disease. Exposure to novel agents may contribute to extramedullary spread of myeloma cells and altered expression of adhesion molecules. Further study on other adhesion molecules is needed. Disclosures: No relevant conflicts of interest to declare.

Identification of Tight Junction Protein (TJP)-1 As a Modulator of Sensitivity to Doxorubicin and Cisplatin in Models of Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3962-3962
Author(s):  
Xing-Ding Zhang ◽  
Robert Z. Orlowski ◽  
Lin Yang
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Hela Cells ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Cancer Center ◽  
In Vivo Models ◽  
Junction Protein ◽  
Rpmi 8226

Abstract Abstract 3962 Background: Therapeutic advances in multiple myeloma have improved the outcomes of patients with this malignant plasma cell disorder, but the disease course is still strongly influenced by both innate, or primary, as well as acquired, or secondary mechanisms of drug resistance. Identification and validation of genes that may mediate these phenotypes is therefore of importance, since they could be useful prognostic markers, and also potential targets to overcome the emergence of resistance, or possibly preclude its emergence altogether. Methods: To identify non-redundant determinants of chemoresistance, we designed a robust, high-throughput RNA interference (RNAi) screen targeting 9610 human genes. The screen involved retroviral-mediated transduction first of HeLa cervical carcinoma cells with either the RNAi library, or with non-targeting retrovirus particles. After infection, cells were selected with puromycin, and treated with different concentrations of doxorubicin and cisplatin. Doxorubicin (Dox) treatment led to 33 surviving colonies from the cells transduced with the shRNA library, cisplatin (Cis) treatment led produced 22 surviving colonies, while non-targeting retrovirus-infected cells failed to form colonies after treatment. Screening was performed to identify the shRNA target gene(s) in each colony, and genes that were identified in both Dox- and Cis-treated HeLa cells, and that were expressed in myeloma cells, were selected for further study. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results: TJP1 (zona occludens (ZO)-1) was identified as one gene whose knockdown promoted survival in Dox- and Cis-treated HeLa cells, and which was expressed in myeloma cell lines and in primary plasma cells. To further examine its potential role in myeloma chemosensitivity, we performed mRNA and protein expression profiling in a panel of 11 cell lines and observed that TJP1 expression was silenced in 3 cell lines (ARP-1, INA-6, and MOLP-8), while it was moderately to highly expressed in 7 cell lines (including RPMI 8226, MM1.S, and U266). Comparing TJP1-positive MM1.S cells to TJP1-null MOLP-8 cells, the latter displayed a significantly higher median inhibitory concentration to Dox and Cis. Knockdown of TJP1 in RPMI 8226 and U266 cells, which produced a >75% target suppression, was sufficient to reduce the proportion of apoptotic cells in the sub-G1 fraction after treatment with Dox or Cis compared to control cells. Conversely, MOLP-8 cells transfected with human TJP1 cDNA exhibited an increase in the sub-G1 population in response to Dox and Cis treatment compared to vector controls. Conclusion: Taken together, these studies support the hypothesis that TJP1 expression mediates myeloma cell resistance to the DNA damaging agents doxorubicin and cisplatin. Further studies are underway to determine the mechanism by which TJP1 influences chemosensitivity, and to validate its impact using in vivo models. Disclosures: No relevant conflicts of interest to declare.

The Expression Of CD200 As a Prognostic Factor In Newly Diagnosed Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3082-3082
Author(s):  
Yi Li ◽  
Wenjun Wu ◽  
Jingsong He ◽  
Xiaoyan Han ◽  
Gaofeng Zheng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Progression Free Survival ◽  
Staging System ◽  
Newly Diagnosed ◽  
New Approach ◽  
Myeloma Cells ◽  
Targeting Therapy ◽  
Molecular Therapeutic

Abstract Introduction multiple myeloma (MM) is currently an incurable hematological malignancy. Discovering molecular therapeutic targets is a new approach to improve the outcome in the treatment of the malignant disease. As CD200 is a type Ⅰmembrane glycoprotein expressed on myeloma cells, we asked if the expression of CD200 could serve as a prognostic marker for MM patients. Our data indicated that the expression level of CD200 is indeed correlated with the prognosis of the MM patients. Methods bone marrow samples from 96 newly diagnosed MM patients from April 2011 to July 2013 were evaluated by flow cytometry, using PE-conjugated anti-CD200 mAb, FITC-conjugated anti-CD138 mAb, and PE-Cy7-conjugated anti-CD45 mAb. PE-or FITC-conjugated normal mouse IgG was used as isotype-matched controls. Results 96 MM patients were investigated in the present study, including 60 men and 36 women, with a median age of 63 years (range 34–86 years). 81/96(84%) MM patients were CD200 positive with a median Mean Fluorescence Intensity (MFI) of 127 as analyzed by flow cytometry, which was consistent with the previous studies. While in 15 of 96 patients, CD200 expression was undetectable. Among the CD200 positive ones 7.40% patients were classified as stage Ⅰ, 12.35% were stage Ⅱ, and 80.25% were stage Ⅲ according to Durie–Salmon staging criteria. 40.74% patients were stageⅠ, 22.22% were stage Ⅱ, and 37.04% were stage Ⅲ, according to the International Staging System (ISS). Analysis of the CD200 positive patients revealed the MFI<127 group had a better progression free survival (PFS) (p=0.046) (Fig 1A) and overall survival (OS) (p=0.069) compared to those with MFI≥127. In the patients with age ≥65 years old, PFS (p=0.023) (Fig 1B) and OS (p=0.044) (Fig 1C) were much shorter in the MFI≥127 group, compared to the MFI<127 ones. Conclusions Our study demonstrated that the expression and MFI of CD200 on primary multiple myeloma cells is correlated with the prognosis of the MM patients. The better PFS and OS were observed in the MFI<127 group compared to the patients with MFI≥127, especially in the patients with age≥65 years old. Improved PFS in CD200 positive ones is likely due to the immune suppression mediated by CD200. Our study suggests that targeting therapy against CD200 may become a new approach to the treatment of MM in clinical practice. Disclosures: No relevant conflicts of interest to declare.

The Effect of Proteasome Subunit Beta Type 1 P11A Polymorphism on the Survival of Multiple Myeloma Patients Treated with First Line Bortezomib Based Chemotherapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1765-1765
Author(s):  
Gergely Varga ◽  
Gabor Mikala ◽  
Gergely Szombath ◽  
Katalin Balassa ◽  
Katalin Piroska Kiss ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Conflicts Of Interest ◽  
Proteasome Inhibitors ◽  
Predictive Marker ◽  
Recessive Model ◽  
First Line ◽  
Proteasome Subunit ◽  
Bortezomib Treatment ◽  
Dismal Prognosis

Abstract Background: Proteasome inhibitors have a fundamental role in the treatment of multiple myeloma however there are still patients who fail to achieve a good response and have a dismal prognosis. Markers predicting the outcome of individual patients are needed to identify those who would benefit more from a different approach. Proteasome subunit beta type 1 (PSMB1) rs12717 single nucleotide polymorphism (C/G substitution resulting in a P11A change) was reported recently to be associated with greater clinical benefit in relapsed follicular lymphoma patients treated with bortezomib containing combination (Coiffier et al Clin Cancer Res 2013). To the best of our knowledge there is only one report with limited number of myeloma patients showing no association between PSMB1 P11A genotype and survival (Lichter et al Blood 2012). Aims and Methods: We analyzed the association of PSMB1 P11A polymorphism and treatment outcome of 220 consecutive myeloma patients having had first line chemotherapy in our unit. PSMB1 P11A polymorphism was tested using LightCycler melting analysis. Statistical analyses were performed using the SPSS (version 20.0) software package. Results: The distribution of presentation prognostic markers such as age, ISS and FISH were even among the three genotype groups. 149 patients had bortezomib-based chemotherapy, mainly VTD (n=76) and MPV (n=47); 63% of these patients had ASCT consolidation. 71 patients had non-bortezomib-containing induction with either thalidomide, lenalidomide or other protocols including MP and VAD; only 28% of these patients had ASCT. In a recessive model the median PFS was 727 (635-818) days in carriers of the wild type C allele either in homozygous of heterozygous form (C/C&C/G, n=188), and only 491 (366-615) days in the homozygous variant G allele carriers (G/G, n=32, p=0.01). In multivariate analysis, C allele carriers had favorable PFS compared to G/G genotype patients with a hazard ratio of 1.402 (1.093-1.799), p=0.008 besides age, ISS, FISH, bortezomib treatment and ASCT. When we analyzed separately the bortezomib-treated and non-bortezomib-treated patients the benefit in PFS was only present in the bortezomib group [Fig 1, PFS 747 (656-837) days in C/C&C/G and 448 (279-616) days in G/G patients, p=0.002], and not in patients treated without bortezomib (p=0.559). We performed statistical interaction testing which showed significant interaction between bortezomib exposure and PSMB1 P11A (p=0.035). Conclusion: PSMB1 P11A is a novel predictive marker in multiple myeloma which could help to identify patients having a suboptimal response to bortezomib who might benefit from alternative management. Disclosures No relevant conflicts of interest to declare.

scholarly journals TRIM33 Loss in Multiple Myeloma Impairs the DNA Damage Response Resulting in Sensitivity to PARP and ATR Inhibitors

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1570-1570
Author(s):  
Roisin M McAvera ◽  
Jonathan J Morgan ◽  
Ken I Mills ◽  
Lisa J Crawford
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Damage Response ◽  
High Throughput ◽  
Chromosomal Instability ◽  
Conflicts Of Interest ◽  
Parp Cleavage ◽  
Structural Variants ◽  
Damage Response ◽  
The Impact

Abstract Introduction Chromosomal instability is a hallmark of Multiple Myeloma (MM), with most patients displaying cytogenetic abnormalities which can arise due to DNA damage response (DDR) defects. TRIM33 is an E3 ligase and transcriptional co-repressor located on chromosome 1p13.2, a region frequently deleted in MM. Previous studies have shown that TRIM33 plays a role in the DDR and can regulate chromosomal stability, but its precise function remains unknown. In this study we investigated the impact of TRIM33 loss in MM on genomic stability and DDR pathways and whether this could be exploited therapeutically. Methods The CoMMpass dataset (IA15 release) was screened to identify patients with copy number (CN) loss of TRIM33 and this was correlated with overall survival (OS) and structural variants. TRIM33 shRNA knockdown models were established in JJN3 and U266 cells. The effect on DDR signalling was determined by western blotting and immunofluorescence. The Selleckchem DNA Damage/Repair Compound Library was screened on the JJN3 model in a high-throughput manner using the CellTox™ Green cytotoxicity assay. Validation of selected compounds was performed using CellTiter® Glo viability assay or clonogenic assays. Combination indices (CI) were calculated using CompuSyn software. Results Data on CN, OS and structural variants were available for 730 newly diagnosed MM patients and of these, 69 (9.5%) were identified to have a CN loss of TRIM33. These patients have poorer OS compared to those without TRIM33 loss (52.3 months vs 72.6 months; p&lt;0.0001). Moreover, they exhibit a significantly higher median number of structural variants (deletions, duplications, inversions, and translocations; 38 vs 26; p&lt;0.0001), indicative of increased chromosomal instability. Our data in MM cell lines has shown that TRIM33 is rapidly recruited to chromatin within 5 minutes of induced DNA damage. TRIM33 knockdown led to an increase in 53BP1 foci formation and endogenous γH2AX (P&lt;0.001) indicating unrepaired DNA double-strand breaks (DSBs) typical of a DDR defect. In response to these DSBs both ATM and ATR kinases were activated as demonstrated by increased pKAP1 Ser824 and pCHK1 Ser345 respectively (p&lt;0.001). Additionally, we observed a reduction in RAD51 (p&lt;0.05) indicative of a potential defect in the DSB repair pathway homologous recombination (HR). To identify therapeutic vulnerabilities relating to TRIM33 loss, we performed a high-throughput screen to assess sensitivity to 160 unique DNA damaging compounds. TRIM33 knockdown cells exhibited increased sensitivity to 27 compounds across a range of drug classes. Additional studies confirmed that compared to control cells, TRIM33 knockdown sensitized cells to the PARP inhibitor Olaparib and ATR inhibitors BAY-1895344 and VE-821. Further investigation with VE-821 demonstrated that whilst treatment induced PARP cleavage and DSBs in both control and knockdown cells within 48 hours, knockdown cells exhibited significantly more pCHK1 Ser345 inhibition (p&lt;0.01). Furthermore, combining VE-821 with bortezomib yielded synergistic effects in TRIM33 knockdown cells across a range of doses (CI range 0.57-0.9) while no synergy was observed in control cells (CI&gt;1 for all combinations). Conclusion We have identified a subset of MM patients with TRIM33 loss who display high-risk disease characterized by chromosomal abnormalities and defective DDR. Alongside this we have identified PARP and ATR inhibitors as therapeutic vulnerabilities in cell line models of TRIM33 loss. Moreover, we demonstrate that ATR inhibition increases the efficacy of bortezomib in TRIM33 knockdown cells. Further investigation into these compounds could lead to novel therapies for patients with TRIM33 loss. Disclosures No relevant conflicts of interest to declare.

scholarly journals Downregulation of DCC sensitizes multiple myeloma cells to bortezomib treatment

2019 ◽  
Author(s):  
Dorival Rodrigues‑Junior ◽  
Tha�s Biassi ◽  
Gabriela de Albuquerque ◽  
Viviane Carlin ◽  
Marcus Buri ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Myeloma Cells ◽  
Bortezomib Treatment
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