Identification of ATM Mutations in Multiple Myeloma Tumours.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1562-1562
Author(s):  
Belinda Austen ◽  
Maria Podinovskaia ◽  
Jane Starcynski ◽  
Giancarlo Barone ◽  
Anne Reiman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Kinase Activity ◽  
Plasma Cells ◽  
Poor Response ◽  
Primary Therapy ◽  
Atm Kinase ◽  
Sequence Change ◽  
Atm Gene ◽  
Atm Protein

Abstract Ataxia Telangiectasia (AT) patients have biallelic inactivation of the ATM gene and exhibit a 200 fold increased frequency of lymphoid tumours, commonly occurring at a young age. In AT families the relative risk for developing myeloma is 4.49 for heterozygous ATM carriers compared to normal controls, and multiple myeloma has been reported in an AT patient with an unusually mild form of the disease associated with prolonged survival. Normal plasma cells have high ATM expression similar to that seen in lymphocytes from the mantle zone of lymph node follicles. Although somatic ATM mutations have been found in a number of adult lymphoid malignancies including T-PLL, mantle cell lymphoma and CLL, there is no data on the occurrence of ATM mutations in multiple myeloma tumours. We screened multiple myeloma bone marrow aspirates for ATM expression by immunohistochemistry and for ATM mutations using denaturing high performance liquid chromatography analysis (HPLC). By immunohistochemistry, markedly reduced ATM expression was seen in the malignant plasma cells in 3 out of 45 tumours. These three tumours were analysed for mutations by HPLC but only one of the three patients had an identified mutation. In this tumour with the lowest ATM expression, the mutation 7181C/T (S2394L) was identified. We next modelled this mutation in an in vitro system to confirm that the S2394L substitution led to a form of ATM protein that had defective ATM kinase activity. We cloned the ATM gene, carrying the sequence change, into ATM negative cell lines and measured ATM kinase activity by assessing the phosphorylation of ATM targets following DNA damage with ionising irradiation. By this method, we were able to confirm that the S2394L sequence change resulted in a form of ATM protein that has absent kinase activity. A further 42 multiple myeloma tumours were analysed for ATM mutations by HPLC. We identified 4 more tumours that had mutations in the ATM gene, namely 8053T/A (S2685T), 6995T/C (L2332P), 4724G/A (R1575H) and IVS40-1G/C. Notably, the changes 8053T/A and 6995T/C are located within highly conserved ATM protein domains and the change IVS40-1G/C has been found in AT patients. Following DNA damage, deficiency of ATM leads to increased levels of unrepaired DNA breaks leading to genomic instability and also to defects in p53 dependent apoptosis. Loss of ATM activity through mutations may be contributing to multiple myeloma development and this idea is supported by the increased incidence of myeloma in AT carriers. ATM is essential for the activation of p53 dependent apoptosis in response to chemotherapy, and ATM mutations are associated with a poor outcome and chemoresistance in CLL patients. Four of the myeloma patients with ATM mutations in this study had aggressive disease including three with a poor response to primary therapy. In summary, we have shown for the first time that ATM mutations can occur at a low frequency in sporadic multiple myeloma tumours, identifying 5 patients in this study. Preliminary data suggests they may be associated with a poor response to treatment although this needs further confirmation.

scholarly journals DNA Damage-Induced Acetylation of Lysine 3016 of ATM Activates ATM Kinase Activity

2007 ◽  
Vol 27 (24) ◽  
pp. 8502-8509 ◽  
Author(s):  
Yingli Sun ◽  
Ye Xu ◽  
Kanaklata Roy ◽  
Brendan D. Price
Keyword(s):  
Dna Damage ◽  
Kinase Activity ◽  
Histone Acetyltransferase ◽  
Kinase Domain ◽  
Atm Kinase ◽  
Lysine Residues ◽  
Atm Protein ◽  
Fatc Domain

ABSTRACT The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-lysine 3016 demonstrate rapid (within 5 min) in vivo acetylation of ATM following exposure to bleomycin. Furthermore, lysine 3016 of ATM is a substrate in vitro for the Tip60 histone acetyltransferase. Mutation of lysine 3016 does not affect unstimulated ATM kinase activity but does abolish upregulation of ATM's kinase activity by DNA damage, inhibits the conversion of inactive ATM dimers to active ATM monomers, and prevents the ATM-dependent phosphorylation of the p53 and chk2 proteins. These results are consistent with a model in which acetylation of lysine 3016 in the FATC domain of ATM activates the kinase activity of ATM. The acetylation of ATM on lysine 3016 by Tip60 is therefore a key step linking the detection of DNA damage and the activation of ATM kinase activity.

scholarly journals ATM kinase activity modulates cFLIP protein levels: potential interplay between DNA damage signalling and TRAIL-induced apoptosis

2010 ◽  
Vol 31 (11) ◽  
pp. 1956-1963 ◽  
Author(s):  
Venturina Stagni ◽  
Michele Mingardi ◽  
Simonetta Santini ◽  
Danilo Giaccari ◽  
Daniela Barilà
Keyword(s):  
Dna Damage ◽  
Kinase Activity ◽  
Atm Kinase ◽  
Protein Levels ◽  
Induced Apoptosis ◽  
Dna Damage Signalling

scholarly journals Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Kenta Yamamoto ◽  
Jiguang Wang ◽  
Lisa Sprinzen ◽  
Jun Xu ◽  
Christopher J Haddock ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Therapy ◽  
Animal Models ◽  
Dna Adducts ◽  
Kinase Domain ◽  
Missense Mutations ◽  
Atm Kinase ◽  
Dna Damage Responses ◽  
Atm Protein ◽  
Kinase A

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (AtmKD/-) is more oncogenic than loss of ATM (Atm-/-) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate AtmKD/-, but not Atm-proficientor Atm-/- leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

scholarly journals Characterization of ATM Expression, Localization, and Associated DNA-dependent Protein Kinase Activity

1998 ◽  
Vol 9 (9) ◽  
pp. 2361-2374 ◽  
Author(s):  
Dennis P. Gately ◽  
James C. Hittle ◽  
Gordon K. T. Chan ◽  
Tim J. Yen
Keyword(s):  
Dna Damage ◽  
Protein Kinase ◽  
Ataxia Telangiectasia ◽  
Kinase Activity ◽  
Human Fibroblasts ◽  
Protein Kinase Activity ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Atm Protein

Ataxia telangiectasia–mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA–dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.

Correction of ATM mutations in iPS cells from two ataxia-telangiectasia patients restores DNA damage and oxidative stress responses

2020 ◽  
Vol 29 (6) ◽  
pp. 990-1001 ◽  
Author(s):  
Dmitry A Ovchinnikov ◽  
Sarah L Withey ◽  
Hannah C Leeson ◽  
U Wang Lei ◽  
Ashmitha Sundarrajan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Stress Responses ◽  
Ataxia Telangiectasia ◽  
Compound Heterozygous ◽  
Gene Correction ◽  
Atm Kinase ◽  
Atm Protein ◽  
Induced Apoptosis ◽  
And Oxidative Stress

Abstract Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.

IgM Multiple Myeloma: A Rare Subtype Highly Sensitive to Lenalidomide

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5220-5220
Author(s):  
Alvaro Moreno-Aspitia ◽  
Antony Charles ◽  
Tejal Patel ◽  
Celine Bueno ◽  
Abba Zubair ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transient Response ◽  
Low Dose ◽  
Plasma Cells ◽  
Poor Response ◽  
Response To Therapy ◽  
Bone Lesions ◽  
Best Response ◽  
M Spike

Abstract Background: IgM multiple myeloma (MM) are very rare plasmaproliferative disorders representing 0.5–1.2% of all cases of MM and < 0.2% of all IgM monoclonal gammopathies. Clinical criterion are not always helpful in differentiating IgM MM from Waldenstrom macroglobulinemia. However, the presence of lytic bone lesions, absence of lymphadenopathy and/or hepatosplenomegaly, presence of translocation of the immunoglobulin heavy chain locus at 14q32 [t(11;14), t(14;16), t(4;14)], and strong expression of CD138 by the plasma cells are useful in the diagnosis of IgM MM. It has been our experience and of others that these cases have an aggressive behavior at presentation, shorter survival than IgG and IgA MM and poor response to therapy for lymphoplasmacytoid lymphomas. We present here 2 cases of IgM MM with a dramatic response to Lenalidomide and low dose dexamethasone (Rev/Dex) Results: Baseline patient characteristics at time of diagnosis of IgM MM and therapy outcome are presented in the following 2 tables: Table 1. Case 1 2 Age and sex 72 (F) 73 (F) Serum M-spike (g/dL) 5.3 6.2 Urine M-spike (mg/dl/24 hrs) 72 412 Serum IgM (mg/dL) 8,590 11,000 BM plasma cells percentage 90 20 Plasma cell immunophenotyping CD138+++, partial CD20, CD56− CD138+++, partial CD20, CD56− Cytogenetics (Standard and/or FISH) Standard: normal FISH: not done on initial biopsy. On follow up there were insufficient number of plasma cells to perform test Standard: of 20 metaphases, 6 had a complex hypotetraploid karyotype with relative loss of 13q, 14, 15, 16, 20, and 22, and numerous unbalanced rearrangements. FISH: a plasma cell clone with monosomy 13 and IGH/c-MAF fusion, t(14;16). In addition, approximately 60% of plasma cells had a tetraploid clone with the same anomalies as well as relative loss of p53 Bone lesions Multiple non-traumatic spinal fractures and of stenum Several lytic lesions of long bones Renal insufficiency No No Anemia (Hbg g/dL) Yes (8.7) Yes (8.1) Hypercalcemia (Ca mg/dL) Yes (12.5) Yes (11.4) Beta 2 microglobulin (mg/dL) 5.79 8.51 Serum viscosity (cpoise) 5.9 4.8 Table 2. Best Response to therapy Case Therapy Best Response Comments 1 Rituxan, then Fludarabine based therapy Transient response Rapid progression after partial and transient response to each therapy 1 Lenalidomide + LD-Dex sCR after cycle #6. Currently on CR 18 months later IgM declined from 8,590 to 43 mg/dL after 4 cycles of Rev/Dex. 2 Lenalidomide + LD-Dex VGPR after cycle #2 IgM declined from 11,000 to 463 mg/dL after cycle 3. Complete disappearance of M-spike in serum; BM to be done after cycle #4 Conclusions: This is the first report that we are aware of a rapid and dramatic response to lenalidomide and low dose dexamethasone in these rare cases of IgM MM with poor response to NHL-type treatment. Lenalidomide-based therapy might abrogate poor prognosis cytogenetics in this unusual subtype of MM (case #2), however, follow up for this patient is still very short.

CLL Tumours with a Deletion of 11q Form Two Functional Subgroups Based on the Mutation Status of the Remaining ATM Allele.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 710-710
Author(s):  
Belinda Austen ◽  
Maria Podinovskaia ◽  
Claire Almond ◽  
Graham Fews ◽  
Anne Gardiner ◽  
...  
Keyword(s):  
Dna Damage ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Fish Analysis ◽  
Wild Type ◽  
Atm Gene ◽  
Atm Protein ◽  
11Q Deletion

Abstract Deletions in chromosome 11q are an established prognostic marker in CLL. One copy of the ATM gene is deleted in these tumours, as detected by FISH analysis. However, it remains unclear whether the ATM gene is the main tumour suppressor gene that is accounting for the poor outcome in tumours with 11q deletions. We have recently reported that patients whose tumours have mutations in the ATM gene have an impaired overall and treatment free survival. In our large cohort of 155 patients, tumours with an ATM mutation only partly correlated with tumours with an 11q deletion. We have therefore investigated the relationship between 11q deletions and mutations in the ATM gene. Using the highly sensitive DHPLC method, we have screened the 60 ATM coding exons for mutations in a cohort of 46 tumours, all with a deletion of chromosome 11q. We have found ATM mutations in 19 tumours, indicating a prevalence of 41%. The ATM protein is vital in the cell’s response to DNA damage including that induced by chemotherapy. ATM acts upstream from p53 and defects in ATM function, like p53, lead to impaired DNA damage induced apoptosis. Furthermore, we have previously shown that loss of ATM function is associated with both in vitro and in vivo chemo-resistance. Therefore, we next assessed whether the status of the remaining ATM allele in the 11q deleted tumours affected the response to DNA damage. Firstly we induced DNA damage with irradiation and measured both the phosphorylation of ATM protein targets and the induction of p53 dependent transcription responses in representative samples. We found that 11q deleted tumours with a remaining wild type ATM allele had responses that were similar to those seen in tumours with two wild type ATM alleles. In contrast, 11q deleted tumours with a mutation in the remaining ATM allele had defective DNA damage induced responses. We then analysed the effects of in vitro treatment with Fludarabine. First we demonstrated that fludarabine induces ATM dependent phophosphorylation responses in CLL tumours. Then we analysed its effect in the two 11q deleted CLL subgroups. We showed defective phosphorylation responses to fludarabine in the tumours with a mutation in the second ATM allele, but normal responses in those with a second wild type ATM allele. In summary, we have shown that approximately 40% of CLL tumours with an 11q deletion have a mutation in their remaining ATM allele. Furthermore, we have demonstrated that the 11q deleted tumours appear to form two functional subgroups based on the presence of a mutation in the remaining ATM allele. In contrast to the subgroup with a wild type ATM allele, CLL tumours with a mutant ATM allele have defective in vitro responses to DNA damage with both irradiation and fludarabine. We expect that the functional differences between the two 11q deleted subsets will translate into differences in clinical outcome.

Gene Expression Profiling of Myeloma Cells To Predict Response to Primary Therapy with Thalidomide-Dexamethasone for Newly Diagnosed Multilple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1494-1494
Author(s):  
Abderrahman Abdelkefi ◽  
John de Vos ◽  
Said Assou ◽  
Tarek Ben Othman ◽  
Jean-Francois Rossi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Gene Expression Profiling ◽  
Treatment Strategy ◽  
Expression Profiling ◽  
Plasma Cells ◽  
Gene List ◽  
Microarray Platform ◽  
Primary Therapy ◽  
Newly Diagnosed

Abstract Background: Thalidomide which represents an effective treatment strategy for relapsed/refractory multiple myeloma, actually represents a standard of care also for newly diagnosed multiple myeloma patients. Methods: In the present study, we adopted a gene expression profiling (GEP) strategy in an attempt to predict response (> 50% reduction in serum M protein) to primary therapy with thalidomide-dexamethasone for newly diagnosed multiple myeloma. Plasma cells (CD138+) were purified from bone marrow aspirates from 17 patients at diagnosis, before initiation of treatment with thalidomide-dexamethasone. GEP was performed using the Affymetrix U133 Plus_2 microarray platform. The Affymetrix output (CEL files) was imported into Genespring 7.3 (Agilent technologies) microarray analysis software, where data files were normalized across chips using GCRMA and to the 50th percentile, followed by per gene normalization to median. Criteria of response were those established by Bladè et al. Results: After sufficient follow-up, responders (n=9) and nonresponders (n=8) were identified, and gene expression differences in baselines samples were examined. Of the 11000 genes surveyed, Wilcoxon rank sum test identified 149 genes that distinguished response from non response. A multivariate step-wise discriminant analysis (MSDA) revealed that 14 of the 149 genes could be used in a response predictor model (see table). Of interest, the gene list encompasses WXSC1, known to be involved in the chromosomal translocation t(4;14) (p16.3;q32.3) in multiple myeloma. Conclusion: These results could be the first step to adopt microfluidic cards, in an attempt to select at diagnosis patients who will respond favourably to a particular treatment strategy. List of 14 genes able to predict response to primary therapy with thalidomide-dexamethasone for newly diagnosed multiple myeloma. Gene ID Gene Name Chromosomal location 212771_at C10orf38 10p13 229874_x_at LOC400741 1p36.13 219690_at U2AF1L4 19q13.12 202207_at ARL7 2q37.1 243819_at GNG2 14q21 203753_at TCF4 18q21.1 235400_at FCRLM1 1q23.3 211474_s_at SERPINB6 6p25 226785_at ATP11C Xq27.1 215440_s_at BEXL1 Xq22.1–q22.3 209054_s_at WXSC1 4p16.3 227168_at FLJ25967 22p12.1 213355_at ST3GAL6 3q12.1 223218_s_at NFKBIZ 3p12–q12

Evidence for Ongoing DNA Damage in Multiple Myeloma as Revealed by Constitutive Phosphorylation of H2AX.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2817-2817
Author(s):  
Denise K. Walters ◽  
Renee C. Tschumper ◽  
Xiaosheng Wu ◽  
Kimberly J. Henderson ◽  
Angela Dispenzieri ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Dna Damage ◽  
Cell Lines ◽  
Plasma Cells ◽  
Clonal Evolution ◽  
S Phase ◽  
Intratumor Heterogeneity ◽  
H2ax Phosphorylation ◽  
Γh2ax Foci

Abstract Abstract 2817 Poster Board II-793 Abnormal plasma cells (PC) present in patients with multiple myeloma (MM) and its precursor condition, monoclonal gammopathy of undetermined significance (MGUS), characteristically possess multiple chromosomal abnormalities. Moreover, both stages of disease exhibit considerable intratumor heterogeneity, which often becomes even more complex during disease progression. The precise mechanism(s) underlying this process remains unknown. However, we hypothesize that DNA double-strand breaks (DSBs) and compromised repair of these deleterious lesions may underlie intratumor heterogeneity and clonal evolution in the monoclonal gammopathies. In this regard, H2AX, a member of the H2A family of histones, plays a particularly important role in the DSB response and prevention of cancer. Immediately following DSB formation, one or more of the PI3K-like kinases become activated and rapidly phosphorylate H2AX on a conserved serine residue. Phosphorylated H2AX (γH2AX) is then rapidly recruited to the DSB site and is readily detectable as DNA damage foci by immunohistochemistry. The precise function of γH2AX has yet to be determined, however, it is hypothesized that γH2AX may recruit DNA repair proteins to the DSB site and may aid in keeping severed DNA ends in place in order to avoid erroneous end joining. Despite the functional uncertainty of γH2AX, the presence of γH2AX nuclear foci serves as an excellent indicator of DSBs. Therefore, the goal of our study was to assess MM cells for evidence of DSBs. We began our studies using a panel of 8 human MM cell lines. Of note, the number of foci was found to vary among the MM cell lines and to vary from cell to cell with the number of γH2AX foci per cell ranging from 0 to 28. The presence of γH2AX in these cells was also confirmed via flow cytometry and western blotting. We also wished to determine if primary MM and MGUS PCs displayed evidence of DSBs. Among primary patient samples, freshly isolated PCs from 13/18 MM patients and 1/3 MGUS patients exhibited evidence of γH2AX foci. Taken together with the MM cell line data, the number of γH2AX foci was found to increase across the disease spectrum of MGUS to MM patient sample to MM cell line. Endogenous γH2AX foci have previously been detected in a variety of tumor cell lines. Although these foci have been hypothesized to derive from multiple factors, the extent of phosphorylation has been shown to be associated with the number of chromosomal aberrations as well as the phase of the cell cycle. In general, S and G2/M phase cells tend to demonstrate higher levels of H2AX phosphorylation, which is most likely due to doubling of histone content during the cell cycle and the fact that chromatin condensation during DNA replication can also trigger H2AX phosphorylation. Thus, it remained possible that the γH2AX displayed by the cell lines simply reflected cells in the S phase of the cell cycle. To address this possibility, we labeled cells with BrdU and then measured levels of γH2AX in cells in the G1, S and G2/M phases of the cell cycle. However, we observed nearly equal levels of γH2AX in G1 and S phase cells suggesting some level of γH2AX foci was independent of DNA replication. These results were also consistent with our observation that there is no correlation between the plasma cell labeling index and the number of γH2AX foci in CD138+ plasma cells isolated from 18 MM patients. Thus, endogenous γH2AX in MM cells does not appear to be primarily attributed to cycling cells and may be indeed reflective of DSBs. Finally, to further demonstrate that the γH2AX foci genuinely reflected sites of DSBs, we performed double staining for γH2AX foci and 53BP1, a protein that is known to be recruited to DSB sites following DNA damage. Results revealed clear colocalization of γH2AX and 53BP1 in both MM cell lines and MM patient samples. Given that DSBs can lead to genomic instability and tumor progression, our observations that primary MGUS and MM PCs display evidence of DSBs at isolation are intriguing and suggest a mechanism whereby clonal evolution occurs in the monoclonal gammopathies. The presence of a higher frequency of γH2AX foci in MM cell lines is consistent with their derivation from MM patients with aggressive disease. Collectively, these studies suggest MGUS/MM PCs may display an impaired ability to repair DNA damage and studies designed to examine this possibility are underway. Disclosures: Dispenzieri: Celgene: Research Funding.

Targeting the CD28-B7 Pro-Survival Pathway In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 132-132 ◽  
Author(s):  
Jayakumar Nair ◽  
Louise Carlson ◽  
Cheryl H Rozanski ◽  
Chandana Koorella ◽  
Megan Murray ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Kinase Activity ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Flow Cytometric Analysis ◽  
Myeloma Cell ◽  
Serum Starvation ◽  
Myeloma Cells ◽  
Survival Signals

Abstract Abstract 132 Multiple myeloma (MM), an incurable neoplasia of terminally differentiated plasma cells, are critically dependent on their interactions with bone marrow stromal cells (BMSC) for essential survival signals, growth and immunosuppressive factors. Very little is known about the specific BM cell type or the molecular elements in these interactions, an understanding of which could provide novel targets that could be interdicted to enhance conventional chemotherapy. A potential MM surface protein that could be involved in these interactions is CD28, based on its known pro-survival role in T cells. Clinical studies have shown that expression of CD28 in multiple myeloma highly correlates (p=0.006) with myeloma tumoral expansion. Moreover, CD28+ MM cells invariably express the CD28 ligand CD86. A survival role for MM-CD28 might involve interactions with BM cells that express B7 (CD80/CD86) such as dendritic cells (DCs, that are known to be closely associated with MM cells in the BM) or with CD86+ MM cells themselves. We had previously shown (ASH2008, #I-769) that blocking CD28-CD86 interactions between myeloma cells with high affinity B7 ligand CTLA4Ig (Abatacept®) sensitized myeloma cells to chemotherapy. Now we show that myeloma cells co-cultured with myeloid DCs in vitro derive both direct and indirect survival signals from DCs, and this can be partially blocked by commercially available reagents. Our data show that flow cytometric analysis of mononuclear cells (MNC) from BM aspirates of myeloma patients with increased CD138+ plasma cell populations (9-58%), show an increased CD11b+ (myeloid) population (20-37%) as well, which is in contrast to healthy transplant donor controls (12-15% CD11b+, 4–6% CD138+). Moreover, a larger fraction (11-47%) of the myeloma CD138+ plasma cells expressed CD28 compared to healthy control (3.3-7.7%). Also, when we analyzed gene expression datasets (NCBI #GSE5900 and GSE4204) from plasma cells (PC) of normal donors, monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SM) and newly diagnosed multiple myeloma (MM), we found a progressive increase in patients showing CD28 expression with increasing severity of disease (normal<MGUS<SM<MM) (Fig 1A). When we sorted the highest scoring MM group (n=538) into 8 genetic subgroups as defined earlier, CD28 expression was found to peak in the MF subgroup (typically associated with poor survival in myeloma patients) (Zhan et al. 2006, Blood 108, pp. 2020) relative to total population (p<0.0001) (Fig 1B). Antibody mediated activation of MM-CD28 over 48 hrs increased viability of myeloma cell line MM.1S cultured under serum starvation (3.7%) or with drugs ATO (1.9%), melphalan (18%) or dexamethasone (3.3%) to 66%, 21%, 33% and 11% respectively. Viability of MM.1S cells or primary CD138+ plasma cells (isolated from myeloma BM aspirates) cultured under serum starvation was enhanced >3 fold (p<0.001) when co-cultured with monocyte derived DCs, and in MM.1S this was partially reversed when either MM-CD28 or DC-B7 was blocked (Fig 2). Similar protection of MM.1S was also observed against a gradient of dexamethasone or melphalan. CD28 activation was accompanied by rapid tyrosine phosphorylation of CD28, association of p85 (PI3K), activation of Vav-1 and increase in CD28 associated tyrosine kinase activity, as shown by immunoprecipitation, western and kinase activity assays. We had previously shown that MM-CD28 interaction drive DC production of pro-survival factor IL-6 and immunosuppressive factor IDO via DC-B7 “backsignaling” (ASH2008 #I-769). Now we show that MM induced DC production of IL-6 (8 ng/ml) was partially inhibited in presence of CD28 blocking αCD28(Fab) fragments (3 ng/ml) or with protein kinase C (PKC) inhibitor Bisindolylmaleimide-I (2.1ng/ml). Activity of the immunosuppressive enzyme IDO in these co-cultures was completely inhibited in the presence of a novel IDO inhibitor from Incyte corporation, and this helped partially reverse IDO mediated suppression of T-cell proliferation in proliferation assays using co-culture supernatants. In conclusion, our data characterizes CD28-B7 pathway and DCs in the BM as vital for myeloma survival and also as possible targets to include in future strategies in the treatment of myeloma. FIGURE 1 FIGURE 1. FIGURE 2 FIGURE 2. Disclosures: Boise: University of Chicago: Patents & Royalties.

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