Molecular and Clinical Characterization of Patients with Myeloid Neoplasms Carrying the 12p Deletion

Abstract Background: Cytogenetic abnormalities have been described in almost 50% of patients with MDS and area strong and independent risk factor for prognosis. The interstitial deletion in the short arm of the chromosome 12 [del(12p)], is a characteristic but rare abnormality in MDS patients. Del(12p) abnormality has been described in approximately 1-5% of patients as a sole anomaly and is also found in up to 4% of patients along with an additional cytogenetic alteration. Isolated del(12p) is classified as a good risk abnormality according to the Revised International Prognostic Scoring Systems (IPSS-R). The commonly deleted region between 12p12.2 and 12p13.1 encompasses the ETV6 gene. To date, besides mutations in the transcriptional factor ETV6 and in the cell signaling KRAS gene, no other molecular mutations have been associated with del(12p). Murine studies have highlighted a role of the transcriptional factors ETV6 and RUNX1 in the impairment of both erythroid and platelets maturation. Here we investigated the presence of alternative molecular factors associated with del(12p) possibly influencing clinical outcomes and disease phenotypes. Methods: We studied the molecular and clinical data of a total of 2834 patients with myeloid neoplasms and found that 3% (93/2834) had alterations in chromosome 12. The median age was 67 years (24-84), with a male: female ratio of 56:37. Del(12p) occurred in 71% of cases (66/93); among them 14% (9/66) had isolated del(12p), 9% (6/66) had del(12p) + 1 additional alteration and 77% (51/66) carried a complex karyotype. The additional alteration to del(12p) included -7/del 7q (N=3), del(5q) (N=1) and t(X;20) (N=1). Cases with del(12p) were also classified according to disease type (MDS=40, AML=16; MDS/MPN=10; P=.057) and according to MDS risk group [lower-risk (33%, 22/66) and higher-risk (45%, 30/66)]. We applied whole exome sequencing and a targeted deep sequencing panel of 64 most frequently mutated genes in myeloid neoplasms. The ETV6 (12p13.2) gene was deleted in 55% (36/66) of cases while the KRAS (12p12) gene was deleted in 32% (21/66) of cases. One-third (32%, 21/66) of patients had deleted both genes. Two patients were hemizygous for KRAS. Results: Comparing patients with del(12p) (isolated, +1 alteration) to patients without alterations in chromosome 12 (n=2741), those with del(12p) had lower hemoglobin levels compared to patients without 12p aberrations (9.2 g/dL (6-16) vs. 9.7 g/dL (3-17); P=.009) and lower platelets counts (47 x109/L (8-577) vs. 73 x109/L (2-2336); P=.04). We noted that patients with isolated del(12p) had a longer median OS compared to patients with del(12p) associated with a complex karyotype [14 months (1-27) vs. 7 months (5-8)] although this difference was not significant. We then analyzed the mutational profile of the del(12p) cohort (isolated, +1 alteration) and compared their mutational spectrum with that of cases diploid for 12p. The most recurrently mutated genes in cases with del(12p) compared to cases diploid for 12p included RUNX1 (27% vs. 7%; P=.01) and DNMT3A (27% vs. 9%; P=.04). When we analyzed all the cases with del(12p) abnormalities (isolated, +1 alteration and complex) the significantly mutated genes were the transcriptional factors TP53 (38% vs. 4%; P=.0001) and RUNX 1 (14% vs. 7%; P=.04) and the histone modifier ASXL1 (21% vs. 10%; P=.01) We then analyzed the gene expression profile of patients carrying the del(12p) abnormality and found that KRAS mRNA expression levels of patients with MDS with del(12p) had a 2-fold reduction compared to the levels of healthy subjects (P=.017). Similarly, we observed also a decrease in ETV6 mRNA expression levels in patients with del(12p) (P=.07). Conclusions: Patients with del(12p) had lower levels of hemoglobin and platelets counts compared to patients without this cytogenetic abnormality. Mutations in transcriptional factors such as RUNX1 were commonly detected in this cohort, suggesting a possible mechanism contributing to the role of ETV6 in the impairment of erythroid and megakaryocytic cell maturation. Disclosures Sole: Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees.

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Quantitative Assessment of Indoleamine 2,3-Dioxygenase (IDO) Expression at Diagnosis Predicts Clinical Outcome in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood-2018-99-115908 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5261-5261

Author(s):

Sarah Parisi ◽

Simone Ragaini ◽

Darina Ocadlikova ◽

Mariangela Lecciso ◽

Giovanni Marconi ◽

...

Keyword(s):

Risk Factors ◽

Clinical Outcome ◽

Mrna Expression ◽

Board Of Directors ◽

Induction Chemotherapy ◽

De Novo ◽

Advisory Committees ◽

Secondary Aml ◽

Expression Levels ◽

Mrna Expression Levels

Abstract Introduction Indoleamine 2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that catalyzes the initial rate-limiting step in tryptophan degradation along the kynurenine pathway. IDO is physiologically expressed by a wide variety of human cells in response to several stimuli and it is known to have a crucial role in the induction of immune tolerance during pregnancy, infections, transplantation, autoimmunity and tumors. IDO-mediated tryptophan degradation results in inhibition of T-cell proliferation, increase of T-cell apoptosis and T-reg induction. Several studies demonstrated that IDO production can induce the increase of Regulatory T-cells (Tregs) directly through the conversion of CD25- into CD25+ T cells, even in acute myeloid leukemia (AML) patients. IDO expression can be considered a novel mechanism of leukemia escape from immune control and its inhibition may represent an antileukemia therapeutic strategy. Aim of our work is to analyze IDO mRNA expression in a cohort of AML patients and to investigate the presence of any significant correlation between IDO expression and standard prognostic factors or clinical outcome. Methods We analyzed a cohort of 68 adult patients aged 18 years or older, who were diagnosed with de novo or secondary AML. IDO mRNA expression was evaluated by Real-Time (RT)-PCR in blood bone marrow and peripheral blood samples at diagnosis. Patients were then retrospectively stratified according to standard risk factors at diagnosis and to IDO mRNA expression levels. Results Median age of analyzed patients was 57 years (range 21-76). Fifty-nine out of 68 patients (87%) had de novo AML, whereas 9 out of 68 patients (13%) had secondary AML. A comprehensive risk assessment was available for 61 patients. Among these 61 patients who were evaluable for risk stratification, 13 patients (21%) resulted to have a favorable risk AML, 30 (49%) had an intermediate risk AML and 17 patients (30%) were stratified as high-risk AML. Sixty out of 68 patients received intensive, standard, induction chemotherapy regimens. The remaining 8 patients were not candidate to receive intensive chemotherapy mainly because of comorbidities. Twenty-three out of 68 patients (34%) were considered eligible for allogeneic stem cells transplantation (alloSCT) as consolidation therapy, after obtaining complete remission with standard chemotherapy. IDO expression in peripheral blood (PB) samples was between 0.07 and 4272.26 (median 5.60). Conversely, IDO expression in bone marrow (BM) samples was between 0.17 and 243.16 (median 1.21). Our data did not establish any significant correlation between IDO expression and leukemia risk factors at diagnosis, in particular cytogenetics, de novo or secondary AML, leukocytosis. Among the 60 patients who received induction chemotherapy, 35 achieved morphological complete remission (CR), 24 did not respond and 1 patient was not evaluable for response. Response to induction chemotherapy was not influenced by IDO mRNA expression levels. Interestingly, among patients undergoing alloSCT, high levels of IDO mRNA expression in PB samples negatively correlated with patients' overall survival. In particular, high IDO expression of more than 10 was associated with worse overall survival after alloSCT even when adjusted by patients' age and disease status at transplant (log rank P=0.02) (Fig.1). With the limitations of the low number of patients, these results from the group of transplanted patients were not likely due to differences in the incidence and severity of graft-versus-host-disease, whereas high IDO mRNA expression level was predictive of increased incidence of relapse. Conclusions This work suggests that IDO mRNA expression levels can be considered as predictive of AML outcome, independently from other risk factors at diagnosis. In our set, higher level of IDO mRNA expression at diagnosis was correlated with worse clinical outcome in patients undergoing alloSCT. Larger studies are warranted in order to establish the real predictive role of IDO mRNA expression in influencing AML outcome. Disclosures Cavo: Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Decreased PERP Expression on Peripheral Blood Mononuclear Cells from Patient with Rheumatoid Arthritis Negatively Correlates with Disease Activity

Clinical and Developmental Immunology ◽

10.1155/2013/256462 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Yanchun Du ◽

Lin Deng ◽

Yan Li ◽

Lu Gan ◽

Yantang Wang ◽

...

Keyword(s):

Disease Activity ◽

Mrna Expression ◽

Mononuclear Cells ◽

Cell Types ◽

Healthy Controls ◽

Peripheral Blood Mononuclear ◽

Expression Levels ◽

Cell Type Specific ◽

Mrna Expression Levels

Background. PERP, p53 apoptosis effector related to PMP-22, is a p53-dependent apoptosis in diverse cell types and has cell type-specific roles in p53-mediated apoptosis. However, its role in PBMCs of RA patients has remained largely unclear.Objectives. The aim of this study was to detect the expression levels of PERP on PBMCs of RA patients and healthy controls and analyze the role of PERP in the pathogenesis of RA.Methods. The mRNA expression levels of PERP and IL-17 were detected by real-time PCR in PBMCs from patients with RA (n=40) and healthy controls (n=40). The correlations of PERP expression levels to IL-17 transcripts and disease activity parameters were analyzed.Results. The PERP and IL-17 expression levels in the PBMCs were significantly decreased and increased in comparison of which in healthy controls. The mRNA expression levels of PERP in PBMCs from patients with RA were negatively correlated with IL-17 and disease activity parameters DAS28, RF, CRP, and ESR rather than Anti-CCP and ANA.Conclusions. These results demonstrated that PERP might be involved in the pathogenesis and a potential therapeutic target of RA by regulating the expression of IL-17.

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Frequent Epigenetic Inactivation of MSX2 In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.4645.4645 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4645-4645 ◽

Cited By ~ 1

Author(s):

Chen Zhao ◽

Xin Han ◽

Yu H. Zhang ◽

Xiaoyan Huang ◽

Aili Dai ◽

...

Keyword(s):

Mrna Expression ◽

Cell Lines ◽

Myeloid Leukemia ◽

Functional Role ◽

Methylation Status ◽

Dna Hypermethylation ◽

Expression Levels ◽

Mrna Expression Levels ◽

Acute Myeloid

Abstract Abstract 4645 DNA hypermethylation has been implicated in the tumorigenesis and prognosis in acute myeloid leukemia (AML). To identify and validate relevant methylated genes in AML, we have compared expression levels and methylation status of 26 candidate genes. One of the interesting candidates identified in our study is MSX2. MSX2 is a member of muscle segment homeobox gene family. MSX2 plays a role in promoting cell growth under certain conditions and may be an important target for RAS signaling pathways. However, the mechanism of transcriptional regulation and functional role of MSX2 in hematological malignancies, especially AML, are poorly understood. In our study, we determined the methylation status, and analyzed the expression levels of MSX2 in AML cell lines and primary AML cells using RT-PCR and/or Taqman real-time PCR. MSX2 mRNA expression was robust in the normal granulocytes and blasts of human bone-marrow, but was either absent or significantly diminished in 6 of 9 (66.7%) AML cell lines. The expression levels of MSX2 in those 6 AML cell lines were restored after treatment of 5-aza 2′-deoxycytidine. In addition, COBRA (Combined Bisulfite Restriction) analysis demonstrated hypermethylation of MSX2 in those AML cell lines (6 of 9, 66.7%), and partial methylation in 3 of 9 AML cell lines. The methylation status was inversely correlated with the mRNA expression levels of MSX2 in those cell lines. Furthermore, the expression levels and methylation status of MSX2 in human primary AML cells were evaluated. COBRA analysis demonstrated frequent hypermethylation of MSX2 in primary AML patient samples (19 of 32, 59.3%). Importantly, the mRNA expression levels of MSX2 as shown by Taqman real-time PCR in those 19 primary AML patient samples were inversely correlated with the methylation status of MSX2. These findings confirmed the role of frequent DNA hypermethylation in silencing MSX2 in AML. We are in the process of determining the functional role of MSX2 in the pathogenesis of AML. In addition, diagnostic and prognostic values of MSX2 in AML are being pursued. Disclosures: No relevant conflicts of interest to declare.

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Zika virus infection during pregnancy and induced brain pathology in beclin1-deficient mouse model

10.1101/843813 ◽

2019 ◽

Author(s):

Mohan Kumar Muthu Karuppan ◽

Chet Raj Ojha ◽

Myosotys Rodriguez ◽

Jessica Lapierre ◽

M. Javad Aman ◽

...

Keyword(s):

Growth Factor ◽

Mouse Model ◽

Mrna Expression ◽

Zika Virus ◽

Neuronal Injury ◽

Viral Rna ◽

Expression Levels ◽

Inflammatory Molecules ◽

Mrna Expression Levels

ABSTRACTWe investigated the role of the autophagy protein, Beclin1, in the replication and disease of Zika virus (ZIKV) in pregnant dams and their offspring using Beclin1-deficient (Atg6+/−) and wild-type (Atg6+/+) mouse model infected with the Honduran (R103451), Puerto Rican (PRVABC59), and the Uganda (MR766) strains of ZIKV. Pregnant dams infected subcutaneously at embryonic stage (E)9 showed viral RNA in serum harvested at E13 and in various organs removed postmortem at E17. Subcutaneous infections with ZIKV also showed the vertical transmission of ZIKV from the placenta to embryos removed postmortem at E17. From the three isolates, R103451-infected Atg6+/− dams had the lowest mortality rate while 30 % of their offspring containing the hemizygous beclin1 allele (Atg6+/−) were smaller in size and had smaller and underdeveloped brain. Growth impairment in the pups became noticeable after two weeks post-birth. After 21-days, pups were sacrificed and brain tissues removed postmortem showed expression of the envelope (E) and the non-structural (NS)-1 proteins, along with signs of neuronal injury, despite an absence in viral RNA detection. A significant decrease in the mRNA expression levels of the insulin-like growth factor-1 (IGF-1) by 8-fold and a decrease in the mRNA expression levels of several microcephaly related genes along with an increase in the secretion of several inflammatory molecules may have contributed to the observed phenotype. Since autophagy regulates cytokines and chemokines production, a dysregulation in this pathway may have further exacerbated the pathology of ZIKV.IMPORTANCEPups delivered from ZIKV-infected dams showed significant growth impairments in the body and the brain. We believe that the reduction in insulin growth factor together with the increase secretion of inflammatory molecules may have triggered neuronal injury and the downregulation of the microcephalic genes, while reduced expression of the autophagy protein, Beclin1 further exacerbated the pathology. Although the mechanism is still unknown, the autophagy pathway seems to play a key role in ZIKV pathology. It is therefore of great significance to study the role of autophagy during viral infection with the goal to identify potential targets for anti-ZIKV therapeutic intervention.

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BRCA2 Heterozygosity Delays Cytokinesis in Primary Human Fibroblasts

Analytical Cellular Pathology ◽

10.1155/2009/929721 ◽

2009 ◽

Vol 31 (3) ◽

pp. 191-201

Author(s):

Asta Björk Jonsdottir ◽

Maaike P. G. Vreeswijk ◽

Ron Wolterbeek ◽

Peter Devilee ◽

Hans J. Tanke ◽

...

Keyword(s):

Mrna Expression ◽

Live Cell Imaging ◽

Cell Imaging ◽

Human Fibroblasts ◽

Immunofluorescence Staining ◽

Wild Type ◽

Expression Levels ◽

Brca2 Protein ◽

Mrna Expression Levels

Background: Inherited mutations in the tumour suppressor gene BRCA2 greatly increase the risk of developing breast, ovarian and other types of cancers. So far, most studies have focused on the role of BRCA-pathways in the maintenance of genomic stability.In this study we investigated the potential role of the BRCA2 protein in cytokinesis in unmodified primary human fibroblast carrying a heterozygous mutation in the BRCA2 gene.Methods: Cell divisions were monitored with time lapse live-cell imaging. BRCA2 mRNA expression levels in BRCA2+/− and BRCA2+/+ cells were quantified with quantitative real-time polymerase chain reaction (qRT-PCR). To investigate the localization of the BRCA2 protein during cytokinesis, immunofluorescence staining using antibody directed against BRCA2 was carried out. Immunofluorescence staining was performed directly after live-cell imaging and cells with delayed cytokinesis, of which the co-ordinates were saved, were automatically repositioned and visualized.Results: We demonstrate that unmodified primary human fibroblasts derived from heterozygous BRCA2 mutation carriers show significantly prolonged cytokinesis.A Subset of the BRCA2+/− cells had delayed cytokinesis (40 min or longer) making the mean cell division time 6 min longer compared with BRCA2+/+ cells, 33 min versus 27 min, respectively. Lower BRCA2 mRNA expression levels were observed in the BRCA2 heterozygous samples compared with the BRCA2 wild type samples.The BRCA2 protein localizes and accumulates to the midbody during cytokinesis, and no difference was detected in distribution and localization of the protein between BRCA2+/− and BRCA2+/+ samples or cells with delayed cytokinesis and normal division time.Conclusion: The delayed cytokinesis phenotype of the BRCA2 heterozygous cells and localization of the BRCA2 protein to the midbody confirms that BRCA2 plays a role in cytokinesis. Our observations indicate that in a subset of cells the presence of only one wild type BRCA2 allele is insufficient for efficient cytokinesis.

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Higher Expression of PALB2 Predict Poor Prognosis in AML Patients and Identifies Potential Targets of Synthetic Lethal Therapies

Blood ◽

10.1182/blood-2018-99-118210 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1507-1507

Author(s):

Antonella Padella ◽

Giovanni Marconi ◽

Giorgia Simonetti ◽

Andrea Ghelli Luserna Di Rora ◽

Maria Chiara Fontana ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Low Risk ◽

Age At Diagnosis ◽

Transcriptional Level ◽

Intensive Chemotherapy ◽

Advisory Committees ◽

Synthetic Lethal ◽

Expression Levels

Abstract Background - Partner and localizer of BRCA2 (PALB2) plays a key role in the DNA damage repair (DDR). Genomic alterations of DDR genes rarely occur in AML, while their deregulation at transcriptional level is a known mechanism exploited by leukemic cells in order to sustain the high genetic instability and to continue proliferation. Aim - We aimed to characterize the role of PALB2 in AML by investigating its expression levels and its prognostic value, in order to evaluate its potential as target of therapies based on a synthetic lethality approaches. Methods - Gene expression profiling (GEP, Affymetrix) was performed on bone marrow cells of 7 healthy donors (HD) and 60 AML patients with more than 80% blast cells. K-means clustering of patients according to the expression of PALB2 was performed and differences in survival were assessed d by Kaplan-Meier survival analysis. Results - Our cohort was characterized by a median age at diagnosis of 60 years-old. Twelve out of 43 patients harbored a mutation in FLT3 (27.9%, 17 patients not tested); 8 patients were NPM1 mutated (27.6%, 31 patients not tested); 2 patients were TP53 mutated (3%, all patients tested). According to ELN2017 guidelines, 17 cases were high-risk AML, 38 cases were intermediate, 3 cases had low-risk classification and 2 cases were not classified due to the lack of prognostic markers. We detected variable levels of PALB2 mRNA (range 52.90-244.37) in AML patients and its median expression was higher compared to HD (129.26 vs 67.85, respectively; p=.019). We clustered our patients according to PALB2 expression and we defined 2 groups of patients: cluster H and L with higher and lower expression levels of PALB2, respectively (cluster centers 158.86 and 105.41, respectively; figure A). Notably, HD revealed PALB2 expression values comparable with the cluster L (range 52.90-99.60). No differences were detected in term of incidence mutations in FLT3 and NPM1, white blood cell count at diagnosis, age at diagnosis and prevalence of karyotype alterations. Patients were treated with best supportive therapy (n=11/58, therapy data missing for 2 patients), hypomethylating agents (n=2/58) and intensive chemotherapy ( n=45/58). Within patients treated with intensive chemotherapy, we compared complete remission rate after induction and we found no differences between H and L. However, patients with higher expression of PALB2 had worst overall survival than patients in cluster L (median survival group H=397 days; CI 95%=288.9-505.0, L=not reached; p=.045; figure B). Notably, we confirmed worst prognosis in patients in cluster H when considering 33 out of 45 patients with intermediate/low risk karyotype (i.e. normal karyotype, t(8;21) or less than 3 aberrations according to ELN2017; p=.026). Conclusion - We identified a subgroup of AML patients with higher expression of PALB2, which predicted poorer prognosis in patients treated with curative intent and it associated with poorer prognosis in patients with low/intermediate risk. While patients carrying mutations in PALB2 (and BRCA1/2) are candidate for PARP inhibitors (PARPi) therapies in breast cancers, few clinical trials with PARPi are available in AML and the frequency of mutations is very low. Our data opens a new scenario in which PALB2 may be a target of therapies in AML based on synthetic lethal approaches targeting the DDR pathway. However, a better understanding of the biological role of PALB2 in AML and its interaction with other alterations is needed. Supported by Fondazione Del Monte Figure. Figure. Disclosures Cavo: Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Incyte Biosciences: Consultancy. Martinelli:Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy; Ariad/Incyte: Consultancy; Amgen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Speakers Bureau.

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Role of PARP1 regulation in radiation-induced rescue effect

Journal of Radiation Research ◽

10.1093/jrr/rraa023 ◽

2020 ◽

Vol 61 (3) ◽

pp. 352-367 ◽

Cited By ~ 1

Author(s):

Spoorthy Pathikonda ◽

Shuk Han Cheng ◽

Kwan Ngok Yu

Keyword(s):

Mrna Expression ◽

Positive Feedback ◽

Feedback Loop ◽

Positive Feedback Loop ◽

Fluorescent Intensity ◽

Rescue Effect ◽

Expression Levels ◽

Radiation Induced ◽

Mrna Expression Levels

ABSTRACT Radiation-induced rescue effect (RIRE) in cells refers to the phenomenon where irradiated cells (IRCs) receive help from feedback signals produced by partnered bystander unirradiated cells (UIRCs) or from the conditioned medium (CM) that has previously conditioned the UIRCs. In the present work, we explored the role of poly (ADP-ribose) polymerase 1 (PARP1) regulation in RIRE and the positive feedback loop between PARP1 and nuclear factor-kappa-light-chain-enhancer of activated B cell (NF-κB) in RIRE using various cell lines, including HeLa, MCF7, CNE-2 and HCT116 cells. We first found that when the IRCs (irradiated with 2 Gy X-ray) were treated with CM, the relative mRNA expression levels of both tumor suppressor p53-binding protein 1 (53BP1) and PARP1, the co-localization factor between 53BP1 and γH2AX as well as the fluorescent intensity of PARP1 were reduced. We also found that IRCs treated with the PARP1 inhibitor, Olaparib (AZD2281) had a higher 53BP1 expression. These results illustrated that PARP1 was involved in RIRE transcriptionally and translationally. We further revealed that treatment of IRCs with CM together with Olaparib led to significantly lower mRNA expression levels and fluorescent intensities of NF-κB, while treatment of IRCs with CM together the NF-κB inhibitor BAY-11-7082 led to significantly lower mRNA expression levels as well as fluorescent intensities of PARP1. These results illustrated that PARP1 and NF-κB were involved in the positive feedback loop transcriptionally and translationally. Thus, the results supported the occurrence of a PARP1–NF-κB positive feedback loop in RIRE. The present work provided insights into potential exploitation of inhibition of PARP1 and/or the PARP1–NF-κB positive feedback loop in designing adjuncts to cancer radiotherapeutics.

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Exploring the metastatic role of the inhibitor of apoptosis BIRC6 in Breast Cancer

10.1101/2021.04.08.438518 ◽

2021 ◽

Author(s):

Santiago Manuel Gomez Bergna ◽

Abril Marchesini ◽

Leslie Cinthya Amoros Morales ◽

Paula Nazarena Arrias ◽

Hernan Gabriel Farina ◽

...

Keyword(s):

Breast Cancer ◽

Gene Ontology ◽

Mrna Expression ◽

Differential Expression ◽

Expression Patterns ◽

Mrna Levels ◽

Expression Levels ◽

Apoptosis Protein ◽

Mrna Expression Levels

Breast cancer is the most common cancer as well as the first cause of death by cancer in women worldwide. BIRC6 (baculoviral IAP repeat-containing protein 6) is a member of the inhibitors of apoptosis protein family thought to play an important role in the progression or chemoresistance of many cancers. The aim of the present work was to investigate the role of apoptosis inhibitor BIRC6 in breast cancer, focusing particularly on its involvement in the metastatic cascade. We analyzed BIRC6 mRNA expression levels and Copy Number Variations (CNV) in three breast cancer databases from The Cancer Genome Atlas (TCGA) comparing clinical and molecular attributes. Genomic analysis was performed using CBioportal platform while transcriptomic studies (mRNA expression levels, correlation heatmaps, survival plots and Gene Ontology) were performed with USC Xena and R. Statistical significance was set at p values less than 0.05. Our analyses showed that there was a differential expression of BIRC6 in cancer samples when compared to normal samples. CNV that involve amplification and gain of BIRC6 gene were correlated with negative hormone receptor tumors, higher prognostic indexes, younger age at diagnosis and both chemotherapy and radiotherapy administration. Transcriptomic and gene-ontology analyses showed that, in conditions of high BIRC6 mRNA levels, there are differential expression patterns in apoptotic, proliferation, and metastatic pathways. In summary, our in silico analyses suggest that BIRC6 exhibits an antiapoptotic, pro-proliferative and an apparent pro-metastatic role and could be a relevant molecular target for treatment of Breast Cancer tumors.

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Role of Selective HDAC6 Inhibition On Multiple Myeloma Bone Disease

Blood ◽

10.1182/blood.v120.21.328.328 ◽

2012 ◽

Vol 120 (21) ◽

pp. 328-328

Author(s):

Loredana Santo ◽

Diana Cirstea ◽

Bin Wang ◽

Tso-Pang Yao ◽

Joy Y. Wu ◽

...

Keyword(s):

Mrna Expression ◽

Board Of Directors ◽

Cell Lines ◽

Bone Disease ◽

Research Funding ◽

Advisory Committees ◽

Vitro Data ◽

In Vitro Data

Abstract Abstract 328 In multiple myeloma (MM), deregulated osteoclast (OC)/osteoblast (OB) cross-talk induces osteolytic bone lesions. The HDAC6 selective inhibitor, rocilinostat (ACY-1215), in combination with bortezomib has shown potent anti myeloma activity in preclinical studies, which provided the rationale for a clinical trial that is currently recruiting relapsed/refractory MM patients (NCT01323751). However, while the beneficial role of bortezomib in tumor-related bone disease has been previously described, the effect of HDAC6 inhibition is not known. Evidence suggests a positive effect on bone turnover as pan HDAC inhibitors accelerate OB maturation and suppress OC maturation in vitro. Here, we evaluated effects of the selective HDAC6 inhibitor rocilinostat (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib in MM bone disease. Rocilinostat (1 μM) alone and in combination with bortezomib (2.5 nM) inhibited OC differentiation, evidenced by a decreased number of TRAP positive multinucleated cells and bone-resorbing activity. In addition, rocilinostat (1 μM) significantly decreased cell growth of mature OC in co-culture with MM cell lines and in combination with bortezomib inhibited transcription factors implicated in OC differentiation including p-ERK, p-AKT, c-FOS, and NFATC1. Importantly, such an effect was cytokine (RANKL and M-CSF) dependent. Further, rocilinostat, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase (ALP) enzyme activity and alizarin red staining. In addition, we found increased mRNA expression of beta-catenin, osteocalcin, ALP, and RUNX2. Based on this promising in vitro data, we used the xenograft model of disseminated human MM in SCID mice to study the effect of rocilinostat, alone and in combination with bortezomib, on MM bone disease. MM.1S-GFP-Luc cells were injected intravenously, and MM disease progression was followed by bioluminescence imaging. A significant decrease in tumor burden was observed in mice following three weeks of treatment with rocilinostat, alone or in combination with bortezomib. Isolating serum from control and treated mice, we also observed a significant decrease of TRAPc5b levels, a marker of bone resorption, as well as a significant increase in osteocalcin levels, a marker of bone formation, in the serum of the combination treated cohort. Cells isolated from the calvaria from the combination treated group compared to the control group showed a significant increase in the mRNA expression of ALP, RUNX2, and osterix, as well as a significant decrease in the mRNA expression ratio of RANKL/OPG. To elucidate the role of HDAC6 inhibition on bone turnover, we used HDAC6 knockout mice. Cells were isolated from femurs, tibia, and spine of 2 month-old wild type (WT) and HDAC6 knockout (KO) mice and mRNA expression for osteocalcin, ALP, RUNX2 and osterix was assessed by qPCR. We observed a significant increase in osteocalcin mRNA expression without significant changes in the mRNA expression of ALP, RUNX2 and osterix. Bone marrow stromal cells (BMSCs) differentiated from WT and KO mice were co-cultured with MM murine cell lines and, notably, the proliferative advantage conferred by BMSC isolated from HDAC6 KO mice to MM cell lines was significantly decreased compared to WT BMSCs. These data suggest that a microenviroment lacking HDAC6 reduces MM cell proliferation. Moreover, treatment with rocilinostat (1mM) for 24 h inhibited proliferation of MM cells cocultured with WT BMSCs to levels observed in MM cells cultured with KO BMSC lacking endogenous HDAC6. Finally, the effect of co-treatment with rocilinostat (1μM) and bortezomib (2.5 nM) on proliferation of MM cells co-cultured with WT-BMSC was similar to that observed when bortezomib was added to MM cells in cocultures with KO BMSC. In conclusion, the in vitro data and the in vivo results from the xenograft models of human MM in SCID mice, as well as data in the HDAC6 KO mice, indicate a potential beneficial role of HDAC6 inhibition on MM-related bone disease. We are currently performing dynamic and static histomorphometric analysis to confirm this effect on bone remodeling at the tissue level. These effects on bone remodeling are an added benefit for MM patients and will be assessed prospectively in our ongoing clinical trial. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

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Molecular Predictors of Response in Patients with Myeloid Neoplasms Treated with Lenalidomide

Blood ◽

10.1182/blood.v126.23.2853.2853 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2853-2853 ◽

Cited By ~ 1

Author(s):

Eiju Negoro ◽

Chantana Polprasert ◽

Tomas Radivoyevitch ◽

Vera Adema ◽

Naoko Hosono ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Somatic Mutations ◽

Snp Array ◽

Low Risk ◽

Clinical Parameters ◽

Complex Karyotype ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Response Expression

Abstract Up to 70% of patients with del(5q) MDS may respond to Lenalidomide (LEN). However, the success rates in non-del(5q) cases, while substantial, are much lower (ranging from 20-40% depending on selection criteria). Aside from the presence of del(5q), up front identification of potentially responsive patients is difficult, particularly as the mechanistic underpinnings of LEN response are still under investigation. Initial attempts to prospectively predict LEN sensitivity resulted in a description of response expression signatures, but they have not been robust enough to serve as an actionable diagnostic test. In the outset of this study, we stipulated that apart of clinical selection (low risk MDS, transfusion-dependence, normal/low risk cytogenetics, etc.), analyses of molecular lesions including somatic mutations and chromosomal defects may help to predict LEN responsiveness. To that end, we performed deep targeted NGS (using multiamplicon panel of the top most commonly mutated genes in MDS). In total we analyzed 143 cases of myeloid neoplasms (MDS, MDS/MPN, or MPN) treated with LEN (median duration 6 months) for whom annotated clinical outcomes were available (83 responders vs. 60 refractory cases). Clinical parameters including IPSS-R, cytogenetics (FISH, SNP-array or metaphase cytogenetics) were used to characterize patients, whose responses were assessed by 2006 IWG criteria. Initially, in a combined analysis, we included both del(5q) (N =37) and non-del(5q) patients (N =106). Very low/low, intermediate, high/very high IPSS-R scores were found in 47%, 23%, 34% cases, respectively. Of 143 LEN-treated patients, regimens included LEN (80%), or LEN+5-Aza (20%). Any hematologic improvement (HI), partial response (PI), and complete response (CR) were achieved in 44%, 14% and 42%, respectively. Responses were associated with a better survival (median survival time 6.2 yrs. vs. 3.7 yrs. in refractory cases; P =.003). Non-responders showed significantly lower platelet levels compared to responders (median 169 vs. 89 K/uL; P =.007) but intricate analysis of clinical parameters (age, other blood counts, blasts and IPSS-R score) failed to identify other factors that would help to select potential responders. As expected, when sub-analysis of patients with del(5q) was performed, combined response was achieved in 78% (OR 13.14 [4.34-39.75]; PR 16%, CR 35%) of patients, respectively, while in non-del(5q) the responses were as predicted lower at 51% (P =.004). Of note is that both del(5q) involving and excluding commonly retained regions (CRR; q11.1-q14.2 and/or q34-qter) also was associated with sensitivity (CRR affected; OR=9.9 [1.4-102], vs. CRR not affected; OR=6.3 [1.3-37.6]). When we analyzed impact of karyotype on LEN sensitivity, -7/del(7q), -20/del(20q), complex karyotype and normal cytogenetics did not correlate with response, but in addition to del(5q); the presence of +8 (7/10 responded; OR 12.25 [1.33-113.06]) was significantly associated with responsiveness. Using targeted deep NGS, we confirmed 168 somatic mutations in responders vs. 142 mutations in non-responders (OR .85; .67-1.07). The number of mutational events per patient did not correlate with responses (P =.38). Among genes sequenced mutations in DDX41 (100% vs. 0%; OR infinity [6.7-infinity]) and RUNX1 mutation+deletion (75% vs. 25%; OR=8.1 [1.1-84.6]) were overrepresented in responders vs. refractory cases while in U2AF1 mutationswere more common among non-responders (20% vs. 80%; OR=.075 [.004-.76]). When reverse analysis was performed DDX41 mutations correlated with LEN response (10% mutant cases among responders vs. 0% in refractory; P =.009), while mutations in U2AF1 correlated with LEN failure (2.4% vs. 13.3% of mutant cases in responders and refractory, respectively; P =.02). The presence of all combined or any of the other spliceosomal mutations (SRSF2, SF3B1, ZRSR2, LUC7L2, and PRPF8) did not influence the results of the therapy. All TP53 mutations were found with complex karyotype with del(5q) and 5/7 (71%) TP53 mutant cases were treatment failures (OR .19 [.01-2.41]). In conclusion, in addition to the presence of del(5q), low platelet count and the presence of various molecular lesions (+8 and RUNX1, DDX41 mutations or wild type status of U2AF1) may help to predict responses to LEN. Disclosures Sekeres: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. List:Celgene Corporation: Honoraria, Research Funding.

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