bluuebunny/crossref_metadata_2025 huggingface dataset:
A Proteomics Screen Identifies MDM2 Binding Partners Specific to Ribosomal Protein Haploinsufficiency
Authors: Raiser David M. , Schenone Monica , Hartman Emily , Carr Steven A. , Ebert Benjamin L.
Published: October 2019

Ribosomopathies such as 5q- syndrome and Diamond-Blackfan anemia are broadly characterized by haploinsufficiency of one or more ribosome-associated proteins leading to an aberrant activation of the p53 pathway. The prevailing hypothesis for the mechanism of p53 stabilization and activation in the context of ribosomal protein haploinsufficiency suggests that increased binding of a subset of free ribosomal proteins (including RPL5, RPL11, RPL23a, and RPS7) to MDM2 abrogates the negative regulatory control of MDM2 on p53. In order to understand more fully how the binding partner profile of MDM2 changes as a consequence of ribosome dysfunction, we performed a SILAC-based IP/MS proteomics screen on MDM2 immunoprecipitates to identify proteins that were differentially bound to MDM2 with and without shRNA-mediated knockdown of RPS14, a commonly haploinsufficient gene in 5q- syndrome. A V5-tagged MDM2 was expressed in A549 cells, and lentivirally-delivered shRNAs were expressed that targeted either luciferase (for control) or RPS14. Immunoprecipitations were performed on whole cell lysates using a V5 antibody and analyzed by mass spectrometry to identify peptides associated with MDM2 in each condition. We found that RPL5, RPL11, and RPL23a were strongly associated with MDM2 in the context of RPS14 knockdown, but were also abundant in MDM2 immunoprecipitates when a control hairpin was used. Another ribosomal protein, RPL38, was also strongly associated with MDM2 in both conditions. In contrast, RPS14 knockdown appeared to abrogate binding of RPS7 to MDM2, which was strongly associated in the control knockdown condition only. These data corroborate previous findings that a subset of 60S subunit ribosomal proteins can physically associate with MDM2, and they further suggest that this binding may commonly occur even in the absence of ribosomal haploinsufficiency. While differential binding of free ribosomal proteins to MDM2 may indeed contribute to p53 pathway activation in ribosomopathies, these data suggest that the difference in binding is subtle and may be a part of a larger set of p53-activating signals. Additional non-ribosomal proteins were identified that were specifically associated with MDM2 in the context of ribosomal protein deficiency, and follow-up experiments are ongoing to interrogate the biological consequences of these context-specific interactions.

Disclosures

No relevant conflicts of interest to declare.
Hepatitis B e Antibody Positive before Rituximab Combination Chemotherapy Is Associated with a Lower Risk of Hepatitis B Virus (HBV) Reactivation in HBV Carriers with Diffuse Large B-Cell Lymphoma
Authors: Cai Qingqing , Chen Kailin , Geng Qirong , Zhong Guangzheng , Li Jianping , Huang Huiqiang , Lin Tongyu , Jiang Wenqi , Li Zhiming
Published: October 2019

Background: With antivirus prophylaxis, reactivation of hepatitis B virus (HBV) also occurred in HBV carriers (hepatitis B surface antigen [HBsAg] positive) undergoing rituximab combination chemotherapy. It was reported that most HBV carriers with improved long-term outcomes were seropositive for hepatitis B e antibody (HBeAb). In this study on HBsAg-positive lymphoma patients with prophylaxis, the objectives were to determine the HBV reactivation rate in patients with HBeAb positive compared with the patients with HBeAb negative.

Methods: We retrospectively analyzed the medical records of 113 HBV carriers with CD20(+) diffuse large B-cell lymphoma (DLBCL) received R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy with antivirus prophylaxis between August 1, 2002 and November 31, 2011 at Sun Yat-sen University Cancer Center, China.

Results: Among 113 HBV carriers with CD20(+) DLBCL, 68(60.2%) were hepatitis B e antibody (HBeAb) positive. There was no significant difference in terms of sex distribution, age, ECOG PS, stage, baseline HBV DNA detectable rate, or ALT, AST, bilirubin, or LDH between the HBeAb positive group and the HBeAb negative group. However, there were a significantly higher proportion of HBeAg seronegativity (94.1%v 24.4%; P< .001), and HBcAb seropositivity (100.0%v 82.2%; P< .001) in the HBeAb positive group.

All the patients received antiviral therapy before chemotherapy. The antiviral treatments were used as follows: lamivudine in 68 (60.2%) patients, entecavir in 18 (15.2%) patients, others in 27 (23.8%) patients. Other antiviral treatments were including adefovir, lamivudine plus entecavir, lamivudine at beginning and then entecavir, and so on. No significant difference was observed in the antiviral drugs between the two groups.

After R-CHOP chemotherapy, HBV reactivation was found in 23.0% of HBV carriers (26/113). And its incidence rate was lower in HBsAg- positive / HBeAb positive patients than in HBsAg-positive/ HBeAb negative patients (16.1% versus 33.3%; P =.034). Multivariate analysis showed that patients positive for HBeAb before chemotherapy was the only significant and independent protective factor associated with hepatitis due to HBV reactivation after chemotherapy.

Chemotherapy was continued when the serum HBV-DNA level reduced or liver function improved. Survival was not affected by the occurrence of HBV reactivation or the status of HBeAb.

Conclusion: HBeAb positive before rituximab combination chemotherapy was associated with a lower risk of HBV reactivation in HBV carriers with CD20(+) DLBCL. A more effective antivirus prophylaxis may be considered in HBV carriers with HBeAb negative in order to reduce HBV reactivation.

Disclosures

No relevant conflicts of interest to declare.
The BCL2 Targeted Deoxyribonucleic Acid Inhibitor (DNAi) PNT2258 Is Active in Patients with Relapsed or Refractory Non-Hodgkin’s Lymphoma
Authors: Harb Wael A. , Lakhani Nehal , Logsdon Ana , Steigelman Mary , Smith-Green Heidi , Gaylor Shari , Rodrigueza Wendi , Woolliscroft Michael , Sooch Mina , Messmann Richard A. , Al-Katib Ayad
Published: October 2019

Background: PNT2258 is a 24-base, single-stranded liposome-encapsulated DNA oligonucleotide that hybridizes to regions of the BCL2 gene, modulating its function. As previously reported, PNT2258 demonstrates antitumor activity in patients with advanced-stage NHL. The updated clinical data that are provided in this abstract (as of 07/25/14) now include anti-tumor activity in patients with DLBCL and Richter’s syndrome.

Methods: All patients had CT-measureable, FDG-PET positive disease with progression prior to study entry, and had previously received rituximab and combination cytotoxic chemotherapy. Refractory tumor status or concomitant medical issues precluded the use of additional cytotoxic therapy in all patients included in the study. PNT2258 induction cycles were administered as a 3-hour IV infusion at 120 mg/m2 days 1-5 of each 21-day cycle for up to 8 cycles. Response evaluation occurred at the end of cycles 2, 6 and 8. Following induction, patients could receive maintenance dosing at 100 mg/m2days 1-2 of each 28-day cycle until PD.

Results: Thirteen patients (median age 65; 8 males, 5 females; ECOG PS 0/1/2: 3/9/1) received all scheduled doses of PNT2258. Regardless of attribution, there were no cycle 1, grade 3/4 AEs. The most common grade 1/2 AEs in cycle 1 included chills, low-grade fever, transient nausea, and tumor or back pain (n=5 for each). There was no evidence of tumor lysis syndrome, febrile neutropenia or significant GI toxicity. Of 4 patients with DLBCL, including 1 with Richter’s syndrome and 1 with Burkitt-like histology, PFS was 9.2 months, range 5.5-12.3 months as of 7/25/14. Best response data (CR/PR/SD/PD) for the DLBCL group was 2/1/1/0. Additionally, in 5 FL patients, best response was 1/1/3/0. Five patients, including 3 DLBCL and 2 FL remain on study as of 7/25/14 receiving maintenance treatment with PNT2258.

Conclusions: PNT2258 is well tolerated and can be administered over prolonged periods of time. Durable single agent activity was previously reported in patients with FL, and has now been observed in patients with aggressive DLBCL. Studies in DLBCL and Richter’s syndrome are being implemented and updated information will be provided at the time of the meeting. Clinical trial information: NCT01733238.

Disclosures

Gaylor: ProNAi Therapeutics: Employment. Rodrigueza:ProNAi Therapeutics: Employment. Woolliscroft:ProNAi Therapeutics: Employment. Sooch:ProNAi Therapeutics: Employment. Messmann:ProNAi Therapeutics: Employment.
Rituximab, Age and High Dose Therapy Followed By Autologus Stem Cell Transplantation Are Independent Prognostic Factors for Survival in the First Line Treatment of Primary CNS-Lymphoma
Authors: Madle Michael , Herth Isabelle , Lehners Nicola , Schwarzbich Mark-Alexander , Wuchter Patrick , Egerer Gerlinde , Ho Anthony D. , Witzens-Harig Mathias
Published: October 2019

Introduction: For patients with diffuse large B-cell lymphoma without the involvement of the CNS, the addition of rituximab to standard chemotherapy has significantly improved survival. Thus far, there have been no prospective randomized trials evaluating the impact of rituximab as part of the primary treatment of PCNSL.

Methods: In this single-center, retrospective analysis, a total of 79 PCNSL-patients treated in our institution between 2000 and 2011 were included. Beside firstline chemotherapy with or without rituximab, we evaluated the impact of age (≤/> 60 years), autologous stem cell transplantation (ASCT +/-) and other factors upon overall survival (OS) and progression-free survival (PFS).

Results: In patients treated with rituximab (n=27), 3-year OS was 77.8% (95%-CI:62-93%). In contrast, in patients treated without rituximab (n=52), 3-year OS was only 39.9% (CI:27-53%, Figure). The difference in OS was significant in the univariate (p=0.002) as well as the multivariate analysis (p=0.049, Hazard ratio (HR)=0.248). In the rituximab group, 80.8% were free of progression at the date of analysis (median not reached), whereas median PFS in the group without rituximab was only 17 months (CI:8-26), (p=0.001). Patients ≤ 60 years of age (n=28) had a 3-year OS of 78.2% (CI:63-94%) and a median PFS of 75 months, in patients > 60 years (n=51) 3-year OS was 38.7% (CI:25-52%) and median PFS was 39 months (CI:6-72). Patients who received high dose therapy and autologous stem cell transplantation (ASCT) had a 3-year OS of 85.2% (CI:72-99%) and 65.1% were alive up to the time of analysis (range 9-131 months). Without ASCT median OS was only 16 months (CI:11-21) and 3-year OS was 35.2% (CI:22-48%). Age and ASCT were significantly associated with better OS in univariate (p=0.002 and p=0.001) as well in multivariate analysis (p=0.004, HR=0.023 and p=0.001, HR=0.014).

Conclusion: Rituximab treatment, ASCT and age are independent prognostic factors for overall survival in the first line treatment of PCNSL.

Figure 1 Figure 1.

Disclosures

Wuchter: Sanofi: Honoraria; ETICHO: Consultancy, Honoraria.
Follicular Lymphoma Survival in Sweden in the Rituximab Era
Authors: Junlén Henna Riikka , Peterson Stefan , Kimby Eva , Lockmer Sandra , Linden Ola , Nilsson-Ehle Herman , Erlanson Martin , Hagberg Hans , Rådlund Anders , Hagberg Oskar , Wahlin Björn E
Published: October 2019

Introduction

Follicular lymphoma (FL) is the most common indolent lymphoma, with 200-250 cases in Sweden per year. FL is considered incurable, because conventional therapy only leads to temporary remissions of varying length. Studies indicate that survival for patients with FL has improved since the introduction of rituximab. In Sweden first-line rituximab was gradually introduced between 2003 and 2007 and the speed of rituximab adoption varied geographically. In the publication of the first national Swedish guidelines for FL in November 2007, rituximab was recommended as first-line therapy and as maintenance after first relapse.

Methods

We conducted a nationwide, population-based registry study to assess patterns of FL survival before and after the introduction of first-line rituximab and to identify prognostic factors in the rituximab era. In the Swedish Lymphoma Registry (SLR) 2641 patients were identified with a FL diagnosed between January 1, 2000 and December 31, 2010. Clinical data, including first-line treatment were collected from the SLR. Overall survival (OS) and relative survival (RS) were estimated by calendar period (the pre-rituximab era 2000-2002, the transitional period 2003-2007 and the established period 2008-2010) and national health care region.

Results

First-line rituximab use increased gradually during the study period (2000-2002, 13%; 2003-2007, 61%; 2008-2010, 86%). During the transitional period, one health care region incorporated first-line rituximab quickly (92% first-line rituximab use) and one slowly (31%), whereas the remaining regions showed a moderate use of first-line rituximab (in total, 62%). OS and RS improved incrementally with each calendar period (P < 0.00005) (Figure 1A). Survival differed, during the transitional period, between regions with different rituximab use (OS, P = 0.039) (Figure 1B). After the national guidelines were published, no regional differences in use of first-line rituximab remained (P = 0.91). The improvement in OS and RS during the study period was greater in women than in men (Figure 1C). Survival improved in all age groups, but most in elderly patients (Figure 2).

Conclusions

The policy of first-line rituximab has resulted in a nationwide improvement in FL survival in Sweden. Women and elderly patients have benefited the most from rituximab.

Figure 1. Cumulative relative survival of follicular lymphoma by (A) calendar period of diagnosis, (B) regional adoption of rituximab during 2003-2007, and (C) calendar period of diagnosis and gender. Figure 1. Cumulative relative survival of follicular lymphoma by (A) calendar period of diagnosis, (B) regional adoption of rituximab during 2003-2007, and (C) calendar period of diagnosis and gender.

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Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
NFATc1 Is Transcriptionally Activated in Chronic Lymphocytic Leukemia (CLL) By Promotor DNA-Hypomethylation Which Correlates with in-Vitro Vulnerability to Calcineurin Inhibitors
Authors: Wolf Christine , Filarsky Katharina , Garding Angela , Claus Rainer , Oakes Christopher C , Idler Irina , Plass Christoph , Lichter Peter , Doehner Hartmut , Stilgenbauer Stephan , Mertens Daniel
Published: October 2019

Chronic lymphocytic leukemia (CLL), the most frequent adult leukemia in Western countries, is characterized by progressive accumulation of mature, monoclonal B lymphocytes in blood, bone marrow, and lymphoid tissues. In the pathogenesis and treatment of CLL, B cell receptor (BCR) signaling plays a crucial role, and aberrations in downstream pathways that become activated in CLL need to be better defined. One downstream target of BCR signaling is NFATc1, a transcription factor with a high oncogenic and transforming potential. Employing a genome-wide comparative DNA methylation analysis the NFATc1 5’ region was identified to be DNA hypomethylated in CLL patient samples. The pilot series comprised CD19+ B-cells from 13 CLL patients without previous treatment (9 with mutated IGHV using a 98% similarity cut-off) and CD19+ B-cells from 6 age-matched healthy donors. DNA methylation was relatively quantified using methyl-CpG immunoprecipitation (MCIp) on custom oligonucleotide promoter arrays covering -8kbp to +2.5kbp of all human transcription start sites. In total 1944 genes showed significant DNA hypomethylation in CLL cells compared to non-malignant B-cells (p<0.05), with NFATc1 ranking at position 36. DNA hypomethylation of the NFATc1 promoter was validated in peripheral blood CD19+ B-cells from 76 CLL patients without previous treatment compared to 14 additional age-matched healthy donors using mass spectrometry based quantitative high resolution measurement of DNA methylation (MASSARRAY). These findings suggest activation of the NFATc1 gene in CLL cells. In fact, NFATc1 mRNA was significantly upregulated in CLL samples compared to non-malignant B-cells, and expression correlated highly significantly with DNA methylation (r=-0.66, p<0.001). Inhibition of NFATc1 in primary CLL cells from patients (n=5) using calcineurin inhibitors like CsA and FK-506 induced significant apoptosis in primary CLL cells in-vitro even when protected by co-culture with bone-marrow stromal cells, suggesting a dependence of CLL cells on the activity of NFATc1. For a mechanistic insight into NFATc1 signal transduction in primary CLL cells and to allow modeling of the signaling network, a panel of phospho-specific antibodies is currently used in flow cytometry. This allows to monitor multiple components of signaling pathways in parallel with high time resolution to understand the impact of NFATc1 upregulation on the cellular signaling networks. The approach will shed further light on how CLL cell survival is regulated how this pathway can be targeted in CLL.

Disclosures

No relevant conflicts of interest to declare.
EZH2 Inhibition and the Combination with Proteasome Inhibition Are Novel Potential Strategies for the Treatment of Multiple Myeloma
Authors: Rizq Ola , Mimura Naoya , Koide Shuhei , Ma Anqi , Jin Jian , Iseki Tohru , Nakaseko Chiaki , Iwama Atsushi
Published: October 2019

PRC2 catalytic component EZH2 is a methyltransferase which induces trimethylation of histone H3 at lysine 27 (H3K27me3) to repress the transcription of target genes and promotes tumor growth in some cancers. Previous reports demonstrated that EZH2 is overexpressed in multiple myeloma (MM) cells and its expression level correlates with the progression of the disease, suggesting that EZH2 acts as an oncogene in MM. However, the detailed function of EZH2 in MM has not yet been elucidated. Therefore in this study, we examined the impact of EZH2 inhibition on MM cells and evaluated the preclinical efficacy of EZH2 inhibitors in the treatment of MM. We first examined the biologic impact of EZH2 knockdown using lentiviral shRNA and observed significant growth inhibition in knockdown cells. We next examined the effect of EZH2 inhibition using a novel selective EZH2 inhibitor UNC1999 (ACS Chem Biol. 2013). UNC1999 induced significant growth inhibition in MM cell lines such as MM.1S, H929, RPMI8226, and DOX40. We confirmed that UNC1999 reduced the levels of H3K27me3 in dose- and time-dependent manners in western blotting. Importantly, UNC1999 in combination with bortezomib demonstrated synergistic cytotoxicity analyzed by MTS assay in these cell lines. Even in the presence of conditioned media derived from bone marrow stromal cells, UNC1999 enhanced bortezomib-induced cytotoxicity in MM.1S cells. Similar synergistic effects were also observed in the combination of another EZH2 inhibitor GSK126 with bortezomib or in the combination of UNC1999 with another proteasome inhibitor carfilzomib. Moreover, apoptosis induced by bortezomib was enhanced by UNC1999 as evidenced by increased annexin V-positive cells in flow cytometric analysis. Apoptosis was further confirmed by cleavage of caspase-8, -9, -3 and PARP in western blotting. To assess the biologic impact of UNC1999 alone or in combination with bortezomib on MM cells, we performed gene set enrichment analysis using our microarray data. We found significantly positive enrichment of polycomb target genes in both UNC1999- and combination- treated MM.1S cells, and also in bortezomib-treated cells. Of interest, we observed that bortezomib alone downregulated EZH2 mRNA and EZH2 protein, which was enhanced by UNC1999, suggesting one mechanism of the synergistic effects of this combination. In order to examine the anti-MM activities of UNC1999, we used the MM.1S subcutaneous xenograft model in NOG mice. Intraperitoneal dosing of UNC1999 (25mg/kg) twice a week for three weeks significantly reduced the MM tumor growth in comparison to the control group (at day 22, p=0.04). In conclusion, these data indicate that selective EZH2 inhibition and the combination with proteasome inhibition are potential novel therapeutic options in MM.

Disclosures

No relevant conflicts of interest to declare.
Role of Erythroferrone in the Pathophysiology of Common Anemias
Authors: Kautz Leon , Jung Grace , Nemeth Elizabeta , Ganz Tomas
Published: October 2019

Introduction: We recently described the erythroid hormone erythroferrone (ERFE) as a critical regulator of hepcidin production during increased erythropoietic activity. Both anemia of inflammation (AI) and beta-thalassemia are associated with elevated levels of erythropoietin, the signal directing ERFE expression, suggesting that ERFE may play a role in the pathogenesis of these disorders. Indeed, Erfe mRNA expression was highly increased in the bone marrow and the spleen of a mouse model of β-thalassemia Hbbth3/+ (Th3/+). We therefore investigated the role of erythroferrone in hepcidin regulation in anemia of inflammation and β-thalassemia. We report that ERFE contributes to the recovery from anemia of inflammation and may be a hepcidin-suppressive factor responsible for iron accumulation in thalassemia.

Methods: We explored the role of ERFE in anemia of inflammation using the heat-killed Brucellaabortus (BA) mouse model of AI. Hepcidin regulation and recovery from anemia was compared between wild-type and Erfe-deficient mice. To determine whether ERFE is the hepcidin-suppressive factor in thalassemia, we generated Erfe-/-/Th3/+ double mutant mice and compared them to their littermate WT, Erfe-/- and Th3/+ mice. Mice were compared at 3, 6 and 12 weeks of age.

Results: Compared to wild-type mice, Erfe-deficient mice did not appropriately decrease hepcidin production during recovery from AI and exhibited greater severity of anemia (figure). These data suggest that compensatory hepcidin suppression during the recovery phase of AI is mediated by ERFE. However, in mice this defect was partly compensated by prolonged stimulation of erythropoiesis and higher reticulocytosis resulting in Erfe-deficient mice reaching wild-type hemoglobin levels by day 28 (figure).

Figure 1 Figure 1.

At the other end of the spectrum, we showed that thalassemic mice exhibited greatly increased expression of Erfe mRNA in the bone marrow and the spleen. Ablation of Erfe in Th3/+ mice restored normal hepcidin levels and significantly reduced serum iron concentration and hepatic iron overload at 6 weeks of age. Comparison of Erfe-/- / Th3/+ and Th3/+ mice at 3, 6 and 12 weeks of age did not show any difference in the severity of the anemia in absence of ERFE suggesting that ERFE regulates hepcidin expression but its absence did not cause iron restriction and did not ameliorate ineffective erythropoiesis.

Conclusion: ERFE is a critical regulator of hepcidin expression during recovery from AI and ERFE agonists may alleviate iron restriction in AI. ERFE may also be the factor responsible for hepcidin suppression and secondary iron overload in β-thalassemia.

Disclosures

Ganz: Intrinsic LifeSciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Keryx Pharma: Consultancy; Merganser Biotech: Consultancy, Equity Ownership.
Molecular Signatures of Tumor-Initiating Cells Unveil Wnt Pathway As a Therapeutic Target in Mantle Cell Lymphoma
Authors: Samaniego Felipe , Sehgal Lalit , Braun Frank K , Berkova Zuzana , Romaguera Jorge E. , Wang Michael , Rodriguez M. Alma , Neelapu Sattva S , Mathur Rohit
Published: October 2019

Introduction

Mantle cell lymphoma (MCL) is an aggressive and incurable form of non-Hodgkin’s lymphoma. Despite initial responses to intense chemotherapy, up to 50% of cases of MCL relapse, often in a chemoresistant form. We hypothesized that the recently identified MCL-initiating cells (MCL-ICs) are the main reason for relapse and chemoresistance of MCL.

Methods

We isolated MCL-ICs from primary MCL cells on the basis of a defined marker expression pattern; CD34-CD3-CD45+CD19-. The MCL-ICs, MCL-non-ICs, and peripheral blood lymphocytes from healthy donors were analyzed for gene expression using the Arraystar platform. The differences in mRNA levels of genes of interest were confirmed by quantitative RT-PCR. The prominent differentially expressed transcripts were analyzed using the Ingenuity Platform. Primary MCL cells were co-culture with mesenchymal stem cells to assess the effects of chemotherapeutic agents such as vincristine, doxorubicin and the newly approved Burton tyrosine kinase inhibitor ibrutinib, and Wnt signaling inhibitors.

Results

Approximately 1% of primary MCL cells are MCL-ICs and they can be maintained in co-culture with mesenchymal stem cells. Comparison of gene expression profiles of MCL-ICs and MCL-non-ICs revealed activation of stem cell-specific pathways in MCL-ICs by expression of Wnt, Notch, and Hedgehog and enhanced expression of Nanog, Oct4, KLF4, ADH1, MT1b and ABCC3. Gene expression microarray data and RT-PCR data suggested predominant activation of the Wnt/Frizzled pathway. Indeed, MCL-ICs were particularly sensitive to Wnt pathway inhibitors. Targeting Wnt-dependent β-catenin‒TCF4 interaction with CCT036477, iCRT14, or PKF118-310 preferentially eliminated the MCL-ICs, reduced the expression of stem cell transcription factors (Myc, Nanog, Oct4, Klf4), and sensitized MCL cells to vincristine, doxorubicin, and ibrutinib. Interestingly, while vincristine, doxorubicin or ibrutinib did kill MCL cells, they did not reduce the percentage of MCL-ICs in treated co-culture.

Conclusion

MCL-ICs are present in primary MCL isolates and they show gene expression pattern of chemoresistant, stem cell-like cells with predominant activation of Wnt signaling. In order to produce durable remissions in MCL patients, treatment strategies should be directed to target MCL-ICs.

Disclosures

Wang: Pharmacyclics, Janssen: Honoraria, Research Funding.
The Inhibitor of NF-KB Kinase, IKKβ Regulates the Transcriptional Activity of GLI1 By Blocking Its Proteasomal Degradation
Authors: Agarwal Nitin Kumar , Kim Chae Hwa , Kunkalla Kranthi , Lossos Izidore S. , Vega Francisco
Published: October 2019

GLI1 is a Hedgehog (Hh) related transcription factor originally discovered as an amplified product in gliomas. Inappropriate activation of the GLI1 has been shown in many cancers including diffuse large B cell lymphoma (DLBCL). We previously showed that GLI1 mediated canonical Hh signaling is constitutive active in DLBCL and contributes to cell survival, proliferation and enhances chemotolerance. Although the importance of GLI1 in tumor development is well recognized, the molecular mechanisms controlling the transcriptional activity of GLI1 are poorly characterized.

To identify regulatory components that participate in the transcriptional activity of GLI1, we explored GLI1 putative interacting proteins by liquid chromatography tandem mass spectrometry following immunoprecipitation of endogenous GLI1. We detected that the inhibitor of NF-KB kinase, IKKβ, is one of the proteins associated with GLI1 transcription factor. Here we investigate the regulatory role of IKKβ in the transcriptional activity of GLI1. We show that IKKβ regulates the transcriptional activity of GLI1 by phosphorylating GLI1 in C-terminal region and modulating its protein stability. Short stimulation of SUDHL4 and DOHH2 cells with TNF-α (20ng/mL) resulted in increased GLI1 protein levels. Similar results were observed in 293T cells transiently transfected with GLI1 and IKKβ kinase constructs. Moreover, silencing of IKKβ using siRNA and shRNAs led to decreased GLI1 protein levels and its transcriptional activity in DLBCL cell lines with constitutive activation of the NF-KB.

Next, we characterized nine probable IKKβ dependent GLI1 phosphorylation sites (S543-S548, S1070, S1071 and S1074 identified by nanospray ion trap mass spectrometry) using mutational and deletions studies. We show that IKKβ phosphorylates GLI1 at Thr1074 and decreases binding between GLI1 and HECT-type E3 ubiquitin ligase (ITCH) resulting in reduced GLI1 polyubiquitination and degradation. Point mutation of Threonine 1074 to Alanine prevents IKKβ-mediated GLI1 phosphorylation and facilitates GLI1-ITCH interaction, polyubiquitination and degradation of GLI1 in the proteasome.

Collectively, our data links IKKβ-mediated NF-kB signaling to the transcriptional activity of GLI1 and illustrates a novel cross talk between these two pathways. This is of clinical interest because activation of the NF-kB pathway is frequent in DLBCL and the connection between Hh and NF-kB pathways may open novel therapeutic avenues for DLBCL.

Disclosures

No relevant conflicts of interest to declare.
Dietary ω-3 Fatty Acid Supplementation As a Potential New Therapy for Vasculopathy in Sickle Cell Disease: Proof of Concept in a Transgenic Mouse Model
Authors: Kalish Brian T , Matte Alessandro , Andolfo Immacolata , Iolascon Achille , Weinberg Olga , Siciliano Angela , Federti Enrica , Puder Mark , Brugnara Carlo , De Franceschi Lucia
Published: October 2019

Sickle cell disease (SCD) is a genetic red cell disorder characterized by hemolytic anemia in association with acute and chronic life-threatening clinical complications mainly related to inflammatory vasculopathy. Promising, but limited, human studies have shown potential therapeutic effects of ω-3 fatty acid supplementation in SCD. Here, we sought to compare the effects of 6 weeks ω-6 (soybean oil diet, SD) vs ω-3 (fish oil diet, FD) diet on vasculopathy in SCD mice (Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow). We treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n=6-7 animals in each group). In SCD mice, FD was associated with a decreased the ratio of ω-6 to ω-3 fatty acids in red cell membranes, as well as a decrease in neutrophil count compared to SD. We then evaluated the effects of FD on cardiovascular system of SCD mice. We found that FD resulted in significantly (i) lower diastolic blood pressure; (ii) reduced cardiac output; (iii) down-regulation of VCAM-1, ET-1 and HO-1 expression in isolated aorta compared to SD, reaching levels similar to those observed in AA mice. In the lung of SCD mice, FD significantly reduced ET-1 expression compared to SD. FD was also associated with reduced bronchoalveolar lavage leukocyte and protein contents compared to SD, suggesting protection against pulmonary leak. We then studied the effect of FD during vaso-occusive crises (VOCs), mimicked by exposing SCD mice to hypoxia/reoxygenation (H/R) stress. In SCD mice, FD significantly (i) ameliorated the H/R-induced reduction in hematocrit and hemoglobin levels; (ii) reduced the fraction of dense red cells and the H/R-induced increase in neutrophil count. Histologic analysis revealed that H/R stress recapitulates the SCD acute organ damage. In the lung of H/R SCD mice, FD reduced inflammatory cell infiltrates, bronchial mucus, thrombosis, and ET-1 and ET1-1R expression. In isolated aorta from SCD mice exposed to H/R, FD prevented the H/R-induced ET-1 and VCAM-1 expression and the H/R-induced up-regulation of anti-oxidant systems compared to SD. These findings provide new insight into the mechanism of action of ω-3 supplementation on the pathogenesis of SCD and strengthen the rationale for ω-3 dietary supplementation as a simple therapeutic intervention to reduce vascular dysfunction in SCD.

Disclosures

No relevant conflicts of interest to declare.
The G-CSF Induced MiR-143 Targets MAPK-Family Proteins and Is a Prognostic Factor for RIC-Transplanted AML Patients
Authors: Hartmann Jens-Uwe , Braeuer-Hartmann Daniela , Gerloff Dennis , Katzerke Christiane , Wurm Alexander Arthur , Müller-Tidow Carsten , Schwind Sebastian , Tenen Daniel G. , Niederwieser Dietger , Behre Gerhard
Published: October 2019

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of defective hematopoiesis of white blood cells and often characterized by limited treatment options and a poor prognosis. MicroRNAs, a class of small non-coding RNAs, have been reported over the last years to play an important role in the regulation of “normal” and pathological processes. By binding to the 3`UTR of their target mRNAs, microRNAs lead to the translational repression of their target gene. Deregulation of a certain microRNA thereby may lead to disrupted signal pathways, such as MAPK-signaling, and to tumorigenesis. However, the role of microRNAs in hematopoietic differentiation and blood cancer development remains largely unknown. In AML only few miRNAs are functionally characterized, and the clinical relevance of these miRNAs still remains to be determined. In this study we identified microRNA-143 (miR-143) to be highly upregulated in G-CSF stimulated CD34+ cells. We could also show the granulopoietic association of miR-143 in several hematopoietic cell line models. In addition, we show that miR-143 is upregulated in APL patients after ATRA treatment. QPCR analysis of pri-miR-143 expression in RIC (reduced intensity conditioning) transplanted AML patients reveal that high expression of miR-143 is associated with high event-free and overall survival. In functional studies, stable overexpression of miR-143 in K562-C/EBPα-p42 cells enhances the C/EBPalpha induced granulocytic differentiation significantly. In contrast, stable knock down of miR-143 reduced the ability of K562-C/EBPα-p42 cells to undergo granulocytic differentiation upon β-estradiol treatment. Moreover, chromatinimmunoprecipitation (ChIP) analysis show the direct binding of C/EBPα to the promoter region of miR-143. By in silico prediction, we found MAPK protein family members (MAPK1, MAPK3 and MAPK7) as predicted targets of miR-143. Western blot analysis of AML patient samples and G-CSF stimulated CD34+ cells clearly show an inverse correlation of miR-143 and MAPK7 (ERK5) protein expression. By transient overexpression of miR-143 we could show a strong downregulation of MAPK protein family member expression in NB4 cells. These studies suggest miR-143 as an essential factor in granulocytic differentiation driven by G-CSF. Deregulation of miR-143 may play an important role in the development of AML. Furthermore, we propose miR-143 as prognostic marker for AML patients undergoing RIC transplantation therapy.

Disclosures

No relevant conflicts of interest to declare.
Identification of the Lymphoma-Initiating Cell Population in a T Cell Lymphoma Mouse Model Resembling Human Anaplastic Large Cell Lymphoma
Authors: Klingeberg Cathrin , Kreutmair Stefanie , Miething Cornelius , Follo Marie , Peschel Christian , Illert Anna Lena , Duyster Justus
Published: October 2019

In 60% of anaplastic large cell lymphoma (ALCL) patients a translocation t(2;5) (p23;q35) is found, which results in NPM-ALK fusion gene expression and constitutive activation of the ALK tyrosine kinase. Immunophenotypic characterization of human ALCLs revealed highly CD30-positive cells of T- or Null-cell-origin. However, the origin of the lymphoma initiating cell population as well as NPM-ALK signal transduction in course of the disease remains unclear.

In this regard, we established a retroviral murine bone marrow transplantation model resembling human ALCL. Therefore we use an inducible Cre/loxP system where NPM-ALK expression is restricted to early T cells. We infected bone marrow of Lck-Cre transgenic mice with our MSCV-Stop-NPM-ALK-IRES-EGFP vector and transplanted it into lethally irradiated recipient mice. With a latency of 4-5 months, these mice developed Thy1.2-positive lymphomas and died from neoplastic T cell infiltration of bone marrow and lymphatic organs. Immunophenotypic analysis confirmed T cell origin of the lymphomas with the characteristic high CD30 expression. Staining of the T cell subpopulations demonstrated high NPM-ALK expression in immature CD4-/CD8- double negative T cells and undifferentiated CD4+/CD8+ double positive T cells. Interestingly, FACS-staining for the proliferation marker Ki-67 as well as the activation marker CD30 revealed highest expression in the CD4-/CD8- double negative T cells. Therefore we hypothesized that the lymphoma-initiating cell must be within this early T cell population.

To substantiate our hypothesis we performed secondary transplantations with sorted T cell subpopulations and indeed, only the CD4-/CD8- double negative population was able to initiate T cell lymphoma in the recipient mice. Immunophenotypic characterization of the lymphoma population of these secondary transplanted mice revealed undifferentiated T cells of all CD4/CD8 subtypes, which argues for the existence of a lymphoma initiating cell population, which can still partly differentiate. Interestingly the CD4-/CD8- double negative lymphoma population aberrantly expressed the T cell receptor alpha/beta chain, which may allow these early T cells to establish a systemic lymphoma. Further analysis of the lymphoma population showed lymphatic precursors (CLP) as well as multipotent progenitors (MMP) and haematopoetic stem cells (LSK), which suggests early bone marrow or thymic progenitor cells as the pool of the lymphoma-initiating cell population.

We therefore were able to prove the existence of lymphoma initiating stem cells in a highly relevant NPM-ALK positive CD30 expressing mouse model of ALCL. Further analysis will give insides into eradication of the identified lymphoma stem cell population by clinical relevant NPM-ALK inhibitors and CD30 immunotoxins.

Disclosures

No relevant conflicts of interest to declare.
Targeting Polo-like Kinase in Acute Myeloid Leukemia
Authors: Cher Chae Yin , Man Cheuk Him , Lam Stephen S.Y. , Ho Eric S.K. , Ng Nelson K.L. , Hospital Marie-Anne , Tamburini Jerome , Leung Anskar Y.H.
Published: October 2019

Acute myeloid leukemia (AML) carrying fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) is associated with poor prognosis when treated with conventional chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT). Despite much interest in tyrosine kinase inhibitor (TKI) targeting FLT3 activation, drug resistance is invariable and acquisition of secondary point mutation in the tyrosine kinase domain (TKD) of FLT3 was frequently reported. We have shown that genes encoding cell cycle regulators including polo-like kinase 1 (PLK1), cell division cycle 25 homolog A (CDC25A), cyclin B2 (CCNB2), and cyclin E1 (CCNE1) were up-regulated in sorafenib-resistant primary AML samples. In particular, PLK1, a member of the polo-like kinase family, has been found to express at several checkpoints critical for cell cycle progression. PLK1 inhibitors have recently been exploited for the treatment of both solid organ and haematological cancers. We hypothesized that aberrant cell cycle progression mediated by increased PLK1 expression might confer survival advantage to drug resistant AML cells and be targetable by PLK1 inhibition. In vitro treatment with PLK1 inhibitors volasertib and BI 2536 significantly inhibited the growth of 8 AML cell lines (KG-1, ML2, MOLM-13, MV4-11, Kasumi-1, NB4, THP-1 and OCI-AML3) with IC50 (all in nM) ranging from 48.7 - 98.4 (volasertib) and 35.1 - 81.6 (BI 2536). The growth inhibitory effects of PLK1 inhibition on two FLT3-ITD+ cell lines, MOLM-13 and MV411, correlated with induction of apoptosis and cell cycle arrest at G2/M phase. Moreover, both PLK1 inhibitors significantly suppressed the growth of a sorafenib resistant MOLM-13R cell line and sorafenib naïve MOLM-13N cell line. Introduction of FLT3-ITD alone or FLT3-ITD and TKD double mutations into Ba/F3 cell lines sensitized them to the growth inhibitory effects of PLK1 inhibitors. Primary FLT3-ITD+ AML cells obtained from patients at TKI resistance were shown to be more sensitive to PLK1 inhibitors than those obtained before treatment. The results suggested that the TKI resistant clones could be effectively targeted by PLK1 inhibition, providing an insight to the design of combination treatment with FLT3 inhibitors in FLT3-ITD+ AML.

Disclosures

No relevant conflicts of interest to declare.
A Systematic Review of Definitions and Reporting of Bleeding Outcome Measures in Hemophilia
Authors: Chai-Adisaksopha Chatree , Hillis Christopher M. , Thabane Lehana , Iorio Alfonso
Published: October 2019

Background: Bleeding frequency is an important outcome commonly used in hemophilia studies. There is variation in practice in how bleeding is measured and defined. We therefore performed a systematic review of the studies reporting bleeding frequency in hemophilia patients.

Methods: MEDLINE, EMBASE, and the Cochrane Central Register of Control Trials were searched from January 1990 to January 2014. We included all published studies that included patients with hemophilia A and/or B and reported some measure of bleeding. Two reviewers independently performed title and abstract screening, full-text review and data abstraction of the identified studies.

Results: A total of 118 studies fulfilled the inclusion criteria. Study designs were randomized controlled trials [RCT] (16/118: 14%), cohort (80/118: 68%), cross-sectional (6/118: 5%) and others design (13/118: 11%). The median duration of follow-up (first quartile [Q1], third quartile [Q3]) was 20 (7.9, 50) months. Joint bleeding is the most common site of reported bleeding (66.9%). We found 10 different bleeding outcomes reported (absolute number of bleeding 60 (50.8%) studies, annualized bleeding rate 60 (50.8%) studies, bleed per month 10 (8.5%) studies and others (bleed per patient-year, bleed per week, bleed per season, etc.) 11 (9.3%) studies). Of these, 32 studies (27%) reported only mean or median without dispersion and 33 (28%) studies did not report any measures of central tendency (dispersion).

Conclusions: There is substantial variation in definitions and measures of bleeding outcomes in the hemophilia literature. This creates difficulty and limitations in comparing the outcomes between studies and in performing meta-analysis. The hemophilia research community needs to develop a consensus definition of bleeding and how to address the limitations associated with variations in measures of bleeding between centers and studies.

Table 1. Measures of bleeding frequency used in the studies Bleeding outcome measures n , (%) 1. Bleeding frequency# Absolute number of bleeds 60 (50.8) Annualized bleeding rate 60 (50.8) Bleed per month 10 (8.5) Bleed per 100 days 4 (3.4) Others (bleed per patient-year, bleed per week, bleed per 5 years, etc.) 11 (9.3) 2. Central tendency and dispersion# Mean with standard deviation 33 (28.0) Median with range or interquartile range 35 (27.0) Only mean or median 32 (27.1) Not reported 33 (28.0)

#Studies could include more than one level of characteristics.

Disclosures

No relevant conflicts of interest to declare.
The Stimulatory Role of CD300a in Diffuse Large B-Cell Lymphoma
Authors: Jiang Lei , Zhou Jeff Xiwu , Morse Herbert , Xu Yulian
Published: October 2019

CD300a is a type I transmembrane receptor protein which has shown inhibitory effect on B-cell receptor mediated signals. In an analysis of publicly available data on diffuse large B-cell lymphoma (DLBCL), however, we found that the expression levels of CD300A mRNA were inversely correlated with the overall survival of DLBCL patients. When analyzing the transcript levels of CD300a in human tissues, we found that CD300a mRNA levels were significantly greater in DLBCL tissues than benign lymphoid tissues (P<0.05). To decipher the role of CD300a in DLBCL, we used shRNA system to knock-down the expression levels of CD300a in DLBCL cell lines, and found that decreased levels of CD300a significantly inhibited cell proliferation of OCI-Ly1 cells, but not of VAL, OCI-Ly10 or SUDHL-8 cells. Mechanistically, reduced expression of CD300a resulted in a marked attenuation of Akt phosphorylation in OCI-Ly1 cells. Pharmacologic inhibition of PI3K by LY294002 displayed a similar inhibitory effect on cell proliferation, indicating the possible involvement of PI3K/Akt signaling pathway in CD300a’s effect. Furthermore, using a xenograft animal model, we found that decreasing expression levels of CD300a in OCI-Ly1 cells significantly inhibited tumor formation of these cells in vivo. Collectively, our results suggested a stimulatory role of CD300a in DLBCL which could serve as a potential biomarker and therapeutic target for this malignance.

Disclosures

No relevant conflicts of interest to declare.
Primary CLL Xenograft: A Model System to Study the Role of T-Cells in CLL Biology and Therapeutic Response
Authors: Oldreive Ceri E , Skowronska Anna , Agathanggelou Angelo , Parry Helen M , Krysov Sergey , Packham Graham K , Zbgniew Rudzki , Pratt Guy , Taylor A. Malcolm R , Moss Paul , Stankovic Tatjana
Published: October 2019

The interaction between chronic lymphocytic leukaemia (CLL) cells and T-cells is an important aspect of CLL biology. CLL cells require T-cell support for their proliferation and in addition induce proliferation of regulatory and cytotoxic (CD8+) T-cells. T-cell number and repertoire are both markedly affected by CLL therapy and there is considerable interest in how current treatments modulate the interaction between T-cells and the tumour clone. In this study we investigated whether this relationship was maintained in a xenotransplantation model.

CLL engraftment in NOG mice was facilitated by humanisation of the murine microenvironment by allogeneic CD34+ umbilical cord cells or CD14+ monocytes. Accelerated engraftment of both CLL and T-cell compartments was observed in xenografts derived from patients with progressive CLL, suggesting that the biological properties of both subsets are maintained in the murine model. Furthermore, the distribution of helper (CD4+), cytotoxic (CD8+) and regulatory (CD4+CD25+FoxP3+) T-cells was maintained within the xenografts, including retention of the CD4:CD8 ratio. Interestingly, the anergic PD-1+CD160+CD244+TIM3+ T-cell phenotype reported in CLL patients was also evident in T-cells expanded in xenograft models. Consistent with an anergic T-cell phenotype, T-cells from CLL xenografts lacked anti-tumour activity in vitro. Importantly, such anergic cells were observed when T-cells were reconstituted from allogeneic cord blood cells as well as autologous cells, suggesting that CLL cells have the ability to shape T-cell populations of different origin in diverse microenvironments. Finally, to investigate the interaction between specific T-cell subsets and engrafted CLL cells, CD4+, CD8+, and CD25+ T-cells were depleted prior to generation of xenografts. CD8+ T-cell depletion significantly prolonged CLL engraftment (p≤0.01) whereas neither depletion of CD4+ nor CD25+cells had a significant impact.

In summary, our results demonstrate that the relationship between CLL tumour cells and reactive T-cells is accurately maintained in a murine xenograft model. Such models will be of great value for investigation of aspects of T-cell function in CLL biology.

Disclosures

No relevant conflicts of interest to declare.