bluuebunny/crossref_metadata_2025 huggingface dataset:
PRL-3 Mediates Survival of Primary Myeloma Cells
Authors: Rø Torstein Baade , Slordahl Tobias Schmidt , Abdollahi Pegah , Vansemb Esten , Hagen Lars , Baranowska Katarzyna Anna , Misund Kristine , Slupphaug Geir , Borset Magne
Published: October 2019

Introduction:Phosphatase of regenerating liver-3 (PRL-3) is a marker of aggressive and metastatic disease in a number of solid tumors. PRL-3 is expressed in primary multiple myeloma (MM) cells and MM cell lines, but not in normal plasma cells. We have previously shown that PRL-3 is induced in MM cells by interleukin (IL)-6 and other growth factors, and that PRL-3 promotes MM-cell migration (Fagerli et al, Blood, 2008). A study by Broyl et al (Blood, 2010) identified PRL-3 as a marker gene for a subgroup of patients with MM. In this study we examined the effect of the small molecular inhibitor PRL-3 Inhibitor I on primary MM cells, and we examined possible cellular targets of PRL-3 using a proteomics approach.

Methods: Co-cultures of primary myeloma cells and pooled bone marrow stromal cells from several MM patients were grown for 3 days in the presence or absence of PRL-3 Inhibitor I. Apoptosis was determined by staining cells with YO-PRO-1. To examine cellular targets of PRL-3 we made PRL-3 overexpressing cells from the human MM cell line INA-6 by retroviral transduction (INA-6-PRL-3) and control cells (INA-6-MOCK). Stable isotope labeling by amino acids in cell culture (SILAC) was used for in vivo incorporation of 12C-Lysine (“light”) and 13C-Lysine (“heavy”) in these transduced cell lines, preparing them for Orbitrap mass spectrometry based quantitative proteomics. The experiments were performed three times. The results were analyzed using Ingenuity Pathway Analysis software (Quiagen).

Results: PRL-3 Inhibitor I significantly reduced viability in all 9 primary MM cell samples tested, ranging from 5-70 % absolute reductions in viability at the top concentration of 40 µM. Proteomics analysis was performed on INA-6-PRL-3 and INA-6-MOCK cell lines at baseline and following incubation in IL-6-free media for 12 hours to deplete INA-6-MOCK cells of PRL-3. At 12 hours, a total of 6747 proteins were identified. 481 proteins were upregulated and 22 proteins downregulated by more than 2-fold in the INA-6-PRL-3 cells compared to INA-6-MOCK cells in 2 out of 3 experiments. Several proteins known to be important for cell survival and migration were upregulated in INA-6-PRL-3 cells, like CD44, Myc assosciated factor X (MAX) and Lyn. Pro-apoptotic proteins Caspase-1 and Caspase-2 were downregulated. Among canonical signaling pathways, there were most proteins belonging to the protein ubiquitination pathway (9,8 % of proteins; p<0,001).

Conclusion: PRL-3 Inhibitor I induced apoptosis in primary MM cells. Quantitative proteomics analyses comparing PRL-3 overexpressing INA-6 cells to normal INA-6 cells identifies several proteins important for MM cell migration and survival. This study indicates that PRL-3 could be important in the pathogenesis of MM and a potential target in the treatment of MM.

Disclosures

No relevant conflicts of interest to declare.
Engineered T Cell Receptor-Mimic Antibody, (TCRm) Chimeric Antigen Receptor (CAR) T Cells Against the Intracellular Protein Wilms Tumor-1 (WT1) for Treatment of Hematologic and Solid Cancers
Authors: Rafiq Sarwish , Dao Tao , Liu Cheng , Scheinberg David A. , Brentjens Renier J
Published: October 2019

Adoptive transfer therapy of T cells expressing chimeric antigen receptors (CAR) against tumor-associated antigens has been shown to be clinically successful in a limited set of leukemia. However, novel antigen targets for both hematological and solid malignancies are required. Most CARs described thus far are targeted against external antigens on particular cell types. We have designed and engineered the first CAR T cell against a human intracellular protein, WT1. WT1 is overexpressed in many cancers, including acute and chronic leukemias and numerous solid tumors. Our TCRm CAR, derived from the ESK1 TCRm mAb, termed WT1 28z, is reactive with the RMFPNAPYL peptide of the WT1 protein that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1 28z expressing T cells have high expression of the CAR on their surface. They are cytotoxic in standard 51Cr assays against a range of cancer cell lines, including the megakaryoblastic cell line SET2, the acute myeloid leukemia (AML) cell line AML14, the multiple myeloma cell line KARPAS, and the ovarian cancer line, OVCAR3, as compared to CAR T cells against an irrelevant antigen. The WT1 28z CAR T cells are also cytotoxic against primary AML bone marrow blasts in this assay. When co-cultured with these primary cells or cancer cell lines, the WT1 28z CAR T cells have enhanced production of proinflammatory cytokines such as IFN-g, IL-2, and GM-CSF, as compared to irrelevant CAR T cells. Importantly, WT1 28z T cells are specific for the WT1-HLA-A*02:01 complex. The cells do not show cytotoxicity against cell lines or primary cells that are not both HLA-A*02:01- positive and WT1 positive. WT1 28z T cells are currently being tested alongside irrelevant antigen CAR T cells in AML and ovarian cancer murine models in vivo to assess efficacy, with the ultimate goal of translating this novel approach into the clinical setting for both hematological and solid cancers. The data provide the proof-of-concept that CAR T cells also may be directed at intracellular antigens.

Disclosures

Dao: Novartis: Patents & Royalties. Liu:Eureka: Employment, Inventor Other. Scheinberg:Novartis: Patents & Royalties. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.
An Epigenomic Map of Multiple Myeloma Reveals the Importance of Polycomb Gene Silencing for the Malignancy
Authors: Agarwal Prasoon , Kalushkova Antonia , Enroth Stefan , Alzrigat Mohammad , Österborg Anders , Nilsson Kenneth , Öberg Fredrik , Jernberg-Wiklund Helena
Published: October 2019

Multiple myeloma (MM) is characterized by accumulation of post-germinal center, isotype-switched, long-living plasma cells with retained proliferation capacity within the bone marrow. MM is highly heterogeneous and remains fatal. This heterogeneity has hampered identification of a common underlying mechanism for disease establishment and the development of targeted therapy. We recently provided proof-of-principle that gene silencing associated with H3K27me3 contributes to the malignancy of MM. Here we present the first epigenomic map of MM for H3K27me3 and H3K4me3 derived by ChIP- and RNA-sequencing from freshly-isolated bone marrow plasma cells from four patients. We compile lists of targets common among the patients as well as unique to MM when compared with PBMCs. Indicating the clinical relevance of our findings, we find increased silencing of H3K27me3 targets with disease progression and in patients presenting with a poor prognosis. Bivalent genes further significantly correlated to under-expressed genes in MM and were unique to MM when compared to PBMCs. Furthermore, bivalent genes, unlike H3K27me3 targets, significantly associated with transcriptional activation upon Polycomb inhibition indicating a potential for drug targeting. Thus, we suggest that gene silencing by Polycomb plays an important role in the development of the malignant phenotype of the MM cell during tumor progression.

Disclosures

No relevant conflicts of interest to declare.
Functional Dissection of the C Terminus of CBFβ-SMMHC Indicates a Critical Role of the Multimerization Domain during Hematopoiesis and Leukemogenesis
Authors: Zhao Ling , Alemu Lemlem , Cheng Jun , Zhen Tao , Friedman Alan D. , Liu Pu Paul
Published: October 2019

The recurrent chromosome 16 inversion (inv(16)) in acute myeloid leukemia (AML) subtype M4Eo results in a fusion between CBFB and MYH11 genes, which encodes a chimeric protein CBFβ-SMMHC (core binding factor β - smooth muscle myosin heavy chain). We previously generated mouse CBFB-MYH11 knock-in models that mimic the human inv(16) AML and demonstrated that the CBFβ-SMMHC fusion protein blocks RUNX1 and CBFβ function during definitive hematopoiesis and plays a driving role in leukemogenesis. Our recent studies indicated that the C-terminus of CBFβ-SMMHC, which contains domains for multimerization and transcriptional repression, is important for leukemogenesis by CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013). In this study we generated a new CBFB-MYH11 knock-in mouse model to determine the role of the multimerization domain of CBFβ-SMMHC during hematopoiesis and leukemogenesis.

Previous studies have dissected the assembly competence domain (ACD) of the CBFβ-SMMHC C-terminus to identify the critical amino acid residuals for multimerization (Zhang et al., Oncogene 25:7289, 2006). Among them, mutations in helices D and E are the ones that affect multimerization the most. Importantly, the helices D and E mutations do not interfere with the repression function of CBFβ-SMMHC. Therefore, we generated knock-in mice expressing CBFβ-SMMHC with mutated helices D &E in the ACD of the C-terminus (mDE) to determine the role of multimerization for the in vivo function of CBFβ-SMMHC.

The embryonic hematopoietic phenotype in the mDE knockin embryos is very similar to what we have observed in the knockin embryos expressing C-terminally-deleted CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013), i.e., heterozygous embryos (Cbfb+/mDE) were viable and showed no defects in fetal liver definitive hematopoiesis, while homozygous embryos (CbfbmDE/mDE) showed hemorrhage in the central nervous system and died around E12.5, as seen in the full length CBFβ-SMMHC heterozygous knockin mice and the Cbfb-/- and Runx1-/- mice. Analysis of peripheral blood from adult Cbfb+/mDE mice showed decreased B cell population and increased T cell population, while the myeloid compartment was unchanged. Preliminary findings suggest that leukemogenesis is at least delayed in the Cbfb+/mDE mice as compared to mice expressing full-length CBFβ-SMMHC. Therefore the multimerization function of CBFβ-SMMHC is critical for its ability to induce defects in embryonic hematopoiesis and for leukemogenesis.

Disclosures

No relevant conflicts of interest to declare.
Ibrutinib Sensitizes AML Cells to ROS Inducers Via a BTK-Independent Mechanism
Authors: Rotin Lianne E , Gronda Marcela , Maclean Neil , Lin Feng-Hsu , Wrana Jeff , Datti Alessandro , Barber Dwayne L , Moran Michael F , Minden Mark D , Slassi Malik , Schimmer Aaron D
Published: October 2019

Ibrutinib is a small-molecule Bruton’s tyrosine kinase (BTK) inhibitor yielding impressive clinical responses in B-cell malignancies, where BTK contributes to disease development and progression. We noted that BTK is expressed and constitutively active in acute myeloid leukemia (AML) cell lines and in a subset of AML patients. We therefore sought to determine BTK’s potential role in AML. Using BTK-pY223 expression on immunoblot as a marker of BTK activity, we demonstrated that ibrutinib doses as low as 1μM were sufficient to inhibit BTK activity in the AML cells TEX and OCI-AML2. Yet, these cell lines were insensitive to ibrutinib compared to the B-cell lymphoma Daudi cell line (IC50 10.4μM and 23.7μM versus 1.1μM, respectively). Although inactive as a single agent, we sought to identify drug combinations that sensitized AML cells to pharmacologically relevant concentrations of ibrutinib by conducting a combination chemical screen with our in-house known drug library in TEX and OCI-AML2 cells. According to excess-over-Bliss additivism criteria, we determined that in both cell lines, the poly(ADP-ribose) glycohydrolase inhibitor ethacridine lactate was the most synergistically cytotoxic, producing an 8-fold reduction in the IC50 of ibrutinib. Synergistic cytotoxicity was also observed in a subset of primary AML cells, but not normal hematopoietic cells. Mechanistically, the combination’s synergistic cytotoxicity resulted from excessive ROS production (>10-fold increase, versus a <2-fold increase with either drug alone in TEX and OCI-AML2), since α-tocopherol addition to combination-treated cells rescued cell viability from 24% to 85% and from 4% to 84% in TEX and OCI-AML2, respectively. Combining ibrutinib with other ROS-inducing agents such as parthenolide and the first-line AML therapy daunorubicin produced similar synergistic effects in AML cells,with α-tocopherol also rescuing cell viability. However, the synergistic effects were likely not related to inhibition of BTK, as knockdown of BTK with shRNA did not sensitize TEX cells to ethacridine or daunorubicin and thus did not recapitulate the effects of ibrutinib. We therefore conclude that the BTK inhibitor ibrutinib lacks single agent anti-AML activity, but synergizes with ROS-inducing agents including daunorubicin by inhibiting targets beyond BTK. Thus, clinical trials of ibrutinib in combination with standard chemotherapy could be warranted in AML.

Disclosures

No relevant conflicts of interest to declare.
FOXM1 Is Overexpressed in B-Acute Lymphoblastic Leukemia (B-All) and Its Inhibition Sensitizes B-All Cells to Chemotherapeutic Drugs
Authors: Consolaro Francesca , Basso Giuseppe , Viola Giampietro , Lam Eric W-.F.
Published: October 2019

B-lymphoblastic leukaemic (B-ALL) patients that respond poorly to glucocorticoid therapy are predicted to relapse. An understanding of the biological mechanism underlying this poor responsiveness is therefore crucial for the development of more effective diagnostics and therapies. Forkhead box protein M1 (FOXM1) is a key transcriptional factor that regulates the expression of several genes that promote cell cycle progression, proliferation, DNA repair. Its expression is up-regulated in most cancer cells and is often linked to high proliferation rates and poor responsiveness to the therapy. In this context, we studied the role of FOXM1 in B-lymphoblastic leukaemia (B-ALL) in order to understand if FOXM1 could be a key target for leukaemia therapy. Our results showed that FOXM1 expression is higher in both B-ALL patients and cell lines compared to PBMC or CD19+ cells from healthy donors (Figure 1 A, 1 B).

Figure 1A: FOXM1 overexpression in both B-ALL patients (A) and cell lines (B). Figure 1A:. FOXM1 overexpression in both B-ALL patients (A) and cell lines (B). Figure 1B Figure 1B.

Furthermore FOXM1 protein levels were higher in glucocorticoid-resistant cell lines (REH, MHH-CALL2, SEM) when compared to their glucocorticoid-sensitive counterparts (RS4;11, NALM-6), suggesting that FOXM1 may have a role in mediating chemotherapeutic drug sensitivity and resistance in B-ALL. Furthermore, depletion of FOXM1 activity in B-ALL cell lines by either transient knockdown or treatment with a FOXM1 inhibitor, thiostrepton, significantly decreases the cell viability of cells that poorly respond to glucocorticoid treatment (REH). The decrease of cell viability was accompanied by an induction of G2/M arrest of the cell cycle along with a reduction of the S phase. Moreover thiostrepton synergises with common chemotherapeutic agents used in B-ALL therapy increasing their efficiency and overcoming drug resistance.

All this data suggest that FOXM1 could be an important therapeutic target for overcoming the resistance to the conventional chemotherapeutic drugs.

Disclosures

No relevant conflicts of interest to declare.
Genetic Characterization of Patients with Monoallelic and Biallelic CEBPA Mutations Using a Targeted Sequencing Approach Reveals Differences in the Spectrum of Cooperating Mutations
Authors: Konstandin Nikola P , Metzeler Klaus H. , Dufour Annika , Greif Philipp A , Herold Tobias , Ksienzyk Bianka , Schneider Stephanie , Berdel Wolfgang E , Büchner Thomas , Woermann Bernhard J. , Sauerland Cristina , Braess Jan , Hiddemann Wolfgang , Bohlander Stefan K , Spiekermann Karsten
Published: October 2019

Mutations in the CCAAT/enhancer binding protein alpha (CEBPA) are detected in about 10% of patients with cytogenetically normal acute myeloid leukemia (CN-AML). CEBPA mutation can either affect both CEBPA alleles (biallelic, biCEBPA) or only one allele (monoallelic, moCEBPA). We and others have shown that only patients with biCEBPA mutations have favorable outcomes when compared to other CN-AML patients (Dufour et al, JCO 2009; Green et al, JCO, 2010). Interestingly, biCEBPA mutations are rarely associated with other known prognostic mutations (e.g. FLT3-ITD, NPM1).

In this study we aimed to characterize the mutational spectrum of CN-AML patients with mo- and biCEBPA mutations using a targeted amplicon sequencing approach. We analyzed 45 biCEBPA patients and 35 moCEBPA patients. 55 of these patients (26 biCEBPA and 29 moCEBPA) were enrolled in a multicenter trial of the German AML Cooperative Group (AMLCG-1999).

Our amplicon resequencing panel included 42 genes which are known to be frequently mutated in AML (Haloplex, target region 62 kilobases). Out of these 42 genes we identified 23 different mutated genes in the biCEBPA subgroup and a total of 28 different mutated genes in the moCEBPA cohort. The mean number of mutated genes per moCEBPA patient was significantly larger (4.37±1.6) than in biCEBPA patients (2.96±1.22) (P<0.05). The two groups also differed remarkably with regard to the genes that were mutated. In the moCEBPA group FLT3 (46%), NPM1 (46%), TET2 (37%) and DNMT3A (26%) were the most frequently mutated genes, whereas the biCEBPA group showed frequent mutations in TET2 (40%), GATA2 (36%) and FLT3 (18%). Thus there was a strong association of NPM1 (P<0.0001), FLT3 (P=0.01) and IDH2 (P=0.04) mutations with the moCEBPA group. GATA2 mutations were significantly associated with biCEBPA mutations (P=0.0003). NPM1 and biCEBPA mutations were mutually exclusive.

In this large and well characterized CEBPA-mutated patient cohort we identified distinct mutational landscapes in patients with moCEBPA and biCEBPA mutated CN-AML. The lower number of mutated genes within the biCEBPA group suggests that biallelic CEBPA mutations may act as a strong driver. In almost all cases, patients with biallelic mutations of CEBPA have a C-terminal mutation in one allele of CEBPA and an N-terminal mutation in the other allele. These date provide further insight into the genetic background of CEBPA mutated CN-AML. We are currently analyzing the prognostic impact of the associated mutations.

Disclosures

No relevant conflicts of interest to declare.
Outcome of Patients with Beta-Thalassemia Major Given Either Bone Marrow and Frozen Cord Blood of Same Sibling or Bone Marrow Transplantation from Sibling Donors
Authors: Wu Xuedong , Wen Jianyun , Fuyu Pei , Chen Libai , Liao Jianyun , He Yuelin , Li Qi , Ruan Yongsheng , Li Chunfu
Published: October 2019

Background: Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for thalassemia major (TM). Bone marrow (BM) and cord blood (CB) are biologically different stem cell sources.

Methods: We analyed the results of a retrospective study of HSCT in 29 chlidren (median age at transplantation was 6 years old) with β-TM after the combined infusion of G-CSF primed bone marrow (BM) and cord blood (CB) from the same transplantation to outcomes in children with β-TM who had received BM (n=26).Patients treated with bone marrow transplant (BMT)were closely matched to the co-transplant group in terms of age, human leucocyte antigen (HLA) matching and duration of follow-up.Compared to BMT group, the donors in co-transplant group were younger (median age 2 vs. 4 years old, p=0.015)

Results: In the co-transplant group,the mean total nucleated cells (TNC) was 2.63×108/kg(range,1.26-3.72×108/kg) and the CB was 0.39×108/kg(range,0.27-0.71×108/kg), respectively.The mean TNC (3.02 vs. 2.79×108/kg, p=0.532) and CD34+cells (7.55 vs. 6.94×106/kg, p=0.227) were insignificantly difference between the co-transplant group and BMT group. Of the 53 patients who had successful engraftment,patients who received a co-transplant had a lower incidence of ≥ grade II acute (3.3 vs. 20.8, p=0.047) and chronic(0vs.16.7%,p=0.022) graft versus host disease (GVHD) compared to BM transplant (BMT) recipients. There was no graft rejection (GR) after co-transplant, but GR happened two patients (7.7%) in BMT group(p=0.132).We found insignificant difference in neutrophils (18.7vs.19.9 days, p= 0.956) and platelet (24.7vs. 26.2 days, p=0.235) engraftment time between the co-transplant and BMT group. All patients were followed up until june 30, 2014, the 5 year probability of overall survival (OS), transplant free survival (TFS) and transplant-related mortality (TRM) were similar for the two groups. The 5-year probability of OS and TFS were 89.7% and 89.7% in the co-transplant group, 92.3%and 84.6% after BMT (P=0.740 and 0.573, respectively).

Conclusions: Our data suggest that the lower risk of GVHD is retained with co-transplant group. The incidence of GR lower in the co-transplant group, although a larger cohort of patients will be needed to confirm this inital obser-vation.Here,we suggest transplantation of G-CSF primed BM a,nd CB of same sibling appears to be a feasible and effective strategy to further optimize outcomes of HSCT for TM with decreasing the risk of the occurrence of GVHD.

Disclosures

No relevant conflicts of interest to declare.
Influence of Btk Inhibitors on Antitumor Activity of Natural Killer Cells
Authors: Siernicka Marta , Bojarczuk Kamil , Bobrowicz Malgorzata , Dwojak Michal , Pyrzynska Beata , Olive Daniel , Winiarska Magdalena
Published: October 2019

Small molecule Bruton's tyrosine kinase (Btk) inhibitors are extensively studied in preclinical investigations and clinical trials in the treatment of hematological malignancies derived from B-cells. Ibrutinib, due to its safety and efficiency, has been recently approved for the treatment of patients with Chronic Lymphocytic Leukemia (CLL) who received at least one prior therapy. Moreover, another Btk inhibitor AVL-292 is currently tested in clinical trials in patients with B Cell Non-Hodgkin's Lymphomas, CLL and Waldenstrom Macroglobulinemia. Despite a huge therapeutic potential of Btk inhibitors we have recently demonstrated that both natural killer (NK) cells cytotoxicity and degranulation are significantly impaired upon ibrutinib treatment (Bojarczuk et al., Leukemia 2014). Since NK cells are effectors of innate immune system capable to kill tumor cells directly and in the mechanism of antibody dependent cell-mediated cytotoxicity (ADCC), which constitutes one of the major mechanism of monoclonal antibodies widely used in hematooncology, in our ongoing studies we are focused to determine in details the influence of various Btk inhibitors on NK cells cytotoxicity, degranulation, cytokine secretion and expression of activatory/inhibitory receptors.

All experiments were performed fully in vitro using human primary NK cells isolated from PBMC of healthy donors as well as NK92 and NK92.CD16 cell lines.

To determine cytolytic activity of NK cells CFSE/PI flow cytometry assay was used. Cytokine secretion and NK cells degranulation, evaluated as the expression of CD107a on the surface of NK cells, were determined with flow cytometry upon incubation with target cells for 4 h. Phenotype of NK cells pre-incubated with tested compounds was determined with flow cytometry using antibodies conjugated with fluorochromes.

The initial results of our studies show that various Btk inhibitors differentially regulate NK cells antitumor activity. Ibrutinib and AVL-292 which covalently bind to cysteines at the position 481 significantly inhibit NK cells cytotoxicity. Interestingly, we have observed that pre-incubation of NK cells with ibrutinib as well as AVL-292 results in down-regulation of CD25 and CD69. This effect was not observed upon treatment of NK cells with CGI-1746, a reversible Btk inhibitor which blocks phosphorylation of BTK in Y551 and Y223. Expression of NK cells activatory receptors such as NKp30, NK44, NKp46, NKG2D, DNAM-1 and CD16 remained unchanged.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Reversible Tumor Suppression By Ikzf1/Ikaros in Mouse Models of BCR-ABL1+ B-ALL
Authors: Witkowski Matthew T , Hu Yifang , Cimmino Luisa , McKenzie Mark D , Liu Grace J , Smyth Gordon K , Dickins Ross A
Published: October 2019

Background: Loss-of-function mutations in the transcription factor IKZF1 (IKAROS) correlate with poor prognosis in B-progenitor acute lymphoblastic leukemia (B-ALL), and are particularly prevalent in the high-risk BCR-ABL1+ and BCR-ABL1-like disease subtypes. While recent studies using mouse models of Ikaros-deficient B-ALL have uncovered Ikaros-regulated genes, the mechanisms by which IKAROS loss promotes leukemogenesis and confers treatment resistance remain unclear.

Results: We have generated a novel transgenic mouse model that allows tet-regulated, shRNA-mediated Ikaros knockdown or restoration in normal lymphocytes and leukemias in vivo. Ikaros knockdown significantly decreases disease latency in mouse models of B-ALL driven by transgenic or retroviral expression of the BCR-ABL1 fusion oncogene, recapitulating a common genetic interaction in high-risk pediatric B-ALL. Remarkably, we find that restoring endogenous Ikaros expression in established BCR-ABL1+ ALL causes rapid disease regression and sustained remission despite ongoing expression of BCR-ABL1, indicating that disabled Ikaros remains a critical disease driver in this context. Using integrated in vivo RNA-seq analysis we have identified a novel set of genes that are (1) differentially expressed in Ikaros-low versus Ikaros-wildtype leukemias and (2) concordantly differentially expressed upon acute Ikaros restoration in established Ikaros-low leukemias. We are now performing in vitro and in vivo loss-of-function genetic screens to interrogate these high confidence Ikaros-regulated genes, focusing on potential roles for Ikaros-activated genes in tumor suppression and Ikaros-repressed genes in promoting BCR-ABL1+ ALL self-renewal.

Conclusions: Our results demonstrate that B-ALL driven by expression of BCR-ABL1 and Ikaros loss remains dependent on ongoing Ikaros suppression, suggesting that re-engaging or inhibiting critical components of the Ikaros-regulated gene expression program may provide new therapeutic avenues in this high-risk B-ALL subtype.

Disclosures

No relevant conflicts of interest to declare.
Secondary Primary Cancers in Nodular Lymphocyte Predominant Hodgkin’s Lymphoma
Authors: Shameem Raji , Hamid Muhammad Saad , Hossain Nasheed Mohammad , Shani Dana
Published: October 2019

Background: Nodular Lymphocyte Predominant Hodgkin’s Lymphoma (NLPHL) is an uncommon subtype of Hodgkin’s Lymphoma. The risk for secondary primary cancers is not well understood.

Methods: The Surveillance Epidemiology and End Results (SEER) database was used to identify cases of NLPHL diagnosed up to 12/31/2011. The Standardized Incidence Ratio (SIR) was calculated as the ratio of observed to expected cases of secondary primary malignancies based on incidence data in the general United States population. The latency exclusion period from the date of diagnosis was 60 months. We also investigated for possible modifying effects of gender, age at diagnosis (20-39 years of age and ≥ 40 years of age), use of radiation therapy, and latency periods (5-10 years and >10 years) after initial diagnosis of NLPHL.

Results: A total 947 cases of NLPHL were present in the database. Secondary primary cancer cases occurred in 64 cases with 36 (56.3%) solid tumors and 28 (43.8%) hematological malignancy cases. Radiation was administered in 38 (59.4%) of secondary primary cancer cases. Females had an increased risk of Non-Hodgkin’s Lymphoma (NHL) (SIR: 13.86, p<0.05), “All Solid Tumors” (SIR: 1.89, p<0.05), Colon and Rectum (SIR: 4.39, p<0.05), and Urinary System (SIR: 2.01, p<0.05) tumors. Comparatively, males had an increased risk of NHL (SIR: 26.02, p<0.05) exclusively. The risk of developing NHL (SIR: 60.23, p<0.05), and “All Solid Tumors” (SIR: 2.47, p<0.05) was increased in individuals between 20-39 years of age. Whereas, increased risk of Urinary System (SIR: 3.78, p<0.05), and NHL (SIR: 12.59, p<0.05) were observed in the ≥ 40 years of age cohort. NHL (SIR: 21.0, p<0.05), “All Solid Tumors” (SIR: 1.65, p<0.05), and Colon and Rectum (SIR: 3.68, p<0.05) tumor risk was increased in cases treated with radiation therapy, while NHL (SIR: 21.0, p<0.05) was the only significantly increased secondary malignancy in non-radiated cases. Latency period of >10 years, after the initial diagnosis, was associated with increased risk of “All Solid Tumors” (SIR: 2.62, p<0.05), Thyroid cancer (SIR: 16.64, p<0.05), and NHL (SIR: 35.68, p<0.05).

Conclusion: NLPHL is associated with an increased risk of secondary primary cancers, both solid and hematological malignancies. Thyroid cancer and NHL were the most frequent tumors observed in patients ten years after initial NLPHL diagnosis. Radiation treatment for NLPHL increased the risk for solid tumors, including colorectal cancer. Further research is necessary to clarify optimal surveillance technique in patients after treatment of NLPHL.

Disclosures

No relevant conflicts of interest to declare.
Second Cancer Incidence Among Chronic Lymphocytic Leukemia (CLL) Patients: A French Population-Based Study
Authors: Cornet Edouard , Jégu Jérémie , Mounier Morgane , Maynadié Marc , Monnereau Alain , Velten Michel , Troussard Xavier
Published: October 2019

Introduction. The risk of developing a second primary cancer (SPC) is increased in patients with CLL. The mechanisms explaining this association could be related to lifestyle, environment, host factors or interactions or other influences. We conducted an epidemiological study based on 10 French registries and evaluated the risk of developing SPC in patients with CLL.

Methods. Data from French population-based registries were used to establish a cohort of all patients diagnosed with a first cancer between 1989 and 2004 and followed-up until December 31, 2007. The person-year approach was used to estimate Standardized Incidence Ratios (SIRs) and Excess Absolute Risks (EARs) of metachronous SPC. Multivariate Poisson regression modules were then used to model SIRs and EARs separately by gender, adjusting for age, year of diagnosis, follow-up and first cancer site. All patients with CLL did not have HIV/AIDS-related disease.

Results. Among 288,967 patients, 21,226 patients (7.5%) developed SPC. Among 4,148 CLL patients (Male: 2,336, Female: 1,812, median age: 70 years), 479 patients (11.5%) developed SPC after a median time of 54 months (2-199). EARs of SPC are different between male and female. In male, localizations of SPC are lung, bronchus and trachea, colorectum, colon, stomach and Hodgkin's disease. Their respective EARs are shown in table 1 and no excess absolute risk was observed for 24 other localizations. In female, localizations of SPC are rectum, stomach and melanoma of skin. Their respective EARs are shown in table 2 and no excess absolute risk was observed for 21 other localizations.

Table 1: Excess Absolute Risks (EARs) of second primary cancer (SPC) for male Male SIR SIR IC95% EAR EAR IC95% Lung, bronchus and trachea 2.08 1.61 2.64 26.1 14.8 39.8 Colorectum 1.54 1.15 2.03 13.2 3.7 25.1 Colon 1.63 1.11 2.3 9.3 1.6 19.2 Stomach 1.91 1.07 3.15 5.4 0.4 12.7 Hodgkin's disease 8.71 2.34 22.29 2.7 0.5 7.4

Table 2: Excess Absolute Risks (EARs) of second primary cancer (SPC) for female Female SIR SIR IC95% EAR EAR IC95% Rectum 2.17 1.19 3.64 6.7 1.1 15.2 Stomach 3.04 1.45 5.58 6 1.3 13.4 Melanoma of skin 2.59 1.04 5.34 3.8 0.1 10.4

Conclusion. The risk for developing SPC after CLL is increased for male and female, especially solid cancers. Except Hodgkin's disease for male patients, there is no increase for other malignant hematological diseases, including acute leukemia and myelodysplastic syndrome. CLL survivors face a high risk of new malignancies and the excess risk of SPC increases with follow-up. As the SPC risk is closely tied to patient's characteristics, a personalized surveillance is required to allow optimal SPC prevention and early detection strategies.

Disclosures

No relevant conflicts of interest to declare.
Combined Inhibition of Wee1 and Poly(ADP Ribose) Polymerase1/2 Synergistically Inhibits Acute Myeloid Leukemia Cells By Enhancing DNA Damage and the Induction of Apoptosis
Authors: Snedeker Jonathan C , Burleson Tamara M , Tibes Raoul , Porter Christopher C.
Published: October 2019

Introduction: Successful treatment of AML remains dependent upon cytotoxic chemotherapy. However, traditional regimens are not well tolerated by older patients who are at highest risk of disease, and salvage rates after relapse are low, necessitating novel therapeutic strategies. Our groups identified Wee1 as a potential therapeutic target in AML, particularly in the context of concomitant treatment with cytarabine (Tibes et al, Blood, 2012; Porter et al, Leukemia, 2012). Wee1 inhibits CDK1&2 via phosphorylation thereby stalling cell cycle progression. One consequence of Wee1 inhibition/CDK1 activation is impairment of DNA repair via homologous recombination (Krajewska et al, Oncogene, 2013). Cells in which HR is impaired are dependent upon Parp1/2 function, and HR deficient cells are particularly sensitive to Parp1/2 inhibition. Therefore, we hypothesized that combined Wee1 and Parp1/2 inhibition may result in greater inhibition of AML cell proliferation and survival than either alone.

Methods: Human AML cell lines, MV4-11 and Molm-13, and a mouse AML that expresses MLL-ENL/FLT3-ITD were cultured with various concentrations of a Wee1 inhibitor (AZ1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry. In some experiments, cells were split into fresh media to recover for 72 more hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots were used to confirm inhibition of CDK1/2 phosphorylation and to measure DNA damage induction (gamma-H2AX).

Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 3 cell lines tested, with combination index values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Western blots for gamma-H2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone.

Discussion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML cell proliferation, DNA damage and apoptosis than either drug alone. Future studies will include experiments with primary patient samples, as well as in vivo trials combining Wee1 inhibition with Parp1/2 inhibition. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in those for whom conventional chemotherapeutics may not be well tolerated.

Disclosures

No relevant conflicts of interest to declare.
The F-BAR Protein PACSIN2 Regulates Platelet Intracellular Membrane Architecture and in Vivo Hemostatic Functions
Authors: Jönsson Terese , Pluthero Fred G , Jurak Begonja Antonija , Kormann Jan , Demers Mélanie , Wagner Denisa D , Plomann Markus , Hartwig John H , Kahr Walter H , Falet Hervé
Published: October 2019

Proteins of the Bin-amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) families bind and deform membranes, promoting in cells tubular invaginations reminiscent of the platelet open canalicular system (OCS) and megakaryocyte (MK) demarcation membrane system (DMS). Here we investigated the role of the F-BAR protein PACSIN2 in platelets and MKs. Spinning disk laser fluorescence confocal microscopy showed that PACSIN2 co-localized with the von Willebrand factor receptor subunit GPIbα at the openings of the OCS in platelets. Endogenous and over-expressed PACSIN2 associated with anastomosing tubular structures reminiscent of the pre-DMS in mouse bone marrow MKs. Pacsin2-null mice had mild thrombocytopenia and polycythemia, MK hyperplasia and splenomegaly. Pacsin2-null mice had a bleeding disorder, as evidenced by significantly prolonged tail bleeding and delayed in vivo thrombus formation following FeCl3 vascular injury, which was normalized by injection of WT platelets. However, Pacsin2-null platelets expressed P-selectin and activated the integrin αIIbβ3 normally in response to stimulation with thrombin and the GPVI-specific collagen-related peptide. In contrast, Pacsin2-null platelets had abnormal flattened morphology, with more and narrower OCS channels on their surface, compared to WT platelets. Together, the data indicate that the F-BAR protein PACSIN2 orchestrates the intracellular membrane architecture of platelets, thereby regulating their in vivo hemostatic functions.

Disclosures

No relevant conflicts of interest to declare.
TMEM16F Deficiency Causes a Strain-Dependent Lethality in Mice and Reduced Phosphatidylserine Exposure on Heterozygous As Well As Homozygous Deficient Mouse Platelets
Authors: Kretz Colin A , Novakovic Valerie A , Kiseleva Anna , Cleuren Audrey C.A. , Khoriaty Rami , Adams Elizabeth , Ginsburg David , Gilbert Gary E.
Published: October 2019

Scott Syndrome is a rare bleeding disorder characterized by a defect in platelet phosphatidylserine (PS) exposure. The syndrome has recently been linked to mutations in TMEM16F, a Ca++-activated ion channel. Tmem16f-/- mice were recently reported to be viable with a prolonged tail snip bleeding time but no spontaneous bleeding (Yang et al, Cell 151: 111-122; 2012). We now report analysis of an additional gene targeted Tmem16f allele generated in C57BL/6 ES cells.

JM8 ES cell were obtained from EUCOMM, and successful Tmem16f gene targeting in intron 1 was confirmed by PCR and sequencing. Genotyping of 120 Tmem16f+/gt (+/gt) intercross progeny identified no surviving Tmem16fgt/gt (gt/gt) mice at weaning (p<0.001). However, +/gt intercrosses generated the expected Mendelian genotype ratios at both E10.5 and E17.5, with gt/gt embryo’s exhibiting no morphological abnormalities on gross or routine histologic examination. Though complete deficiency of TMEM16F is lethal in the C57BL/6J genetic background between E17.5 and birth, an F2 intercross of +/gt mice outcrossed one generation to 129x1SvJ resulted in gt/gt mice surviving to weaning, though at reduced numbers (6/75 total progeny compared to ~19 expected, p <0.002). Progeny testing of surviving gt/gt mice suggest a single autosomal dominant 129x1SvJ-associated genetic modifier. Preliminary genetic analysis of these mice appears to map this locus to the proximal region of chromosome 3.

Tail bleeding times for gt/gt were >10min, whereas littermate +/gt and +/+ mice bleeding ceased at 8 ± 1 min and 6 ± 0.8 min, respectively, each significantly different than gt/gt (p<0.05). Notably, platelets from +/gt mice exhibited a trend toward reduced PS exposure, detected with FITC-labelled lactadherin, in response to PAR4 agonist peptide, whereas gt/gt mice had significantly reduced PS exposure (p < 0.05). gt/gt platelets showed a trend toward reduced PS exposure in response to A23187, as well as prolonged platelet rich plasma clotting times, and less efficient lactadherin inhibition of platelet clotting time.

These data suggest the existence of a viability-determining genetic modifier of TMEM16F in the 129x1SvJ mouse strain. Identification of the responsible gene may uncover novel functions for TMEM16F and the regulation of hemostatic function. The observation of a PS exposure and hemostatic phenotype in +/gt mice also suggests the possibility that heterozygous TMEM16F mutations may subtly influence hemostasis or thrombosis in humans.

Disclosures

No relevant conflicts of interest to declare.
Platelet-Derived Proteases ADAM10 and ADAM17 Impair NK Cell Immunosurveillance of Metastasizing Tumor Cells By Diminishing NKG2D Ligand Surface Expression
Authors: Raab Stefanie , Kropp Korbinian Nepomuk , Steinle Alexander , Klein Gerd , Kanz Lothar , Kopp Hans-Georg , Salih Helmut R
Published: October 2019

Introduction: NK cells are cytotoxic lymphocytes the reactivity of which is governed by the principles of ‘missing-self’ and “induced-self’ recognition. This implies that cells with low or absent expression of MHC class I and/or stress-induced expression of ligands for activating receptors like NKG2D (NKG2DL) are preferentially eliminated by NK cells. We and others reported previously that NKG2DL undergo proteolytic cleavage (i.e. shedding) which promotes evasion from NKG2D-mediated tumor immunosurveillance. Notably, the reactivity of NK cells is also influenced by various other components of the hematopoietic system, and we and others provided evidence for the involvement of platelets in tumor immune evasion including impairment of NKG2D-mediated immune surveillance (e.g., Kopp et al., Cancer Res 2009, Raab et al., Blood 2013 122:3488). Here we extend our recent findings and provide further data how platelets affect immunostimulatory NKG2D-NKG2DL interaction. Methods: Tumor cells were incubated with platelets from healthy donors or treated with platelet-derived soluble factors (releasate) obtained by stimulation of platelets with known platelet agonists including ADP and thrombin. NKG2DL and ADAM10 as well as ADAM17 surface expression on tumor cells and platelets, respectively, was measured by FACS, while release of NKG2DL was determined by ELISA. ADAM10 and 17 protein levels were assessed by immunoblotting. NK cell lysis of tumor cells in the presence or absence of coating platelets or platelet releasate was determined by chromium release assays. Results: We found that interaction of platelets with tumor cells resulted in substantially reduced NKG2DL expression on the surface of the malignant cells, which was paralleled by enhanced release of soluble NKG2DL. Similar albeit weaker effects were observed upon treatment of tumor cells with platelet releasate, indicating that platelet-derived factors mediate NKG2DL shedding from the tumor cell surface. Notably, ADAM10 and ADAM17, the known sheddases of NKG2DL, were found to be expressed on the platelet surface, and sheddases could also be detected in platelet releasate, pointing to an involvement in platelet-mediated NKG2DL shedding. Diminished NKG2DL surface expression resulted in reduced NKG2D-mediated NK cell cytotoxicity as revealed by blocking experiments using NKG2D antibody and F(ab)2 fragments specific for the modulated NKG2DL. Conclusion: We propose that induction of NKG2DL shedding constitutes a novel mechanism by which the interaction of platelets with metastasizing tumor cells impairs NK cell immunosurveillance.

Disclosures

No relevant conflicts of interest to declare.
uPAR Directly Interacts with LDLR-like Proteins to Induce Angiogenic Endothelial Cell Behavior
Authors: Pausz Clemens , Mawas Rula , Unseld Matthias , Chilla Anastasia , Novotny René , Prager Gerald W
Published: October 2019

In this study we characterized a conserved motif of domain 3 of the urokinase-type plasminogen activator receptor (uPAR) to directly interact with low-density lipoprotein receptor (LDLR)-related protein (LRP) family proteins, thereby affecting endothelial cell motility and angiogenesis in vitro and in vivo.

There is increasing evidence that uPAR plays a central role in growth factor induced endothelial cell activation. Beside its proteolytic role, urokinase-type plasminogen activator (uPA) / uPAR-complex formation induces intracellular signal transduction, which leads to endothelial cell migration and invasion. Since uPAR is a GPI-anchored protein, an interaction with transmembrane proteins - such as members of the LDL-receptor family - is required, inducing signal transduction but also regulating distribution of uPAR via its internalization and recycling to the leading edge. Recently, a direct interaction between uPAR and LRP-family members has been suggested to be sufficient to mediate internalization of uPAR-complex. A crystal structure analysis revealed a small sequence of domain 3 (D3) of uPAR, to be highly exposed upon uPA binding to its receptor.

Applying affinity chromatography analysis as well as mutation expression studies, we identified the sequence as an LRP-binding motif, which affects endothelial cell spreading, migration and invasion upon VEGF in vivo as well as in vitro.

In detail, matrigel-filled angioreactors with embedded retroviral constructs, carrying wild-type or modified uPAR genes, were implanted subcutaneously into uPAR deficient C57BL/6 mice. After explantation, blood vessel in-growth analysis revealed that only angioreactors with reconstituted wild-type uPAR but not reactors with modified uPAR, being deficient in LDLR interaction, showed angiogenesis.

To test a therapeutic impact, peptides mimicking the binding motif and competitive for LDLR binding were used. We found that in a dose dependent manner the peptides did not only block uPAR/LDLR-like protein interaction, but were also capable of blocking VEGF-induced endothelial cell migration in vitro.

In summary, our data show that a conserved motif of uPAR domain 3 is capable to interact with LDLR-like proteins, which is required for efficient growth-factor induced endothelial cell behavior. Preliminary functional data suggest that this extracellular motif might be a potential therapeutic target in angiogenesis dependent diseases such as cancer.

Disclosures

No relevant conflicts of interest to declare.
Bradykinin Receptors Regulate Proliferation, Migration and Tube Formation of Endothelial Progenitor Cells
Authors: Wu Yi , Backdash Nadia , Colman Robert W.
Published: October 2019

Bradykinin is a potent activator of endothelial cells and is known to induce vasodilation, vascular permeability, and nitric oxide production. Bradykinin signaling is initiated by binding to G protein-coupled bradykinin receptors 1 and 2 (B1R and B2R). In this study we investigated whether these two receptors are involved in regulation of endothelial progenitor cell (EPC) biology. First we isolated CD34+ cells from wild-type (WT) and double B1R and B2R knockout (B1RB2R-/-) mice, and compared their capacities of proliferation, migration, as well as tube formation in a 3D cell culture system. Bone marrow-derived B1RB2R-/- EPCs displayed a reduction in proliferation and migration through collagen-coated transwell, compared with EPCs from WT mice. Moreover, implanted B1RB2R-/- EPCs into immunocompromised NOD-SCID mice showed significantly less vasculogenesis in matrigel plug. Human EPCs were isolated from peripheral blood of healthy donors using CD34+ selection. In a concentration-dependent manner, bardykinin stimulated proliferation, migration and tube formation of human EPCs. In contrast to human EPCs, human umbilicial vein endothelial cells (HUVECs) did not display a significant increase in proliferation, migration, and tube formation upon bradykinin stimulation, suggesting that bradykinin selectively enhances the function of EPCs, but not mature endothelial cells. Pre-incubation with a B2R selective antagonist (Icatibant) reduced these functions of human EPCs. When human EPCs were stimulated with [des-Arg9]-BK, a selective B1R agonist, their proliferation and tube formation remained unchanged. Moreover, preincubation of human EPCs with [des-Arg10]-Icatibant, a B1R selective antagonist, did not affect their proliferation, migration and tube formation. As detected by Western blot and real time RT-PCR, B2R was constitutively expressed in EPCs, while the expression of B1R was only induced when stimulated by inflammatory stimuli, such as TNFα and IL-1β. Take together, bradykinin regulates proliferation, migration and tube formation of EPCs through constitutively expressed B2R, whereas B1R is probably involved in inflammatory settings.

Disclosures

No relevant conflicts of interest to declare.