bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.