scholarly journals TFL Deletion Induces Incredible CXCL13 Secretion and Cachexia in Vavp-Bcl2Transgenic Mice

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3908-3908
Author(s):  
Kentaro Minagawa ◽  
Kanako Wakahashi ◽  
Fukui Chie ◽  
Shinichiro Nishikawa ◽  
Noboru Asada ◽  
...  
Keyword(s):  
Weight Loss ◽  
Body Weight ◽  
T Cell ◽  
B Cell ◽  
Mrna Expression ◽  
Cell Lines ◽  
Cell Lineage ◽  
Body Weight Loss ◽  
Wild Type ◽  
B Cell Lymphomas

Abstract Diffuse large B-cell lymphomas (DLBCL) are heterogeneous diseases caused by several genetic aberrations. The novel post-transcriptional regulator gene called transformed follicular lymphoma (TFL) was first identified from t(2;6)(p12;q23), which appeared during the transformation of FL to DLBCL (Minagawa et al. Br J Haematol 2007). Normal human lymphocytes generally express TFL, but it is defective in some leukemia/lymphoma cell lines. TFL overexpression in such cell lines inhibited cell growth, suggesting that TFL functions as a tumor suppressor (Minagawa et al. Mol Cancer Res 2009). TFL locates in mRNA processing body in the cytoplasm and has the unique CCCH-type zinc finger motif functioning as RNase. TFL regulates several cytokines, including IL-2, IL-6, IL-10, TNF-α, and IL-17a, via mRNA degradation. In an experimental autoimmune encephalitis model, TFL null mice (TFL-/-) demonstrated persistent paralysis, resulting from more infiltration of Th17 cells into CNS with markedly increased IL-17a mRNA levels. Therefore, a TFL-driven feedback mechanism for excessive inflammation is indispensable to suppress T-cell-mediated autoimmune diseases (Minagawa et al. J Immunol 2014). TFL deletion examined by FISH using a 110kbp DNA probe containing an entireTFL locus was found in 12.8% of mature B-cell lymphomas (n=86, FL=30, DLBCL=40). However, the pathological significance of TFL deletion has not yet been clarified. To investigate how TFL loss affects lymphoma biology, we developed VavP-bcl2 transgenic (Bcl2-Tg)/TFL-/-mice. Although the survival of TFL-/- was comparable to the wild-type, Bcl2-Tg/TFL-/- died about 19 weeks earlier than Bcl2-Tg (Fig. 1). Both strains developed lymphadenopathy and splenomegaly similarly. No different microscopic finding was noted in lymph nodes, spleen, or bone marrow (BM). No additional malignancy was found in Bcl2-Tg/TFL-/- on autopsy. However, significant body weight loss appeared by 30 weeks in Bcl2-Tg/TFL-/- but not in Bcl2-Tg (Fig. 3). To identify what causes earlier death in Bcl2-Tg/TFL-/-, we carefully examined the phenotypic change of BM lymphocytes. We found a unique B220-IgM+ population in Bcl2-Tg BM, which was not found in wild-type. We speculated that TFL deficiency in this population might drive the deterioration in Bcl2-Tg/TFL-/-. To identify which mRNA was dysregulated by TFL deficiency, we comprehensively analyzed mRNA expression profiles in B220-IgM+ cells in both strains using cDNA microarray chip. Among several genes upregulated at least threefold in Bcl2-Tg/TFL-/- than Bcl2-Tg, we paid attention to CXCL13, the mRNA expression of which in Bcl2-Tg/TFL-/- was 4.19-fold higher than that in Bcl2-Tg (p=0.03). In fact, CXCL13 concentration in BM extracellular fluid as well as plasma in Bcl2-Tg/TFL-/- showed incredible increase in a logarithmic scale (Fig. 2). As a noteworthy event, body weight loss in Bcl2-Tg/TFL-/- followed the increase of CXCL13 in plasma by 30 weeks (Fig. 3). To confirm that TFL post-transcriptionally regulates CXCL13 mRNA through the degradation of its 3′UTR, we performed a reporter assay with a plasmid vector containing 3′UTR of CXCL13 mRNA. Co-transfection with a TFL expression vector showed decreased luciferase activity compared to the control. This suggests that TFL directly regulates CXCL13 mRNA via its 3′UTR degradation. This regulation occurs more prominently in B-cell lineage rather than myeloid or T-cell lineage, whereas IL-2 mRNA regulation occurs promiscuously. CXCL13 secretion was significantly increased in the culture supernatant of BM cells but not spleen cells derived from Bcl2-Tg/TFL-/-. We further sorted several cell populations, including B220-IgM+ in BM, and cultured them for 96 h. CXCL13 secretion from B220-IgM+ population was increased significantly compared to other populations. Thus, we concluded that B220-IgM+ cells in BM are the main producer of CXCL13 in Bcl2-Tg/TFL-/-. Loss of TFL-driven attenuation for excessive inflammation in lymphoma-bearing mice could contribute to the short survival. It is of interest whether high plasma CXCL13 directly affects cachexia and early death in Bcl2-Tg/TFL-/-. TFL deletion in human lymphoma might contribute not only to malignant transformation but also to a major B symptom, i.e., weight loss. Our findings may open a new window for the predictive factor on the prognosis of B-cell lymphoma and/or new therapeutic intervention by targeting CXCL13. Disclosures No relevant conflicts of interest to declare.

scholarly journals The bcl-2 gene is rearranged in many diffuse B-cell lymphomas

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 969-972 ◽  
Author(s):  
AC Aisenberg ◽  
BM Wilkes ◽  
JO Jacobson
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lineage ◽  
Large Cell ◽  
Chromosome Translocation ◽  
Lymphocytic Leukemia ◽  
Chain Gene ◽  
B Cell Lymphomas ◽  
Follicular Lymphomas ◽  
Center Cell

Abstract Southern blotting was used to detect rearrangement of the bcl-2 gene in 104 cases of non-Hodgkin's lymphoma subclassified by the Working Formulation, 24 cases of B cell chronic lymphocytic leukemia (B-CLL) and 14 cases of T cell malignancy. Earlier workers reported rearrangement of this gene (located on chromosome 18) in a major fraction of follicular lymphomas, lymphomas in which a 14;18 chromosome translocation is frequently observed. In the present study, bcl-2 was rearranged in 30% (11 of 37) of follicular lymphomas and 19% (11 of 58) of diffuse lymphomas of follicle center cell lineage. In 18 of 19 samples studied, the rearranged bcl-2 fragment also hybridized with a probe for the joining region of the immunoglobulin heavy chain gene located on chromosome 14, indicating a 14;18 translocation. In lymphomas not derived from follicle center cells, ie, diffuse lymphomas of small B lymphocytes, B-CLL and T cell neoplasms, the bcl-2 gene was always in germline configuration. The frequent rearrangement of bcl-2 in a variety of B cell lymphomas of diffuse morphology (small cleaved cell, large cell, small noncleaved cell and immunoblastic) is noteworthy.

573 A novel human anti-PD1/IL15 bi-functional protein with robust anti-tumor activity and low systemic toxicity

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A607-A607
Author(s):  
Dan Lu ◽  
Zhanna Polonskaya ◽  
Tzu-Pei Chang ◽  
Stella Martomo ◽  
Xenia Luna ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Weight Loss ◽  
Body Weight ◽  
Fusion Protein ◽  
Body Weight Loss ◽  
Systemic Toxicity ◽  
Wild Type ◽  
Functional Protein ◽  
Bifunctional Protein ◽  
Anti Tumor Activity

BackgroundIL-15 is a key cytokine promoting CD8+ T, NK, and NKT cell proliferation and has demonstrated clinical activity in cancer patients without evidence of any Treg stimulation.1 2 However, its short half-life and systemic toxicity limit its clinical utility. Kadmon has established an IL-15 fusion protein platform to extend the IL-15 serum half-life and direct its action to the tumor microenvironment.3 A major asset of this platform is anti-PD1/IL15 bifunctional protein. To test the bifunctionality hypothesis of this fusion protein in murine models, four different formats of the surrogate bi-functional proteins were engineered by fusing mouse IL-15 to a mouse-human chimeric anti-mouse PD1 antibody (m3A7). We presented earlier that the single IL-15 N-terminal fusion to anti-PD1 antibody (1N-IL-15/m3A7) showed significantly stronger anti-tumor activity in vivo mainly due to the cis-presentation to the PD1 and IL2Rβγ co-expressed on TILs. The cis-presentation potentially maximizes the bi-functionality of PD1 blockade and IL-15 stimulation.4 Here, the therapeutic single IL-15 N-terminal fusion antibodies containing a novel human PD1 antagonist antibody (38B2) and either wild-type IL15 (1N-IL-15/38B2) or mutated 65S-IL15 (65S-1N-IL-15/38B2) have been constructed; their anti-PD1 functions, IL15 stimulation and anti-tumor activities were evaluated.MethodsPurified 1N-IL-15/38B2 and 65S-1N-IL-15/38B2 were generated and characterized in vitro.4 The anti-tumor activities were examined in the human-PD-1/PD-L1 transgenic BALB/c mice subcutaneously transplanted with the human-PD-L1 positive CT26 colon carcinoma. The treatment was started when tumors reached 100 mm3 (IP, QW).ResultsAll 1N-IL-15/anti-PD1 fusions showed similar potencies in binding to the soluble and cell expressed human PD1 and blocking the hPDL-1 binding to hPD1. Comparing to wild-type 1N-IL-15/38B2, mutated 65S-1N-IL-15/38B2 showed lower stimulation (>150 folds) in the M07e, CTLL2, mouse spleen cells and hPBMC (mainly CD8T+ T cell) proliferation. When we treated hPDL1-CT26 tumor transplanted hPDL1-hPD1 transgenic mice with 65S-1N-IL-15/38B2 at 6 mg/kg, 80% of tumor growth inhibition (TGI) was achieved with no body-weight loss. Although wild-type 1N-IL-15/38B2 at 3 mg/kg demonstrated similar efficacy, a significant mouse body-weight loss was observed and 1/3 mice died after second injection. No anti-tumor activity was observed for 65S-1N-IL-15 non-target fusion in 6 mg/kg.ConclusionsThe previous observation of robust anti-tumor activity of surrogate 1N-IL-15/m3A7 in PD1 resistant LL2 model was replicated with the therapeutic bifunctional protein in this study. We also found that lower stimulation 65S-1N-IL-15/38B2 showed strong anti-tumor activity with significant low systemic toxicity; suggesting that the 65S mutation increased the therapeutic window of this bi-functional proteinReferencesGoldrath, AW etc. Cytokine requirements for acute and basal homeostatic proliferation of naive and memory CD8+ T cells. J Exp Med 2002; 195:1515–1522.Waldmann TA. Cytokines in Cancer Immunotherapy. Cold Spring Harb Perspect Biol 2018;103. Martomo, S. etc. ESMO 2019 1221P; SITC 2019 P#4854. Polonskaya Z. etc. AACR 2020 #2263

A Meta-Analysis Of Hodgkin Lymphoma Reveals 19p13.3 (TCF3) As a Novel Susceptibility Loc

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 626-626
Author(s):  
Wendy Cozen ◽  
Dalin Li ◽  
Maria Timofeeva ◽  
Arjan Diepstra ◽  
Dennis Hazelett ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Meta Analysis ◽  
Cell Lineage ◽  
Cell Phenotype ◽  
Open Chromatin ◽  
Expression Levels ◽  
Nodular Sclerosis

Abstract Background Recent genome wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified several associations at both HLA and non-HLA loci. However, much of HL heritability remains unexplained. Methods To identify novel risk loci, we performed a meta-analysis of 3 HL GWAS including a total of 1,810 cases and 7,879 controls. Results were replicated in an independent set of 1,163 cases and 2,580 controls, for a total of 3,097 and 11,097 cases and controls combined, respectively. participants in discovery and replication stages were of European descent. quality control and imputation we conducted a meta-analysis addressing 1,004,829 variants (λ= 1.10, λ1000= 1.03). Associations between SNP genotypes and HL risk were evaluated under a log-additive model of inheritance adjusting for sex, study center and significant principal components to control for population stratification. We performed an analysis with all HL cases and then conducted stratified analyses by histological subtype (classical, nodular sclerosis and mixed cellularity), age at diagnosis (nodular sclerosis among those diagnosed at 15- 35 years in all studies, and those diagnosed at 35 and older years in the European Study only) and EBV tumor status (negative and positive). We then used a bioinformatic approach (FunciSNP) to identify potential functional variants associated with HD risk correlated with risk loci of interest. We extracted publically available ENCODE data on biofeatures to identify potential functional motifs associated with the index SNP or correlated SNPs. Finally, we measured expression levels of the two alternative mRNA transcripts in lymphoblastoid cell lines (LCLs) from 49 post-therapy HL patients and 25 unaffected controls. RT-PCR was carried out in triplicate. Relative expression levels were calculated relative to TBP as housekeeping gene. Linear models were used to assess correlation between genotype and TCF3expression levels. Results The meta-analysis identified a novel susceptibility variant at chromosome 19p13.3 (rs1860661) associated with HL risk (Odds Ratio [OR]= 0.78, P=2.0*10-8, I2=0%). variant is located in intron 2 of TCF3 (also known as E2A), a regulator of B- and T-cell lineage commitment. was also significantly associated with HL (OR= 0.85, P=0.002) in the replication series of 1,281 cases and 3,218 controls. the combined analysis consisting of the discovery and replication sets, rs1860661was strongly associated with HL (OR=0.81,=3.5*10-10), with no evidence of heterogeneity between contributing studies (Phom=0.41, I2=0%). The number of G alleles defined by rs1860661 was significantly associated with a reduced risk of each HL subtype except EBV positive HL. rs1860661 and two correlated SNPs, rs10413888 (r2=0.90) and rs8103453 (r2=0.89) identified by FunciSNP analysis map in or near marks of open chromatin and in DNAse hypersensitivity sites in TCF3 in CD20+ B cell lines., the protective minor alleles of these SNPs as defined by the G-G-G haplotype map to the binding sites for ZBTB7a (rs10413888 and rs1860661) and (rs8103453) transcription factors, likely improving the binding efficiency to the sites which may result in increased transcription rates of TCF3. TCF3 is encoded by two alternative transcripts (E12 and E47). Higher expression levels of TCF3-E47, whose transcription start site is located close to rs1860661, was associated with the rs1860661-G allele in controls (P=0.02), but not in HL patients (P=0.22). Conclusion/Discussion TCF3 is essential for the commitment of lymphoid progenitors to both B-cell and T-cell lineage development. A molecular and phenotypic hallmark of classical HL is the loss of the B-cell phenotype in HRS cells, including lack of demonstrable B-cell receptor and most B-cell specific markers such as CD19 or CD20. HRS cells have a low level of TCF3, particularly homodimers of the isoform E47, due to expression of the ABF-1 and ID2 inhibitors that bind to TCF3. Thus, higher TCF3 levels in HRS precursor cells may lead to enhanced retention of the B cell phenotype, thereby conferring a protective effect. These data suggest a link between the 19p13.3 locus including TCF3 and HL risk, indicating that TCF3 could be relevant to HL etiology and pathogenesis. Disclosures: Link: Genentech: Consultancy; Millenium: Consultancy; Pharmacyclics: Consultancy; Spectrum: Consultancy.

scholarly journals P088 TLR3 ameliorates colitis-associated colon tumourigenesis in mice

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S179-S179
Author(s):  
Y K Jun ◽  
S J Koh ◽  
H W Gang ◽  
K Y Chung ◽  
J M Han ◽  
...  
Keyword(s):  
Weight Loss ◽  
Body Weight ◽  
Experimental Model ◽  
Free Water ◽  
Body Weight Loss ◽  
Poly I:C ◽  
Wild Type ◽  
Tumour Development ◽  
Iκb Kinase ◽  
Significant Difference

Abstract Background Toll-like receptor 3 (TLR3) signalling regulates innate and adaptive immune systems by the recognition of dsRNA. Activation of TLR3 signalling by poly(I:C) attenuates dextran sodium sulphate (DSS)-induced murine colitis. However, little information is available on the role of TLR3 signalling in the development of colitis-associated colon tumourigenesis. Methods Wild-type (WT) and TLR3-deficient (TLR3−/−) mice were intraperitoneally injected azoxymethane (AOM) 12.5 mg/kg on day 0 followed by three cycles of 2% DSS for 5 days and 2 weeks of free water consumption. Clinical indices such as weight change, colon length, the number of tumours, and the histologic severity of colitis were evaluated in each experiment. Immunohistochemical or immunofluorescence analyses for phospho-IκB kinase (IKK) and β-catenin were performed in colon tissues. To elucidate the antitumourigenic mechanism by colon inflammation, poly(I:C) or PBS was intraperitoneally injected in the AOM/DSS-induced tumourigenesis model in WT mice. To evaluate direct antitumor effect on tumourigenesis, as first experimental model, both WT and TLR3−/− mice were intraperitoneally injected AOM weekly for 12 weeks without DSS treatment. As the second experimental model, WT and TLR3−/− mice were received 2% DSS mixed with drinking water three times for 5 days every 2 weeks after one intraperitoneal AOM injection. Results TLR3−/− mice exhibited a higher tumour burden compared with wild-type mice. Body weight loss was greater in TLR3−/− mice than in WT mice. However, here was no significant difference in colon length and the severity of colitis between the two groups. Immunoreactivity for β-catenin was markedly increased in TLR3−/− mice. However, there was no difference in IKK expression. Activation of TLR3 by poly(I:C) was not associated with the reduced tumour development in WT mice. However, repeated AOM injections without DSS resulted in greater body weight loss in TLR3−/− mice than in WT mice, which was associated with the increased tumour development in TLR3−/−mice. Conclusion TLR3 signalling attenuated colitis-associated colon cancer development. Based on our experiments, TLR3 signalling inhibits colon tumourigenesis by direct antitumor activity rather than anti-inflammatory effect of colitis.

scholarly journals The bcl-2 gene is rearranged in many diffuse B-cell lymphomas

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 969-972 ◽  
Author(s):  
AC Aisenberg ◽  
BM Wilkes ◽  
JO Jacobson
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lineage ◽  
Large Cell ◽  
Chromosome Translocation ◽  
Lymphocytic Leukemia ◽  
Chain Gene ◽  
B Cell Lymphomas ◽  
Follicular Lymphomas ◽  
Center Cell

Southern blotting was used to detect rearrangement of the bcl-2 gene in 104 cases of non-Hodgkin's lymphoma subclassified by the Working Formulation, 24 cases of B cell chronic lymphocytic leukemia (B-CLL) and 14 cases of T cell malignancy. Earlier workers reported rearrangement of this gene (located on chromosome 18) in a major fraction of follicular lymphomas, lymphomas in which a 14;18 chromosome translocation is frequently observed. In the present study, bcl-2 was rearranged in 30% (11 of 37) of follicular lymphomas and 19% (11 of 58) of diffuse lymphomas of follicle center cell lineage. In 18 of 19 samples studied, the rearranged bcl-2 fragment also hybridized with a probe for the joining region of the immunoglobulin heavy chain gene located on chromosome 14, indicating a 14;18 translocation. In lymphomas not derived from follicle center cells, ie, diffuse lymphomas of small B lymphocytes, B-CLL and T cell neoplasms, the bcl-2 gene was always in germline configuration. The frequent rearrangement of bcl-2 in a variety of B cell lymphomas of diffuse morphology (small cleaved cell, large cell, small noncleaved cell and immunoblastic) is noteworthy.

scholarly journals A Novel HCK Inhibitor Kin-8193 Blocks BTK Activity in BTKCys481 Mutated Ibrutinib Resistant B-Cell Lymphomas Driven By Mutated MYD88

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 40-40
Author(s):  
Guang Yang ◽  
Jinhua Wang ◽  
Li Tan ◽  
Xia Liu ◽  
Manit Munshi ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Target Engagement ◽  
Wild Type ◽  
B Cell Lymphomas ◽  
Oral Dosing ◽  
Activating Mutations ◽  
Ibrutinib Resistance

Abstract Activating mutations in MYD88 alone or in coordination with BCR activating mutations transactivate Bruton's tyrosine kinase (BTK) in WM and ABC DLBCL cells (Yang et al, Blood 2013; Wilson WH et al, Nat Med 2015; Phelan JD, Nature 2018). Ibrutinib is a covalent inhibitor that binds to BTKCys481 and is active in MYD88 mutated WM and ABC-DLBCL (Treon et al, NEJM 2015; Wilson et al, Nat Med, 2015). Acquired resistance to ibrutinib is increasingly being recognized in WM, as well as other B-cell malignancies due to somatic mutations at BTKCys481 that abrogate BTK-ibrutinib binding (Xu et al, Blood 2017). BTKCys481 mutations are usually sub-clonal, but can drive ibrutinib resistance and protect BTK wild-type clones through a paracrine mechanism involving activation of ERK1/2 (Chen et al, Blood 2018). Hematopoietic cell kinase (HCK) which is aberrantly upregulated and transactivated by mutated MYD88, and triggers multiple pro-survival signaling cascades that include activation of BTK, as well as PI3K/AKT and MAPK/ERK1/2 (Yang G et al, Blood 2016). We therefore examined if inhibition of HCK could abrogate BTK activity and overcome ibrutinib resistance driven by BTKCys481 mutations in MYD88 mutated B-cell lymphomas. We performed a screen of 220 clinical and preclinical kinase inhibitors to identify compounds with potent HCK inhibition. Over 100 analogs of three series of promising compounds with HCK activity were synthesized and triaged. Target deconvolution was performed to clarify selectivity, and other important kinase targets. These efforts led to the selection of a lead compound identified as a type-II kinase inhibitor, KIN-8193, with a molecular weight around 600. Single-digit nanomolar (nM) biochemical and double-digit nM cellular potency was demonstrated, with high selectivity (S-score 0.07) in line with that observed with ibrutinib (S-score of 0.03). Live-cell target engagement for HCK was confirmed by competitive ATP-biotin binding assay. DMPK and PK studies showed very high levels of mouse, rat, and human microsomal stability (42.4, 60.2 and 79.3 minutes respectively), and oral bioavailability in mice (48%) and rats (79%). Cmax reached 2.0 µM, while T1/2 was 26.8 hours with 25 mg/kg single oral dosing in rats. Rats toxicology studies showed excellent tolerability in 28-days repeated oral dosing with 25 mg/kg/biw dosing schedule. No relevant inhibition was observed against a panel of 100 other receptor targets, including hERG. AMES was negative up to 100 uM, and Cyp inhibition studies showed acceptable inhibition up to 10 uM. KIN-8193 potently inhibited the phosphorylation of HCK(p-Y411) and its downstream target BTK(p-Y223) in both BTK wild-type and ibrutinib-resistant BTKCys481 mutated WM and ABC DLBCL cell lines driven by activating MYD88 mutations, and primary WM cells (Fig 1A). Target engagement studies showed HCK but not BTK direct binding. In vivo pharmacodynamic (PD) studies using luciferized BCWM.1 cells WM xenograft mouse model showed potent dose-dependent inhibition of HCK(p-Y411) and BTK(p-Y223) at 6 and 24 hours following single dose administration of KIN-8193. Importantly, KIN-8193 showed selective cytotoxicity against MYD88 mutated BTK wild-type and BTKCys481 mutated, ibrutinib-resistant WM and ABC DLBCL cell lines, and primary WM cells, but had no impact on healthy donor B- and T-cells at pharmacologically achievable levels (Fig. 1B). We therefore describe a novel, highly selective and potent HCK inhibitor that is well tolerated in long-term rat toxicology studies and shows selective killing of MYD88 mutated WM and ABC DLBCL cells. Inhibition of HCK by KIN-8193 blocks downstream wild-type BTK and Cys481 mutated BTK activity, and overcomes ibrutinib resistance induced by BTKCys481 mutations. Disclosures Hunter: Pharmacyclics: Consultancy. Castillo:Genentech: Consultancy; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Millennium: Research Funding. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership. Treon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Research Funding; Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses.

scholarly journals Development of First-in-Class Histone Acetyltransferase (HAT) Activators for Precision Targeting of Epigenetic Derangements in Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 37-37 ◽  
Author(s):  
Yuxuan Liu ◽  
Jole Fiorito ◽  
Yulissa Gonzalez ◽  
Elisa Zuccarello ◽  
Elisa Calcagno ◽  
...  
Keyword(s):  
Weight Loss ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Histone Acetyltransferase ◽  
B Cell Lymphoma ◽  
Dependent Manner ◽  
Wild Type ◽  
Cytotoxic Effects ◽  
Inactivating Mutations

Abstract Monoalleleic inactivating mutations in histone acetyltransferase (HAT) enzymes promote lymphomagenesis in germinal center derived B-cell lymphomas, follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), occurring in about 40% of patients. The intact wild-type allele offers an opportunity to leverage the normal enzyme to overcome the pathogenic impact of the mutated allele. We hypothesize that if inactivating mutations in HATs are critical to FL and DLBCL lymphomagenesis, then drugs capable of inducing enhanced function of the wild-type HAT allele product should be cytotoxic in cells harboring HAT mutations. We designed and synthesized a library of new chemical entities with HAT activating properties (N=70). The cytotoxic effects of the compounds (N=29) were evaluated via medium-throughput screening in 4 DLBCL cell lines. IC50 ranged from 3.6 to 43.2 µM. Focusing on 6 analogue compounds, which share the same Nphenylbenzamide scaffold, we evaluated cytotoxicity across an expanded panel of 11 DLBCL cell lines. The median IC50 of 6 analogues tested was lower in the EP300 mutated cell lines (median 9.6 µM, range 5 - 11 µM) compared to the wildtype lines (median, 17 µM, range 15 - 24 µM). YF2 was chosen as the lead compound because it was the most selective of the analogues in inducing cytotoxicity in cell lines harboring EP300 mutations compared to wildtype (IC50 5 µM and 19 µM respectively, p<0.0005). To determine YF2's functional effect on activating p300 in a cell free assay, p300-mediated histone and p53 acetylation was measured by combining recombinant p300, substrate and acetyl-CoA. YF2 increased p300-mediated histone H3 lysine27 acetylation (EC50=38.64 nM) and H3 lysine18 acetylation (EC50=1.656 nM). YF2 also induced acetylation of p53 by 5-fold in a dose dependent manner. In cellular assays, YF2 induces acetylation of histone (H3K27 2-fold and H3K14 1.6-fold) after exposure in the SUDHL-6 cell line (EP300 mutated) as measured by mass spectrometry and confirmed by immunoblot of histone extracts. To assess the pharmacokinetics (PK) and preliminary in vivo efficacy of YF2, SUDHL-6 (EP300-mutated) xenograft bearing mice were treated once daily i.p. for 6 days with YF2 doses of 40mg/kg or 60mg/kg. Serum and tumor samples were collected at sequential time points. The Cmax of YF2 60mg/kg was 2424 ng/mL (5.1 µM) whereas Cmax of 40mg/kg was 2091.96 ng/mL (4.5 µM) which is consistent with the cellular IC50. Both concentrations of YF2, 40mg/kg and 60mg/kg, accumulated in tumor with Cmax 9536.71 and 9858.15 ng/g, respectively. YF2 is rapidly absorbed in the serum (Tmax 0.25 h) and sustained in the tumor (Tmax 4h). Significant effects on tumor size were observed in 13 of 19 mice demonstrating decreased tumor volume following only 6 days of YF2 treatment. Mice treated with YF2 40mg/kg induced H3K27 acetylation in tumor specimens as determined by mass spectrometry.YF2 40mg/kg was well tolerated in SCID/beige mice for 30 days without significant weight loss, while 60mg/kg YF2 led to 20% weight loss during 6-days of treatment. Additionally, YF2 demonstrated cytotoxic effects in 2 primary patient lymphoma samples but were non-cytotoxic to peripheral blood mononuclear cells from healthy donors. Furthermore, we hypothesized that if DLBCL is sensitive to an enhanced acetylation state, then combined targeting of epigenetic machinery with HAT activators and HDAC inhibitors may induce profound epigenetic modification leading to synergistic induction of programmed cell death. The concentration : effect relationship of YF2 and the pan-HDAC inhibitor, romidepsin, was evaluated over time across a panel of lymphoma cell lines (N=7). Synergy was calculated by Excess over Bliss (EOB>10 connotes synergy). Combination of YF2 and romidepsin demonstrated strong synergism in DLBCL lines (EOB = 48). The combination led to enhanced histone acetylation compared to either single agents. The combination is safe in mice and murine xenograft studies of the combination are underway. In summary, YF2 induces HAT-mediated acetylation of histone and p53. It demonstrates selective cytotoxic effects in EP300-mutated DLBCL cell lines, and is both well tolerated and effective in xenograft mouse models of lymphoma suggesting potential clinical application and precision medicine opportunities for patients harboring this mutation. Disclosures O'Connor: Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Celgene: Research Funding.

CD3/Bars: A Novel Bispecific Format for the Treatment of B-Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3516-3516
Author(s):  
Moritz Bewarder ◽  
Klaus-Dieter Preuss ◽  
Natalie Fadle ◽  
Evi Regitz ◽  
Lorenz Thurner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphoma Cell ◽  
Bispecific Antibodies ◽  
B Cell Lymphomas ◽  
Lymphoma Cells

Abstract Background Autoantigens are suspected to play an important role in the pathogenesis of different types of B cell neoplasia. Suggestive of this hypothesis is the restricted usage of a stereotyped IgHv repertoire in CLL, MCL and DLBCL. Further evidence supporting this notion is the identification of specific autoantigens as the target of the B-cell receptor from malignant lymphomas and myelomas, such as paratarg-7 as antigenic target for 15% of paraproteins of MGUS and MM patients. ARS2 was previously identified as the autoantigenic target for the B-cell receptor of approximately 25% of DLBCLs of the ABC type, here termed ARS2 reactive lymphomas. We had recently shown that the B-cell receptor antigens can be used to target B-cell lymphoma cells in vitro and in vivo in an approach designated as BARs (B-cell receptor antigens for reverse targeting), the first therapeutic strategy in oncology with absolute and exclusive specificity for the malignant clone. To test whether BARs can substitute the B-cell binding antibody (e.g. CD19) in T-cell engaging bispecific antibodies, we designed a bispecific CD3/BAR product consisting of a recombinant single chain fragment (scFv) against CD3 linked to ARS2 (CD3-ARS2). One arm of this construct should engage the T cell co-receptor CD3 of human T cells, and the other site should bind to the B cell receptor of ARS2 reactive lymphomas thus specifically redirecting and activating T cells against lymphoma cells. Material and methods VL and VH genes of the CD3 - OKT3 hybridoma and the DNA sequence of the 33 amino acids containing the B-cell receptor binding epitope of ARS2 were cloned into a pcDNA 3.1 vector by standard cloning techniques. VH and VL were linked by a glycine-serine linker, as was VL to the ARS2 epitope resulting in VH-(Gly₄Ser₁)ⁿ-VL-(Gly₄Ser₁)ⁿ-ARS2 peptide chain. The final cloning product was transfected in HEK cells for production of the bispecific construct. Binding capacity to lymphoma cell lines (OCI-Ly3, U2932, HBL-1) and PBMCs was assessed by flow cytometry. Western blot analysis was used for detection of CD3-ARS2 after incubation with the monoclonal anti-His Tag antibody. Cytotoxicity was evaluated by LDH release assay. Results The CD3 - ARS2 bispecific construct bound to CD3 on T cells and the B-cell receptor of ARS2 reactive lymphoma cells. CD3/ARS2 induced rapid cytotoxicity exclusively in ARS2 reactive lymphoma cell lines at concentrations as low as 250 ng/ml with an effector - target ratio of 10:1. Specific T-cell mediated cytotoxicity reached 40% after 4 hours. Lymphoma cell lines with BCRs of a specificity other than ARS2 were not affected. Conclusion The CD3/BAR construct is a novel therapeutic principle for the treatment of B-cell lymphomas, suggesting that BARs might also be useful as part of CAR-T cells. Compared to CD3/CD19 bispecific antibodies the CD3/BARs are exclusively cytotoxic against the malignant clone and spare normal B-cells. This should considerably reduce the acute toxicity of T-cell engaging bispecific constructs and circumvent long-term B cell depletion. Experiments comparing the cytotoxic capacity of CD3/BARs with CD3/CD19 bispecific antibodies are underway, as are analyses evaluating possible synergisms of these constructs. Disclosures No relevant conflicts of interest to declare.

rHuKGF ameliorates symptoms in DSS and CD4+CD45RBHi T cell transfer mouse models of inflammatory bowel disease

2002 ◽  
Vol 282 (4) ◽  
pp. G690-G701 ◽  
Author(s):  
Fergus R. Byrne ◽  
Catherine L. Farrell ◽  
Richard Aranda ◽  
Karen L. Rex ◽  
Sheila Scully ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Weight Loss ◽  
Body Weight ◽  
T Cell ◽  
Large Intestine ◽  
Bowel Disease ◽  
Body Weight Loss ◽  
Cell Transfer ◽  
Inflammatory Bowel ◽  
T Cell Transfer

There is an acute need for effective therapy for inflammatory bowel disease (IBD), particularly at the level of repair of the damaged epithelium. We evaluated the efficacy of recombinant human keratinocyte growth factor (rHuKGF) in both the dextran sodium sulfate (DSS) and the CD4+CD45RBHi T cell transfer models of IBD. Disease was induced either by the ad libitum administration to normal mice of 4% DSS in the drinking water or by the injection of 4 × 105 CD4+CD45RBHi T cells into immunodeficient scid/scid mice. rHuKGF was administered by subcutaneous injection at doses of 1.0 or 3.0 mg/kg in both preventative and therapeutic regimens during both studies. rHuKGF significantly improved survival and body weight loss in the DSS model in both preventative and therapeutic dosing regimens. It also improved diarrhea, hematochezia, and hematological parameters, as well as large intestine histopathology. In the T cell transfer model, rHuKGF improved body weight loss, diarrhea, and levels of serum amyloid A, as well as large intestine histopathology. In both models of IBD, the colonic levels of intestinal trefoil factor (ITF) were elevated by the disease state and further elevated by treatment with rHuKGF. These data suggest that rHuKGF may prove useful in the clinical management of IBD and its effects are likely mediated by its ability to locally increase the levels of ITF.

Author response for "Combining T‐cell‐based immunotherapy with venetoclax elicits synergistic cytotoxicity to B‐cell lines in vitro"

2020 ◽  
Author(s):  
Satsuki Murakami ◽  
Susumu Suzuki ◽  
Ichiro Hanamura ◽  
Kazuhiro Yoshikawa ◽  
Ryuzo Ueda ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Author Response ◽  
Synergistic Cytotoxicity
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