Autologous T cells expressing the oncogenic transcription factor KLF6-SV1 prevent apoptosis of chronic lymphocytic leukemia cells

Abstract Abstract 3886 ROR1 is a receptor-tyrosine kinase like protein expressed on the surface of chronic lymphocytic leukemia (CLL) B cells, but not on normal mature B cells, suggesting that it may be a promising therapeutic target. We have generated a chimeric monoclonal antibody (mAb), UC99961, which binds to an intradomain epitope of human ROR1 (hROR1). UC99961 binds the same epitope as the murine anti-hROR1 mAb, UC D10–001, which has direct cytotoxic effects on hROR1 positive CLL cells. In this study we investigated the in-vivo anti-leukemic activity and tolerability of UC99961 on ROR1+ primary patient CLL cells and human cord-blood-derived B cells and T cells, respectively. For these studies, immunodeficient RAG2−/−γc−/− neonatal mice were reconstituted with a human immune system by intrahepatic xenotransplantation of 1×105 CD34+ human cord blood progenitor cells. Eight to ten weeks post transplantation, cord blood engraftment was verified by peripheral blood screening, at which point the mice received an intraperitoneal transplantation of 2×107 primary patient ROR1+ CLL cells. Twenty-four hours after CLL transplantation, five animals per group were each treated with a single intraperitoneal injection (10mg/kg) of UC99961, UC D10–001, or control IgG. Seven days following mAb treatment, the animals were sacrificed and marrow, spleen, thymus, and peritoneal lavage samples were collected and analyzed by flow cytometry for CLL cells, as well as normal cord-blood-derived B cells and T cells. To confirm mAb administration according to the study design, serial residual ROR1 plasma antibody levels were determined by ELISA. Results from three consecutive experiments using leukemia cells from two different patients showed that the vast majority of CLL B cells remained in the peritoneal cavity of the animals and did not migrate to other hematopoietic organs. Both anti-hROR1 mAbs UC99961 and UC D10–001 significantly reduced the average number of harvested CLL cells in the peritoneal lavage compared to control IgG (99% and 71% reduction respectively), while cord-blood-derived T cells (CD45+3+) in thymus remained unaffected by the mAb treatment. For the majority of cord-blood-derived B cells in marrow and spleen, no significant reduction could be observed after UC99961 or UC D10–001 mAb treatment. A small CD19+ROR1+CD34− cord-blood-derived B cell population was identified in marrow and spleen that was reduced after UC99961 and UC D10–001 mAb treatment. This study demonstrates that the anti-human ROR1 specific mAbs have in vivo anti-leukemic activity with minimal impact on human cord-blood-derived B cells and T cells. From these results, UC99961 appears to be an excellent candidate antibody for future clinical studies for patients with CLL. Disclosures: No relevant conflicts of interest to declare.

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Mutations in the NOTCH1 PEST Domain Enhance the Expression of CCL17 By Displacing the Transcription Factor HDAC1 in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2018-99-116388 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4408-4408

Author(s):

Zhenshu Xu ◽

Shifen Wang ◽

Xiuli Chen ◽

Xueting Liang ◽

Huixin Liang ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Transcription Factor ◽

Chronic Lymphocytic Leukemia ◽

Cd4 T Cells ◽

Conflicts Of Interest ◽

Premature Stop Codon ◽

Lymphocytic Leukemia ◽

Control Group ◽

Multiple Point

Abstract The NOTCH1 is a ligand dependent transcription factor that plays an important role in lymphocyte differentiation and apoptosis . NOTCH1 dysfunction is closely related to the proliferation, differentiation, and apoptosis of tumor cells in chronic lymphocytic leukemia (CLL). With the popularization of next-generation sequencing technology the relationship between NOTCH1 mutations and disease progression in CLL, has attracted increasing attention. Here, we investigated whether the loss of a 2 bp frame shift deletion mutation influences the NOTCH1 pathway and whether this mutation alters the NOTCH1 nuclear mechanism in CLL. The ICN plasmid was engineered by cloning the ICN coding sequence into a pmax-Clover vector. The c.7541-7542delCT mutation (CTdel) was generated by site-specific mutagenesis. The BaF3 cells were transfected with Amaxa Nucleofector technology then sorted. The NOTCH1 protein expression was evaluated by Western blotting using an anti-NOTCH1 antibody, which showed compatible with the ICN. The CTdel mutation resulted in a lower molecular weight band, consistent with the presence of a premature STOP codon. Results from qRT-PCR showed elevated mRNA expression of NOTCH1 in the groups transfected with ICN and CTdel genes. An immunofluorescence assay showed that NOTCH1 was distributed in both the nuclei and cytoplasm in the control cells, while it was located in the nucleus of the cells of the ICN and CTdel groups. Compared with the control group, the activity of the reporter genes in both the ICN and CTdel groups increased, with the highest increase in the CTdel group, as reported by the two-fluorescent enzyme reporting system assay. These results determine the presence or absence of a NOTCH1 mutation, the ICN protein is located in the nucleus, and show that the NOTCH1 pathway is enhanced and the function is more stable in the presence of a NOTCH1 mutation. From the RNA-seq results we found that RT-PCR showed transcription levels of CCL17 in the ICN and CTdel groups were higher than those in the control group, and that CCL17 in the CTdel group was significantly higher than in the ICN group. We collected the culture supernatant of CLL cells for an ELISA assay and found that CCL17 was significantly elevated in the CTdel group, but CCL17 was not detected in the control group and the ICN group. In order to verify the CCL17 function of in CLL with the NOTCH1 mutation, we performed a transwell experiment to detect the ability of mediating activated CD4+ T cell migration by CCL17. The results showed that the number of CD4+ T-cells in the CTdel group that migrated in response to CCL17 was more than in the ICN group. In order to verify that the NOTCH1 mutation changed the ICN binding function, we performed a CO-IP experiment. The results showed that ICN had an interaction with MTA2/HDAC1, but this interaction was weakened with CTdel. Mass spectrometry (MS) analysis suggested that ICN was combined with MTA2 while CTdel peptides were not detected in MTA2 samples. In order to verify the negative regulatory effect of MTA2 and HDAC1 on the NOTCH1 mutation induced by C-terminal truncation in CCL17 transcription, we conducted a CHIP experiment on the nuclear pyrolysis of ICN/CTdel.The results showed that the combination of MTA2/HDAC1 and the promoter of CCL17 and ICN/CTdel was weaker than that of the control group. Because of the multiple-point binding characteristics of transcription factors on gene expression regulation, it can be concluded that CTdel DNA binding is weaker than the binding of ICN. As a result, in the presence of the NOTCH1 protein C-terminal truncation, which has lost MTA2/HDAC1 binding, its inhibition is reduced and the CCL17 expression becomes significantly elevated. In conclusion, it is suggested that the NOTCH1 mutation found in CLL stimulates the NOTCH1 pathway, and is related to the high expression of CCL17. The chemokine CCL17 can cause the migration of CD4+ T-cells and change the microenvironment to favor tumor cell survival. ICN in the nucleus combines with CSL to form activating complexes or recruits transcription factor MTA2/HDAC1 to form inhibiting complexes, and constitutes the balance between the promotion and the inhibition for the downstream gene expression. The NOTCH1 mutation with CTdel could result in loss of this balance, and activate the expression of downstream genes, such as CCL17. Key words: chronic lymphocytic leukemia; NOTCH1; mutation; HDAC1; CCL17; chemokine Disclosures No relevant conflicts of interest to declare.

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Homeostatic chemokines increase survival of B-chronic lymphocytic leukemia cells through inactivation of transcription factor FOXO3a

Oncogene ◽

10.1038/sj.onc.1210519 ◽

2007 ◽

Vol 26 (50) ◽

pp. 7081-7091 ◽

Cited By ~ 56

Author(s):

M Ticchioni ◽

M Essafi ◽

P Y Jeandel ◽

F Davi ◽

J P Cassuto ◽

...

Keyword(s):

Transcription Factor ◽

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Leukemia Cells

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Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells

Journal of Clinical Investigation ◽

10.1172/jci24176 ◽

2005 ◽

Vol 115 (7) ◽

pp. 1797-1805 ◽

Cited By ~ 177

Author(s):

Güllü Görgün ◽

Tobias A.W. Holderried ◽

David Zahrieh ◽

Donna Neuberg ◽

John G. Gribben

Keyword(s):

Gene Expression ◽

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Lymphocytic Leukemia ◽

Leukemia Cells

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Some chronic lymphocytic leukemia cells bearing surface immunoglobulins share determinants with T cells

European Journal of Immunology ◽

10.1002/eji.1830081214 ◽

1978 ◽

Vol 8 (12) ◽

pp. 900-904 ◽

Cited By ~ 97

Author(s):

Laurence Boumsell ◽

A. Bernard ◽

Virginia Lepage ◽

L. Degos ◽

J. Lemerle ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Bearing Surface ◽

Surface Immunoglobulins

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Lenalidomide Induces Immunomodulation in Chronic Lymphocytic Leukemia and Enhances Antitumor Immune Responses Mediated by NK and CD4 T Cells

BioMed Research International ◽

10.1155/2014/265840 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 31

Author(s):

Andrea Acebes-Huerta ◽

Leticia Huergo-Zapico ◽

Ana Pilar Gonzalez-Rodriguez ◽

Azahara Fernandez-Guizan ◽

Angel R. Payer ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Nk Cells ◽

Cd4 T Cells ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Therapeutic Activity ◽

Healthy Donors ◽

Cd20 Expression ◽

Dependent Cell

Lenalidomide is an immunomodulatory drug with therapeutic activity in chronic lymphocytic leukemia (CLL). However, it has pleiotropic effects, and the mechanism of action responsible for its therapeutic activity has not been well defined yet. Herein, we show that lenalidomide treatment does not have an effect on the proliferation of leukemia cells, but it increases the proliferation of B cells from healthy donors. Lenalidomide did not exert a direct effect on the apoptosis of leukemia cells obtained from CLL patients, although it indirectly induced their apoptosis through the activation of nonmalignant immune cells. Thus, lenalidomide markedly increased the proliferation of NK and CD4 T cells. The effect of lenalidomide on NK cells was secondary to the induction of IL-2 production by CD4 T cells. Accordingly, depletion of T cells or blockade of IL-2 activity completely abrogated the proliferation of NK cells. Additionally, lenalidomide enhanced NK and NKT-like cell-mediated natural cytotoxicity against leukemia cells from CLL patients. Lenalidomide also upregulated CD20 expression on leukemia cells and, accordingly, it had a synergistic effect with rituximab on promoting antibody-dependent cell-mediated cytotoxicity against primary leukemia cells. Overall, these observations provide a support for combining lenalidomide with rituximab as a treatment in CLL.

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ICOS Is Induced by B Cell Receptor Signalling on Chronic Lymphocytic Leukemia B Cells.

Blood ◽

10.1182/blood.v104.11.969.969 ◽

2004 ◽

Vol 104 (11) ◽

pp. 969-969 ◽

Cited By ~ 1

Author(s):

Tetsuya Fukuda ◽

Traci L. Toy ◽

Laura Z. Rassenti ◽

Kanti R. Rai ◽

Thomas J. Kipps

Keyword(s):

Flow Cytometry ◽

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Progressive Disease ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Activated T Cells

Abstract Patients with chronic lymphocytic leukemia (CLL) cells that express unmutated immunoglobulin (Ig) heavy chain variable region genes (IgVH genes) generally have a more aggressive clinical course than do patients with leukemia cells that express mutated IgVH. The reason(s) accounting for this are not known. Microarray gene expression analyses revealed that CLL cells that express unmutated IgVH could be distinguished from the leukemia cells that express mutated IgVH via the differential expression of a relatively small number of genes, one of which encodes the zeta-associated chain of 70kD (ZAP-70), which generally is expressed by CLL cells that express unmutated IgVH. Although the expression of ZAP-70 is associated with expression of unmutated IgVH in CLL, this association is not absolute. This was the case for a pair of monozygotic twins who both developed CLL at age 57. Although each of the twins had leukemia cells that expressed mutated IgVH, only one of the twins had leukemia cells that lacked expression of ZAP-70 protein and has indolent, non-progressive disease (Blood100: 4609–14, 2002). We performed microarray analysis using Affymetrix HG-U133A array on the isolated leukemia cells of each twin to define the genes that were differentially expressed between the two. In addition to ZAP-70, we found that the CLL cells of the twin with progressive disease also expressed the inducible co-stimulatory molecule (ICOS), a member of the CD28/CTLA-4 family of immune accessory co-stimulatory molecules that ordinarily only is expressed by activated T cells. Expression of ICOS protein by this leukemia B cell population, but not by the CLL B cells population of the other twin, was confirmed using fluorochrome-labeled anti-ICOS mAb and flow cytometry. We examined the CLL B cells from 58 additional patients for expression of ICOS by flow cytometry and found that 16 (28%) also expressed ICOS. We found that expression of ICOS was associated with expression of ZAP-70, as assessed via flow cytometry and immunoblot analyses. Whereas 14 of the 29 ZAP-70+ cases expressed ICOS, only 2 of the 29 ZAP-70-negative cases expressed this immune co-stimulatory molecule. Nevertheless, we found that nearly all of the 56 of the 58 cases expressed B7h, the ligand for ICOS. The two cases that did not express detectable B7h expressed ZAP-70 and were ICOS+. In preliminary studies, we found that treatment of ICOS-negative, ZAP-70+ CLL cells (n = 2) with goat anti-human Ig could induce expression of ICOS, suggesting that, as on T cells, this molecule also might be inducible in some cases of B cell CLL. Culture of ICOS+ CLL cells with an anti-B7h mAb capable of blocking ICOS-B7h interactions significantly enhanced ICOS surface expression, as assess by flow cytometry, suggesting that B7h may down-modulate ICOS through paracrine/autocrine receptor-ligand interactions. Because of this we evaluated for functional expression of ICOS on CLL B cells. We found that ligation of ICOS could induce enhanced signaling via the PI3K/Akt pathway in isolated CLL B cells, resulting in enhanced phosphorylation and activation of Akt. As such, we speculate that the expression of ICOS and its ligand in B cell CLL may enhance leukemia cell survival and/or proliferation, potentially contributing to the more aggressive disease observed in some patients with this disease.

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