Lenalidomide Inhibits Proliferation of Chronic Lymphocytic Leukemia Cells in Vitro

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1775-1775
Author(s):  
Jessie-Farah Fecteau ◽  
Diahnn Futalan ◽  
Ila Bharati ◽  
Emanuela M. Ghia ◽  
Laura G Corral ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Research Funding ◽  
Single Agent ◽  
Leukemia Cell ◽  
S Phase ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Cell Expression

Abstract Abstract 1775 Promising clinical responses have been observed in chronic lymphocytic leukemia (CLL) patients treated with lenalidomide as a single agent or in combination with other agents, The mechanisms of action of lenalidomide are under study; unlike most other anti-leukemia drugs, lenalidomide has no direct cytoxic effects in vitro on primary CLL cells, which typically are in G0/1 phase of the cell cycle. We examined the activity of lenalidomide on CLL cells that were induced to proliferate in vitro. To induce proliferation, CLL cells were cultured in media containing human interleukin (IL)-4 and IL-10 and with stromal cells (HeLa) made to express CD154. Labeling CLL cells with carboxyfluorescein succinimidyl ester (CFSE), allowed us to monitor for several rounds of induced CLL-cell division via flow cytometry. We found that addition of 0.33–10 micro M lenalidomide to such cultures resulted in a dose-dependent reduction in the number of leukemia cells induced to undergo cell-division. Moreover, we found that lenalidomide could significantly reduce the number of dividing CLL cells in each patient sample tested (n=4) by an average of 1.7 fold, reducing the fraction of dividing cells from 77% ± 27% to 44% ± 22% after 6 days of culture (mean +/− SD, P < 0.05). Evaluation of the DNA content of CLL cells using propidium iodide (PI) and flow cytometry revealed that lenalidomide significantly decreased the percentage of CLL cells in the G2/M phase of the cell cycle from 9% ± 2.7% to 5.1% ± 2.1% (mean +/− SD, n=4; P< 0.05) in control versus lenalidomide-treated cultures, respectively. Furthermore, lenalidomide appeared also to reduce the percentages of CLL cells in S phase from 12% ± 8% to 6.2% ± 3.1%. We found that the capacity of lenalidomide to inhibit CLL cell-division was associated with lenalidomide-induced leukemia-cell expression of p21/WAF/Cip, which can directly inhibit the activity of cyclin-dependent kinases required for progression from G1 into the S phase of the cell cycle. Gene expression analysis of CLL cells (n=10) revealed that lenalidomide induced increased leukemia-cell expression of p21/WAF/Cip at 6h and at 24h, an effect that also was noted for leukemia cells in the blood of patients treated with single-agent lenalidomide. Lenalidomide-induced expression of p21/WAF/Cip was associated with induced expression of the pro-apoptotic protein Bim, a downstream target of of p21/WAF/Cip. However, lenalidomide did not appear to induce leukemia-cell expression of TP53, which can induce p21/WAF/Cip, suggesting that lenalidomide induces p21/WAF/Cip via a TP53-independent mechanism. These results suggest that lenalidomide has effects on CLL cells that are distinct from those induced by CD40-ligation. Moreover, these studies reveal a potential mechanism for the anti-leukemia activity of lenalidomide, which might inhibit factors that potentially drive leukemia-cell proliferation in vivo. Disclosures: Corral: Celgene: Employment. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding. Messmer:Celgene: Research Funding.

Lenalidomide Abrogates the Protective Influence of Nurse-Like Cells on Primary Chronic Lymphocytic Leukemia Cells In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3116-3116 ◽  
Author(s):  
Danelle F. James ◽  
Maryann R. Betty ◽  
Ruzbeh Mosadeghi ◽  
Thomas J. Kipps
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
Cell Survival ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Response To Treatment ◽  
Leukemia Cells ◽  
High Dependency ◽  
Cells Cultured

Abstract Lenalidomide (3-(4-amino-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione)) is an agent approved for treatment of patients with del 5q myelodysplastic syndromes and previously treated multiple myeloma. Lenalidomide has been found in early clinical trials to have potential therapeutic activity in patients with relapsed chronic lymphocytic leukemia (CLL). The mechanism(s) whereby this drug is active in CLL is unknown. In particular, studies to date have not found lenalidomide to have any direct cytotoxic activity on CLL cells in vitro. This has stimulated speculation that this agent might adversely affect the positive influence of the microenvironment on leukemia-cell survival. We and others have observed that cells found in the leukemia microenvironment can support CLL-cell survival in vitro. One such type of cells are nurse-like cells (NLC), which can differentiate from the CD14-positive blood mononuclear cells of CLL patients into large, round adherent cells that can attract and support CLL cell survival in vitro for weeks, if not longer. We evaluated the effects of lenalidomide on primary leukemia-cell survival in vitro when the CLL cells from different patients (N=21) were cultured alone or together with NLC generated as previously described [Tsukada Blood 2002]. We assessed the in-vitro activity of lenalidomide on primary CLL cells from 21 patients, in duplicate in a series of 6 experiments. Lenalidomide at concentrations of 0.1μM-200μM did not significantly impact the survival of CLL cells that were cultured alone for up to 12 days. Analysis of cell surface markers revealed increased expression of CD38 at 36 hours in 5/5 lenalidomide treated CLL samples compared with untreated cells (MFIR 5.7 +/− .86 vs. 3.4 +/− .83 p=.003). We observed sustained upregualtion of CD40 and regulation of CXCR4 in the majority of cells treated with lenalidomide. When cultured with NLC, the survival of CLL cells was comparable to or significantly higher than that of CLL cells cultured alone 62.4% vs. 51% (+/−3% SEM n=21 p [<] 0.0005). The addition of lenalidomide at concentrations of 0.1μM and greater to co-cultures of NLC and CLL cells caused specific reductions in CLL cell survival to levels similar to or lower than that of CLL cells cultured without NLC. In the presence of NLC, lenalidomide at 1μM reduced CLL cell viability compared to control (41.5% vs. 56% +/−4% p [<] 0.0005 paired student t test n=13). For most patients the levels of CLL cell viability on days 4 through 8 in the co-cultures with lenalidomide was significantly lower than those of CLL cells co-cultured with NLC in the absence of lenalidomide. As such, this study reveals that physiologic concentrations of lenalidomide might abrogate the protective influence of NLC on CLL cell survival in vitro and potentially in vivo. Conceivably, those patients who have leukemia cells displaying a high dependency on NLC for survival in vitro also might be most likely to experience a favorable clinical response to treatment with lenalidomide. This hypothesis will be tested in a prospective manner with a planned clinical trial evaluating lenalidomide for treatment of CLL through the CLL Research Consortium.

Wnt5a Induces Association of ROR1 with 14-3-3ζ to Enhance Chemotaxis and Proliferation in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 349-349 ◽  
Author(s):  
Jian Yu ◽  
Liguang Chen ◽  
Yun Chen ◽  
Ling Zhang ◽  
Laura Z. Rassenti ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Research Funding ◽  
Critical Role ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Orphan Receptor ◽  
Binding Motif ◽  
Human Leukemia

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. We found that ROR1 was a receptor for Wnt5a, which could activate Rho GTPases (e.g. RhoA and Rac1) in CLL cells by inducing the recruitment to ROR1 of guanine exchange factors (GEFs), notably ARHGEF2. How ARHGEF2 can complex with ROR1 was not known. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 and identified 14-3-3ζ, a highly conserved, cytoplasmic-protein member of the tetratricopeptide repeat-like superfamily. 14-3-3ζ plays a critical role in cell-signaling pathways, which promote proliferation, adhesion, and survival in a variety of human cancers. We validated the Wnt5a-induced interaction of ROR1 with 14-3-3ζ in primary CLL cells using co-immunoprecipitation studies and immunoblot analyses. We found the capacity of Wnt5a to induce ROR1 to associate with 14-3-3ζ could be blocked by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope in the ROR1-extracellular domain; this mAb is undergoing clinical testing in patients with CLL. Furthermore, we found that 14-3-3ζ could interact with ARHGEF2 in CLL cells. Silencing 14-3-3ζ via RNAi impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance the in vitro exchange activity of ARHGEF2 for RhoA and Rac1, and (3) induce activation of RhoA and Rac1 in primary CLL cells. Consistent with these findings, we found that Difopein, an inhibitor of 14-3-3ζ, also could inhibit Wnt5a-enhanced chemokine-directed migration and proliferation of primary CLL cells in vitro, at low concentrations that did not result in leukemia-cell apoptosis. To examine structure-function relationships, we employed the MEC1 cell line, which was derived from human CLL. Prior studies found MEC1 cells expressed Wnt5a, which induced activation of RhoA and Rac1 in MEC1 cells made to express ROR1 (MEC1-ROR1), but not in parental MEC1 cells, which lacked ROR1. Similar to work on primary CLL cells, we detected 14-3-3ζ in anti-ROR1 immune precipitates via mass spectrometry and immunoblot analyses. We identified a 14-3-3ζ binding motif (RSPS857SAS) in the cytoplasmic domain of ROR1; site directed mutagenesis and transfection of MEC1 with mutant forms of ROR1 determined that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2-dependent activation of RhoA and Rac1, respectively. In addition, we used CRISPR/Cas9 technology to delete 14-3-3ζ (Δ14-3-3ζ) in MEC1 and MEC1-ROR1 cells. We found that MEC1-ROR1 had significantly higher rates of proliferation than MEC1-ROR1-Δ14-3-3ζ cells, which in turn had rates of proliferation comparable to those of MEC1 cells or MEC1-Δ14-3-3ζ lacking expression of ROR1. MEC1-ROR1 also had a significantly greater capacity to migrate in response to chemokine (CCL21) than did MEC1-ROR1-Δ14-3-3ζ cells, which migrated in response to CCL21 as well as MEC1-Δ14-3-3ζ or MEC1 cells lacking expression of ROR1. To examine whether such differences affected leukemia-cell growth in vivo, we engrafted Rag2−/−γc−/− mice each with equal numbers of MEC1, MEC1-Δ14-3-3ζ, MEC1-ROR1, or MEC1-ROR1-Δ14-3-3ζ cells. We found that mice that received MEC1-ROR1 cells had significantly higher levels of engraftment and human leukemia-cell proliferation than did mice that received MEC1, MEC1-Δ14-3-3ζ, or ROR1-Δ14-3-3ζ cells, which had comparable levels of engraftment and proliferation, indicating that 14-3-3ζ was necessary for the engraftment/growth advantage of MEC1-ROR1 over MEC1 cells in vivo. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1-dependent-signaling leading to enhanced migration and proliferation of CLL cells in vitro and in vivo. Disclosures Kipps: Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding.

scholarly journals Cirmtuzumab Targets ROR1 to Inhibit Ibrutinib-Resistant, Wnt5a-Induced Rac1 Activation in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2034-2034 ◽  
Author(s):  
Jian Yu ◽  
Liguang Chen ◽  
Bing Cui ◽  
Christina Wu ◽  
Michael Y. Choi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Research Funding ◽  
Standard Dose ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Synergistic Activity ◽  
Bcr Signaling

Abstract Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). This is underscored by the clinical effectiveness of an inhibitor of Bruton's tyrosine kinase (BTK), ibrutinib, which can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, suggesting that ancillary pathways contribute to CLL growth/survival that are independent of BCR-signaling. ROR1 is a receptor for Wnt5a, which can promote activation of Rac1 to enhance CLL-cell proliferation and survival. We hypothesized that the effects of ibrutinib on blocking BCR-signaling might be offset by non-canonical Wnt-signaling via ROR1. If so, then inhibition of both ROR1- and BCR-signaling might have an enhanced anti-tumor effect. We examined the CLL cells of patients who were taking ibrutinib at the standard dose of 420 mg per day. Freshly isolated CLL cells had activated Rac1, which diminished over time upon culture in serum-free media, unless treated with exogenous Wnt5a, as noted for CLL cells of patients not taking ibrutinib. Moreover, Wnt5a could induce Rac1 activation and enhance proliferation of CLL cells treated in vitro with ibrutinib, even at concentrations that exceeded those required to completely inhibit BTK and BCR-signaling. On the other hand, Wnt5a-induced activation of Rac1 was blocked by treatment of the CLL cells with cirmtuzumab (UC-961), a first-in-class humanized mAb specific for a functional extracellular epitope of ROR1; this mAb is being evaluated in a phase I clinical trial in patients with CLL. To examine the activity of ibrutinib and/or cirmtuzumab, alone or in combination, we transferred human CLL cells into the peritoneal cavity of immune-deficient Rag2−/−γc−/− mice, which subsequently were treated with ibrutinib via oral gavage and/or cirmtuzumab administered iv. Although either agent alone resulted in some leukemia-cell clearance, cirmtuzumab and ibrutinib had apparent synergistic activity when used together in clearing human leukemia cells. We also examined the activity of each agent, alone or in combination, against a ROR1+ mouse leukemia, which we had engrafted in Rag2−/−γc−/− mice. While the engrafted mice treated with cirmtuzumab or ibrutinib alone had significantly smaller spleens and lower proportions of leukemia cells than the engrafted animals that did not receive any treatment, the mice treated with the combination of cirmtuzumab and ibrutinib had significantly smaller spleens and synergistic clearance of leukemia cells. Collectively, this study demonstrates that cirmtuzumab and ibrutinib may have synergistic activity in the treatment of patients with CLL, providing the rationale for clinical trials using cirmtuzumab in combination with ibrutinib, or another inhibitor of BTK, such as acalabrutinib, for treatment of patients with CLL or other B-cell malignancies dependent on non-canonical Wnt5a/ROR1 signaling. Disclosures Kipps: Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding.

scholarly journals Chronic Lymphocytic Leukemia Cells with Mutated Nfkbie Are Positively Selected By Microenvironmental Signals and Display Reduced Sensitivity to Ibrutinib Treatment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 248-248
Author(s):  
Alice Bonato ◽  
Riccardo Bomben ◽  
Supriya Chakraborty ◽  
Giulia Felician ◽  
Claudio Martines ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Pi3k Inhibitor ◽  
Leukemia Cells ◽  
Wild Type ◽  
Clonal Composition

Abstract Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in chronic lymphocytic leukemia (CLL). However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown and requires further study in in vivo models of the disease. To this end, we generated transplantable murine leukemias with inactivating NFKBIE mutations and investigated their impact on leukemia growth and response to ibrutinib (IBR) treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two recently established autoreactive leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines proliferate spontaneously in vitro in a BCR-dependent manner, but also respond with increased proliferation to certain microenvironmental signals, such as those generated by Toll-like receptor (TLR) stimulation (Chakraborty S et al, Blood 2021). To investigate whether NFKBIE mutations can affect the proliferation of these cell lines in vitro, we performed competition experiments with mixed cultures of cells with wild type and mutated NFKBIE. Analysis of the clonal composition after 2 weeks showed no change in the mutant allele frequency (MAF), suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of the immortalized leukemia cells. However, repeated TLR or BCR stimulation of these cells with CpG-DNA, LPS, anti-IgM or autoantigen resulted in a 2-3 fold increase in MAF, suggesting that NFKBIE mutations provide a growth advantage when the cells are exposed to certain microenvironmental signals (n=3 experiments/condition, P&lt;0.05 for each condition). To investigate the impact of NFKBIE mutations on leukemia growth in vivo, the same cells were transplanted by intraperitoneal injection in wild type mouse recipients (n=8) and the clonal composition was determined 3 weeks later by MAF analysis of cells isolated from peritoneal cavity (PC), blood and spleen. A significant increase in MAF was observed only in leukemia cells isolated from the spleen (P&lt;0.05), suggesting that microenvironmental signals that positively select NFKBIE-mutated cells are available only in certain tissue compartments. Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we next investigated whether NFKBIE mutations can also affect the response to IBR treatment. In vitro BrdU-incorporation experiments showed that IBR inhibits the proliferation of cells with mutated NFKBIE to a significantly lesser extent compared to cells with wild type NFKBIE (% proliferating cells with wild type and mutated NFKBIE, respectively, cultured without IBR: 90% vs 88%, P=n.s., with 0.2 μM IBR: 57% vs 73%, P&lt;0.001, with 1.0 μM IBR: 28% vs 53%, P&lt;0.001). Consistent with this finding, positive selection of NFKBIE-mutated cells was observed in the presence of IBR after 14 days in mixed culture competition experiments (mean MAF without IBR 47%, with 0.2 μM IBR 61%, p=0.032, with 1.0 μM IBR 64%, p=0.034). The greater resistance of NFKBIE-mutated cells to IBR was further validated by in vivo competition experiments showing a significantly greater increase in MAF in mice treated with IBR compared to controls in all three investigated compartments (n=4 mice/group, PC: P=0.029, blood P=0.029, spleen: P=0.001). To validate these findings in the clinical setting, we investigated the presence of NFKBIE mutations in a cohort of 84 IBR-treated CLL patients. Mutations of NFKBIE were detected at pre-treatment in 10/84 patients, 7/10 with &gt;10% VAF values. Kaplan Meier analysis showed a trend towards reduced progression-free and overall survival from the beginning of IBR treatment for NFKBIE-mutated cases (Figure 1A). Analysis of an extended cohort of over 200 cases is ongoing and will be presented at the meeting. Finally, to investigate whether leukemic cells with mutated NFKBIE remain sensitive to other BCR inhibitors, we tested their growth in the presence of the PI3K inhibitor idelalisib or SYK inhibitor fostamatinib (Figure 1B). In contrast to IBR, both drugs inhibited the proliferation of NFKBIE-mutated cells in vitro, with a greater effect observed with idelalisib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K inhibitor. Figure 1 Figure 1. Disclosures Marasca: Janssen: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; AbbVie: Honoraria, Other: Travel grants. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Laurenti: Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; BeiGene: Honoraria. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding.

In Vitro Studies of Bortezomib with Daunorubicin and Cytarabine:Sequence of Administration Affects Leukemia Cell Chemosensitivity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4464-4464 ◽  
Author(s):  
Eyal C. Attar ◽  
Emily Learner ◽  
Philip C. Amrein
Keyword(s):  
Cell Cycle ◽  
Proteasome Inhibitor ◽  
Remission Rate ◽  
Single Agent ◽  
Leukemia Cell ◽  
In Vitro Studies ◽  
Leukemia Cell Line ◽  
Sequential Administration ◽  
Synergistic Cytotoxicity

Abstract Induction chemotherapy with cytarabine and an anthracycline has been a standard treatment of newly diagnosed AML for the past 20 years. While the complete remission rate is high, only 20–40% of patients have prolonged leukemia-free survival and thus novel treatments are required. Bortezomib represents a first-in class proteasome inhibitor with activity in a variety of hematologic malignancies. Recent evidence has demonstrated synergistic cytotoxicity between the proteasome inhibitor, MG-132, and the anthracycline, idarubicin, in primitive leukemia cells.(Guzman et al. Proc Natl Acad Sci U S A 99 2002 16220-5) In order to determine if adding bortezomib to the conventional antileukemic therapies daunorubicin and cytarabine enhances cellular cytotoxicity, we conducted in vitro studies on the acute myeloid leukemia cell line, KG-1. Proliferation assays demonstrated that single-agent daunorubicin or single-agent cytarabine impaired proliferation to a greater extent than when either agent was simultaneously combined with bortezomib. Reasoning that treatment with bortezomib resulted in cell cycle arrest and protection from cycle-dependent agents, we treated cells with bortezomib and observed an accumulation of cells in the G2/M phase of the cell cycle, supporting our hypothesis. We then sought to determine whether sequential administration of bortezomib and chemotherapy affected leukemia cell proliferation. Cells were treated with bortezomib either 24 hours before or after idarubicin or cytarabine. Surprisingly, pretreatment of cells with bortezomib followed by chemotherapy resulted in increased proliferation relative to chemotherapy alone while initial treatment of cells with chemotherapy followed by bortezomib resulted in enhanced cytotoxicity. In conclusion, these data indicate that in in vitro leukemia cell cultures the timing of administration of bortezomib relative to conventional anti-leukemic agents critically affects cytotoxicity.

Entinostat, a Novel Histone Deacetylase (HiDAC) Inhibitor Enhances the Anti-Tumor Activity of Bortezomib (BTZ) in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Cell Lines,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3734-3734
Author(s):  
Cory Mavis ◽  
Sarah Frys ◽  
Juan Gu ◽  
John Gibbs ◽  
Myron S. Czuczman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
In Vitro Exposure ◽  
Anti Tumor Activity

Abstract Abstract 3734 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins [histones (class I) and non-histone proteins (class II)], leading to regulation of gene transcription and other cellular processes. Entinostat (MS-275) is a novel and potent DAC class I inhibitor undergoing pre-clinical and clinical testing. In order to better characterize the role of DAC inhibitors in the treatment of refractory/resistant (r/r) B-cell lymphoma, we studied the anti-tumor activity of entinostat as a single agent or in combination with the proteasome inhibitor bortezomib (BTZ) against a panel of rituximab-[chemotherapy]-sensitive cell lines (RSCL), rituximab-[chemotherapy]-resistant cell lines (RRCL), and primary lymphoma cells isolated from patients with treatment-naïve or r/r B-cell lymphoma. In addition, we characterized the mechanisms responsible for entinostat's anti-tumor activity. Non-Hodgkin lymphoma (NHL) cell lines were exposed to escalating doses of entinostat (0.1 to 20uM) +/− BTZ (1–10nM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Changes in cell cycle were determined by flow cytometric analysis. Subsequently, protein lysates were isolated from entinostat +/− BTZ exposed cells and changes in members of Bcl-2 and cell cycle family proteins were evaluated by Western blotting. Finally, to characterize entinostat's mechanisms-of-action, lymphoma cells were exposed to entinostat with or without pan-caspase (Q-VD-OPh, 5mM) and changes in cell viability were detected. Entinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary tumor cells (N=32). In addition, in vitro exposure of lymphoma cells to entinostat resulted in an increase in G1 and a decrease in S phase. Moreover synergistic activity was observed by combining entinostat with BTZ in vitro. The pharmacological interactions between entinostat and proteasome inhibitor could be explained in part by each agent's effects on the expression levels of cell cycle proteins. In vitro exposure of lymphoma cells to entinostat resulted in p21 upregulation and p53 down-regulation, whereas BTZ exposure lead to up-regulation of Bak and Noxa and downregulation of Mcl-1 and Bcl-XL. Caspase inhibition diminished entinostat anti-tumor activity in RSCL but not in RRCL. Together this data suggests that entinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and independent pathways. Our data suggests that entinostat as a single agent is active against rituximab-chemotherapy sensitive and resistant lymphoma cells and potentiates the anti-tumor activity of BTZ. A better understanding in the molecular events (caspase-dependent and -independent) triggered by entinostat in combination with proteasome inhibition is important in order to develop optimal combination strategies using these novel agents in future clinical trials. Disclosures: Czuczman: Millennium: Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy.

In Vitro and in Vivo Single-Agent Efficacy of Checkpoint Kinase 1 (Chk1) and 2 (Chk2) Inhibitor PF-0477736 (Pfizer) in B- and T-Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1496-1496 ◽  
Author(s):  
Ilaria Iacobucci ◽  
Andrea Ghelli Luserna Di Rorà ◽  
Maria Vittoria Verga Falzacappa ◽  
Enrico Derenzini ◽  
Anna Ferrari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Western Blot ◽  
Cell Lines ◽  
Single Agent ◽  
Time Dependent ◽  
Leukemia Cells ◽  
In Vivo Models

Abstract Abstract 1496 Introduction: Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1- negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5–2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 > 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p < 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

BMS-936564 (MDX1338): A Fully Human Anti-CXCR4 Antibody Induces Apoptosis in an in Vitro Model of Stromal – Leukemia Cell Interaction for Chronic Lymphocytic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2887-2887
Author(s):  
Manoj Kumar Kashyap ◽  
Deepak Kumar ◽  
Harrison Jones Jones ◽  
Michael Y. Choi ◽  
Johanna Melo-Cardenas ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Leukemia Cell ◽  
Cell Interaction ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Drug Induced ◽  
Induced Apoptosis ◽  
Cells Cultured

Abstract Abstract 2887 Chronic lymphocytic leukemia (CLL) remains incurable despite advances in the biology and treatment of this disease. Current data support the notion that resistance to therapy is promoted by a “protective” tumor microenvironment in which non-leukemia cells produce factors that enhance the resistance of CLL cells to spontaneous or drug-induced apoptosis. One such factor is the chemokine CXCL12, which interacts with its receptor CXCR4 on CLL cells to promote cancer cell survival. To examine the therapeutic potential of blocking CXCL12-CXCR4 interactions, we studied the effect of BMS-936564, a fully human IgG4 anti-CXCR4 antibody, using an in vitro co-culture model of human bone marrow derived stomal-NKter cells – leukemia cell interaction. Such stromal-NKter cells secrete CXCL12 and enhance the resistance of CLL cells to apoptosis in vitro. We observed that primary CLL cells co-cultured with stromal-NKter cells had significantly greater viability than CLL cells cultured alone (20–60% above baseline at 48 hours). Moreover, CLL cells co-cultured with stromal cells had enhanced resistance to drug-induced apoptosis. We found that BMS-936564 antibody at concentrations of 2–200nM could enhance the rate of apoptosis of CLL cells cultured alone or in the presence of stromal cells. CLL cells that expressed unmutated IgVH genes or ZAP-70 appeared equally susceptible to treatment with BMS-936564 as did CLL cells that lack these adverse prognostic markers, as did CLL cells that harbored deletions in 17p13.2 and that were resistant to chemotherapeutic agents, such a fludarabine monophosphate. BMS-936564 antibody inhibited CXCL12 mediated F-Actin polymerization in CLL cells at lower concentrations (20–200nM) compared to AMD-3100 (Mozobil), a small molecule CXCR4 inhibitor (50–150μM). In addition, AMD-3100 did not induce apoptosis in CLL cells (10–300μM). In summary, we observed that the anti-CXCR4 antibody BMS-936564 inhibited CXCL12 mediated activation of the CXCR4 receptor in CLL cells and induced apoptosis in leukemia cells. The pro-apoptotic activity of BMS-936564 was observed in cells cultured alone or together with stromal cells suggesting that this antibody had direct cytotoxic effect on leukemia cells and that it can overcome the protective tumor microenvironment. More over, the activity of BMS-936564 was independent of the presence of poor prognostic factors such as del(17p) suggesting that its mechanism of action is P53 independent. These findings show evidence that the CXCR4-CXCL12 pathway is a valid therapeutic target in CLL and provide additional biological rationale for ongoing clinical trials in CLL and other hematological malignancies using BMS-936564. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Kipps:Abbott: Consultancy, Research Funding.

scholarly journals The Glutaminase Activity of L-Asparaginase Mediates Suppression of Asns Upregulation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3959-3959 ◽  
Author(s):  
Wai Kin Chan ◽  
Lin Tan ◽  
Karine Harutyunyan ◽  
Di Du ◽  
Leona Martin ◽  
...  
Keyword(s):  
Cell Death ◽  
Amino Acid ◽  
Anticancer Activity ◽  
Single Agent ◽  
Leukemia Cell ◽  
Cell Types ◽  
Leukemia Cells ◽  
E Coli ◽  
Glutaminase Activity

Abstract L-asparaginase (ASNase) is a standard component of treatment regimens used for acute lymphoblastic leukemia and is being tested against other cancer types, including acute myeloid leukemia, lymphoma, and pancreatic cancer. We and others have reported that the anticancer activity of ASNase requires the enzyme's glutaminase activity, but the underlying glutaminase-mediated mechanism(s) that lead to leukemia cell death are unknown. Glutamine, the most abundant amino acid in the blood, is known for pleiotropic roles in numerous biological pathways, including energy metabolism, redox metabolism, nucleotide anabolism, and amino acid anabolism. Many cancer cells have been found to reprogram their metabolic pathways to become highly dependent on glutamine for survival and proliferation. Glutaminase (GLS/GLS2)-mediated conversion of glutamine to glutamic acid provides the latter as a substrate for conversion to α‐ketoglutarate by transaminases or glutamate dehydrogenases (GLUD1/GLUD2) to fuel the TCA cycle. Consequently, targeting glutamine metabolism has become an attractive strategy for anticancer therapy. The enzyme asparagine synthetase (ASNS) mediates resistance to ASNase through synthesis of asparagine. ASNS is expressed in most cell types, and its expression is upregulated in response to a wide variety of cell stresses, including amino acid limitation and endoplasmic reticulum stress. We and others have shown that ASNS-positive leukemia cells capable of synthesizing asparagine de novo are less responsive than ASNS-negative leukemia cells to ASNase therapy (Chan et al., Blood, 2014). Moreover, ASNase resistance has been associated with elevated ASNS expression. In fact, we have shown that ASNS expression is a predictive marker of the in vitro response of leukemia cell lines and some solid tumor cell types to ASNase. The expression of ASNS in most cells in the body poses a serious challenge for successful therapy with ASNase; for example, production of asparagine by the liver and cells (e.g., mesenchymal stem cells and adipocytes) of the tumor microenvironment may contribute significantly to ASNase resistance in vivo. Here we used the high-glutaminase E. chrysanthemi ASNase (Erwinaze®), wild-type E. coli ASNase (ASNaseWT), and the glutaminase-deficient E. coli mutant, ASNaseQ59L, as models of high, medium-, and low-glutaminase, respectively, to explore ASNase glutaminase activity-mediated mechanisms of leukemia cell death. Unexpectedly, we found that increasing glutaminase activity caused an increase in the suppression of ASNS upregulation in vitro (Figure 1A). In NSG mice injected with luciferase-labeled Sup-B15 cells, single-agent ASNaseWT yielded a durable response approximating cure, whereas glutaminase-deficient ASNaseQ59L yielded a complete response but with recurrence. Together, the results suggest that ASNase glutaminase activity is associated with suppression of ASNS upregulation, making durable, single-agent anticancer activity easier to achieve. Overall, the results provide new insight into the mechanism of action of ASNase. Disclosures Konopleva: Stemline Therapeutics: Research Funding. Weinstein:NIH: Patents & Royalties: L-asparaginase. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties.

The Relative Dependency of Chronic Lymphocytic Leukemia (CLL) Cells on Nurselike Cells for in Vitro Survival Is Associated with Overall Survival in Previously Untreated CLL Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4169-4169
Author(s):  
Danelle James ◽  
Sonia Jain ◽  
Maryann Betty ◽  
Harrison Malone ◽  
Andrew Greaves ◽  
...  
Keyword(s):  
Overall Survival ◽  
Chronic Lymphocytic Leukemia ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Cell Microenvironment ◽  
Relative Viability ◽  
Dependency Index ◽  
In Vitro Survival

Abstract Chronic lymphocytic leukemia (CLL) cells often undergo spontaneous apoptosis in vitro unless co-cultured with accessory cells, such as nurselike cells (NLC), which likely also function in the leukemia-cell microenvironment to promote CLL-cell survival in vivo. However, we observed the CLL cells of different patients varied in their relative dependency on NLC for survival, even when examined on NLC derived from the same donor. We hypothesized that the relative dependency on NLC was an intrinsic characteristic for each CLL cell population and that measurement of the relative degree of NLC-dependency might serve to segregate patients into subgroups with different biologic or clinical behavior. To test this hypothesis, we assessed the cyropreserved primary leukemia cells of 45 untreated CLL patients for their relative dependency on NLC for survival in vitro. NLC were generated as described through co-culture of CLL B cells with isolated CD14+ blood mononuclear cells of healthy donors. The viability of CLL cells cultured with or without NLC was determined after two days co-culture and compared with that of the initial CLL cell viability, which typically was greater than 85%. To assess the survival support provided by NLC to the leukemia cells we determined the relative viability of leukemia cells alone was divided by the viability of CLL cells co-cultured with NLC. CLL NLC dependency index NLC was defined as 1-relative viability. We multiplied this result by 100 and then subtracted the product from 1 to derive the NLCdependency index. The NLC-dependency index for the CLL cells from different patients (n =16) ranged from 0.5–63.7 with a median of 26.9. In contrast, we observed relatively little variation in the NLC-dependency index for any one sample when measured in different experiments (2–4 assays/sample). Furthermore, the NLC-dependency index for CLL cells collected at different times from any one patient also was relatively constant, resulting in an intrapatient variation of in NLC-dependency index of only 5.6%. Using recursive partitioning, we examined the relationship between the NLC-dependency index and time from diagnosis to first treatment for 45 patients. This allowed us to determine the optimal threshold for segregating patients into groups with high versus low NLCdependency. A NLC-dependency of 26 was found the optimal cut point to segregate the 45 patients into 2 groups that exhibited different median times from diagnosis to initial therapy. Using this threshold, the NLC dependency index could reliably segregate the patients into the two categories of high versus low NLC-dependency, even when assessed on CLL cells collected at different time points. There was a significant association between a high NLC dependency index and expression of CD38 (spearman’s rho = 0.4, p=0.008). CD38 is noted to be a receptor for CD31, which is found expressed on NLC. Moreover, when we segregated patients into groups of low versus high NLC-dependency based upon the index cut-off of 26, we found a significant association between high NLC-dependency and expression of CD38 [Fisher’s exact test, p=0.017]. Furthermore, we observed that patients with CLL cells that had a high NLC-dependency index experienced a significantly shorter median survival (4137 days) than did those patients with CLL cells that had a low NLC-dependency index (8276 days), using the log rank test (p=0.0037). (See Figure) The overall survival hazard ratio for NLC dependency was 11.5 (Cox proportional hazards regression, p=0.021). This intriguing inverse relationship between the relative dependency of CLL cells on NLC for in vitro survival and overall patient survival suggests that the microenvironment might play a significant role in the pathophysiology and/or progression of CLL and that agents targeting the leukemia-cell microenvironment might be effective in patients with relatively aggressive disease. Figure Figure

Sign in / Sign up

Export Citation Format

Share Document