Lw-218, a New Flavonoid Compound, Exerts Anti-T-Cell Malignancies Effects Via Autophagy-Mediated Apoptosis

Background:T-cell malignancies are characterized by the excessive proliferation of hematopoietic precursor cells of T-cell lineage lymphocytes in bone marrow. T-cell malignancies consist of T-cell lymphoblastic leukemia (T-ALL) and T-cell non-Hodgkin lymphoma (T-NHL). Effective therapeutic options are limited for T-cell malignancies patients. Apoptosis is one of the most important means to cure cancer. Autophagy, a conserved process that sequesters and targets cellular components for lysosomal degradation, also plays highly context-specific roles in mediating cell death. Our research found that LW-218, a new synthetic flavonoid compound, exerted anti-T-cell malignancies cell lines effects by autophagy-mediated apoptosis induction. Methods: In vitrostudy, T-ALL cell lines (Jurkat, Molt4) and T-NHL cell lines (Hut102, Hut78) were used to investigated LW-218-induced apoptosis effects by survival assays (Flow cytometry, Annexin V/PI). A series of experiment were used to assess the autophagy flux triggered by LW-218, by using mCherry-GFP-LC3 construct, Lysotracker Red staining, and determining the expression ratio of LC3-II/LC3-I. Western blot assay, RT-qPCR, transfection with siRNA, and immunofluorescence were used to investigate the underlying mechanism. In vivostudies, the anti-tumor effects of LW-218 were evaluate in T-ALL xenografts which established by subcutaneous injection of Jurkat cells. Results:Mechanistic studies demonstrated that LW-218-induced cell apoptosis in T-cell malignancies cell lines was correlated with activation of caspase 8, caspase 3 and caspases 9, and the substrate PARP1. In addition, LW-218 induced autophagy flux in T-cell malignancies, as increased ratios of LC3B-II/LC3B-I and reductions of P62 expression. The increased numbers of GFP-mCherry puncta and fluorescent intensity also indicated the increase in autolysosomes. The autophagy-mediated cell death must strictly follow the criteria that the inhibition of autophagy, through either genetic or chemical means, prevents cell death. LW-218-induced cell apoptosis and the activation of caspases3/8/9 could be prevented when the autophagy flux was blocked by autophagy inhibitors. mTOR is a master regulator of cellular metabolism and governs the programmed cell death pathways of apoptosis and autophagy. The results showed that LW-218 inhibited the expression of p-mTOR (Ser2448). And the restored activation of mTOR correlated with the inhibition of autophagy flux, which indicated mTOR might be involved in LW-218-induced autophagy. LW-218 also induced cleavage of Bid, the substrate of caspases 8, which indicated that the apoptosis mediated by autophagy might via caspases 8 and Bid activation. Thein vivostudies also verified that LW-218 inhibited the growth of tumors. Conclusions:Our study provides a new insight into the mechanism of LW-218-induced autophagy-mediated apoptosis, suggesting the potency of LW-218 to be a promising agent against T-cell malignancies. Disclosures No relevant conflicts of interest to declare.

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FV-429 induces autophagy blockage and lysosome-dependent cell death of T-cell malignancies via lysosomal dysregulation

Cell Death and Disease ◽

10.1038/s41419-021-03394-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Po Hu ◽

Jubo Wang ◽

Yingjie Qing ◽

Hui Li ◽

Wenzhuo Sun ◽

...

Keyword(s):

Cell Death ◽

T Cell ◽

Single Agent ◽

Tumor Development ◽

Flavonoid Compound ◽

Autophagy Flux ◽

Dependent Cell ◽

T Cell Malignancies ◽

Chemotherapy Agents ◽

Autophagy Inhibition

AbstractIt is widely accepted that lysosomes are essential for cell homeostasis, and autophagy plays an important role in tumor development. Here, we found FV-429, a synthetic flavonoid compound, inhibited autophagy flux, promoted autophagosomes accumulation, and inhibited lysosomal degradation in T-cell malignancies. These effects were likely to be achieved by lysosomal dysregulation. The destructive effects of FV-429 on lysosomes resulted in blockage of lysosome-associated membrane fusion, lysosomal membrane permeabilization (LMP), and cathepsin-mediated caspase-independent cell death (CICD). Moreover, we initially investigated the effects of autophagy inhibition by FV-429 on the therapeutic efficacy of chemotherapy and found that FV-429 sensitized cancer cells to chemotherapy agents. Our findings suggest that FV-429 could be a potential novel autophagy inhibitor with notable antitumor efficacy as a single agent.

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NKL Homeobox Gene MSX1 Reactivates An Oncogenic Network In Lymphoid and Myeloid Malignancies

Blood ◽

10.1182/blood.v122.21.3765.3765 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3765-3765

Author(s):

Stefan Nagel ◽

Stefan Ehrentraut ◽

Corinna Meyer ◽

Maren Kaufmann ◽

Hans G. Drexler ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Molecular Cytogenetics ◽

Homeobox Gene ◽

In Silico Analysis ◽

Reciprocal Regulation ◽

Myeloid Malignancies

Abstract Homeobox genes encode conserved transcription factors (TFs) which regulate fundamental cellular processes during development. Many members of the NKL homeobox gene subfamily are aberrantly expressed in T-cell leukemia and compromise cell differentiation. NKL homeobox gene MSX1 is expressed during embryonic hematopoiesis and its deregulation in Hodgkin lymphoma suggests an oncogenic role of this gene in hematopoietic malignancies. After screening 114 leukemia/lymphoma cell lines by microarray profiling, we detected MSX1 overexpression in three examples each from T-cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL), and one from acute myeloid leukemia (AML). In silico analysis by R-based statistical tools identified conspicuous expression of MSX1 in 11% of pediatric T-ALL patients, and in 3% each of MCL and AML patients. Thus, we found aberrant MSX1 expression in subsets of both lymphoid and myeloid malignancies. Focusing on MCL and AML we excluded chromosomal rearrangements by classical and molecular cytogenetics at the MSX1 locus underlying overexpression in affected cell lines. However, comparative expression profiling data indicted aberrant histone acetylation involving PHF16 and RTN1, together with TFs FOXC1, HLXB9 and TAL1, as activators of MSX1 transcription. Their involvement was confirmed by siRNA-mediated knockdown and overexpression studies. Reciprocal regulation of MSX1 involved CCND1 and NOTCH signalling. Reporter gene analyses demonstrated that CCND1 and CDKN2D are direct transcriptional targets of MSX1 and its repressive cofactor histone H1C. Fluorescence in situ hybridization showed that t(11;14)(q13;q32) in MCL results in detachment of CCND1 from its corresponding repressive MSX1 binding site. In conclusion, we uncovered a regulatory network around MSX1 in leukemia/lymphoma cells, involving factors and pathways implicated in embryonic hematopoiesis. The reciprocal regulation of MSX1 and the NOTCH pathway in B-cells parallels that of MSX2 in T-cells. These data support the view of a recurrent genetic network involved in hematopoietic development which is reactivated in malignant transformation. Disclosures: No relevant conflicts of interest to declare.

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Identification of a New Targeted Therapy for T-Cell Acute Lymphoblastic Leukemia and Studies on Its Mechanism of Action

Blood ◽

10.1182/blood-2019-129823 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2752-2752

Author(s):

Kinjal Shah ◽

Julhash U. Kazi

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Predictive Biomarkers ◽

Protein Kinase Inhibitors ◽

Maturation Stage ◽

Mrna Levels ◽

Cell Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy, of which T- cell acute lymphoblastic leukemia (T-ALL) constitutes an aggressive subset. Due to the advent of new therapies, T-ALL now has a 5-year event-free survival (EFS) rate exceeding 85%. However, some patients still relapse and display resistance to therapy. Moreover, adverse side-effects of intensive chemotherapy worsen the duration of treatment. Therefore, we still need to improve our current treatment beyond that of the chemotherapeutic approaches. It has been shown that the maturation stage of T-ALL decides its dependency on Bcl-2/Bcl-xL. The immature early T cell progenitor ALL (ETP-ALL) rely on Bcl-2 for their survival while all the other stages of T-ALL and primary patient samples depend on Bcl-xL. Bcl-2 inhibitors have thus shown to display promising antitumor activity against ETP-ALL, a subgroup with a high risk of relapse, but with a variable response across these patients. Therefore, there is a need for predictive biomarkers and further investigation towards finding a combination of drugs for the treatment of these patients. Methodology & Aim: We screened 10 different T-ALL cell lines with a combination of Bcl-2 inhibitor and a panel of 378 protein kinase inhibitors and identified polo-like kinase inhibitor as a promising candidate. We thus aimed to study the combined effect of Bcl-2 and PLK1 inhibition in a panel of T-ALL cell lines and in a PDX model of chemo-resistant childhood T-ALL. We also investigated the underlying mechanism of drug synergy by various biochemical assays. Results: Cell viability of 14 T-ALL cell lines was determined after being subjected to Bcl-2 inhibitor (ABT-199) and PLK1 inhibitor (BI-6727). All cell lines responded well to BI6727 with an EC50 of less than 70nM. However, they showed differential response to ABT199 with only 3 cell lines being sensitive with an EC50 of less than 40nM. The mRNA levels of Bcl-2, Bcl-xL and PLK 1, 2, 3 and 4 were determined by qRT-PCR. PLK1 was found to be highly expressed in all the cell lines as compared to the rest of the 3 PLK family proteins. ABT-199-sensitive cell lines showed lower Bcl-xL mRNA levels irrespective of their Bcl-2 expression, and displayed synergy with BI-6727. A higher degree of apoptosis was also observed in the combination treatment as compared to a single drug. Immunoblot analysis revealed cleavage of PARP1 and lower levels of c-Myc and MCL1 expression in the presence of both ABT-199 and BI-6727. Conclusions: Upregulation of the anti-apoptotic BCL2 family members is one of the canonical ways for cancer cells to escape apoptosis. In the past years, several highly selective and potent BCL2 inhibitors have been developed and showed promising efficacy in various cancers. We found that the sensitivity of T-ALL cell lines to ABT-199 is largely determined by the lower levels of Bcl-xL expression. Furthermore, ABT-199 displays synergy with the PLK inhibitor. T-ALL cell lines predominantly express PLK1 and thus the combinatorial effect of ABT-199 and BI-6727 is mediated through the pharmacological inhibition of both BCL2 and PLK1. Currently, we are generating iRFP-expressing T-ALL cell lines which will be used to check drug efficacy in vivo. Furthermore, we have collected chemo-resistant PDX cell lines which will be used to verify the cell line data. Besides its role in cell cycle control, we still have very limited knowledge about the function of PLK1 in leukemia. Thus, studying its role in T-ALL cell lines by knocking down PLK1 with CRISPR/Cas9 technology will provide an important insight. Disclosures No relevant conflicts of interest to declare.

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Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Blood ◽

10.1182/blood.v124.21.3569.3569 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3569-3569

Author(s):

Ye Shen ◽

Chun Shik Park ◽

Koramit Suppipat ◽

Takeshi Yamada ◽

Toni-Ann Mistretta ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Tumor Suppressor ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Brdu Incorporation ◽

Jnk Pathway

Abstract Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies. KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL. To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status. Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. Disclosures No relevant conflicts of interest to declare.

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Sequential Induction of Cell Cycle Arrest and Mitotic Catastrophe Followed By Apoptosis Via Activation of PTEN By GL-V9 in Human T-Cell Malignancies

Blood ◽

10.1182/blood.v128.22.5146.5146 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5146-5146

Author(s):

Hui Hui ◽

Jingyan Xu

Keyword(s):

Cell Death ◽

T Cell ◽

Conflicts Of Interest ◽

Mitotic Catastrophe ◽

Anticancer Activities ◽

High Concentration ◽

Lymphoid Tumor ◽

Western Blot Assay ◽

Human T Cell ◽

T Cell Malignancies

Abstract GL-V9, a newly synthetic flavonoid derivative, is an active cytotoxic component and exhibits anticancer activities. Here, we demonstrated the sequence of death reponses to different concentration of GL-V9 in human T-cell malignancies cell Jurkat and HuT78. Responses included transient accumulation of cells at the G2/M border followed by also transient rise in several mitotic markers and mitotic attempts were largely abnormal, resulting in numerous micronucleated and multinucleated cells in low drug concentration (2 μM), as shown by flow cytometre analyses and Giemsa staining. These events, indicative of mitotic catastrophe, were not associated with immediate cell death. However, MTT assay showed that high concentration of GL-V9 (4, 8, 16 μM) causes near complete cell growth inhibition at 12 h. Also, the classical manifestations of apoptosis (including phosphatidylserine externalization, mitochondrial dysfunction, and caspase activation) were marginal at 24 h, as shown by Annexin V-PI staining, JC-1, and western blot assay. The mechanism research reveals that low dose of GL-V9 induced mitotic catastrophe via decreasing EEA1, which is required for cytokinesis, and increasing the expression of PTEN. Besides, high concentration of GL-V9 induced cell apoptosis by up-regulating the expression of apoptosis-related proteins caspase 3 and Bim in lymphoid tumor cell lines and primary ALL cells. Collectively, these findings show that different concentrations of GL-V9 can induce mitotic catastrophe and ultimate cell death event of human T-cell malignancies respectively, which would be a potential therapeutical compound for treating human lymphoid tumor. Disclosures No relevant conflicts of interest to declare.

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New LMO2 Translocations in T-Cell Acute Lymphoblastic Leukemia, Involving STAG2 at Xq25 and MBNL1 at 3q25: A Positive Change?.

Blood ◽

10.1182/blood.v114.22.2641.2641 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2641-2641

Author(s):

Suning Chen ◽

Stefan Nagel ◽

Bjoern Schneider ◽

Maren Kaufmann ◽

Ursula R. Kees ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

B Cell Lymphoma ◽

Small Sample ◽

Upstream Region ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2641 Poster Board II-617 Background: In T-cell acute lymphoblastic leukemia (T-ALL) the LMO2 transcription factor locus is juxtaposed with T-cell receptor (TCR) genes by a recurrent chromosome translocation, t(11;14)(p13;q11). Recent molecular cytogenetic data indicate that unlike classical TCR rearrangements, t(11;14) operates synonymously with submicroscopic del(11)(p13p13) by removing a negative upstream LMO2 regulator (Dik et al., Blood 2007;110:388). The combined incidence of both LMO2 rearrangements is ∼10-15% (Van Vlierberghe and Huret, Atlas Genet Cytogenet Oncol Haematol, November 2007). However, aberrant LMO2 expression occurs in nearly half of all T-ALL cases, a discrepancy which may indicate a significant contribution by cryptic chromosome alterations. We attempted the extended characterization of the LMO2 genomic region in T-ALL cell lines to look for such rearrangements. Cells and Methods: We investigated a panel of 26 well characterized and authenticated T-ALL cell lines using parallel fluorescence in situ hybridization (FISH) with a tilepath BAC/fosmid contig and both conventional and quantitative reverse transcriptase (Rq)-PCR. Global gene expression was additionally measured in some cell lines by Affymetrix array profiling. Results: LMO2 rearrangements were detected in 5/26 (19.2%) cell lines including both established rearrangements, t(11;14) and del(11)(p13p13) in one cell line apiece (3.8%). Interestingly, we found two novel LMO2 translocations: t(X;11)(q25;p13) in 2/26 (7.7%), and t(3;11)(q25;p13) in 1/26 (3.8%) cell lines, respectively. Comparing transcription levels in cell lines with and without genomic rearrangements showed that LMO2 expression was significantly higher in T-ALL cell lines carrying LMO2 rearrangements (P<0.001). Rq-PCR revealed that 5 of the top 10 (50%) LMO2 expressing cell lines carry cytogenetic rearrangements at this locus, compared to 0/16 remaining examples. Loss of a recently defined LMO2 negative regulatory element was identified in the del(11)(p13p13) cell line but no other deletions were detected. Two genes STAG2 at Xq25 and MBNL1 at 3q25 were identified as novice LMO2 partners in t(X;11) and t(3;11), respectively. In both genes breakpoints lay at intron 1 close to deeply conserved noncoding regulatory regions. Both t(X;11) cell lines displayed conspicuous silencing of the ubiquitously expressed STAG2 gene highlighting the transcriptional significance of the region displaced. Unlike t(11;14)/del(11)(p13p13) both new rearrangements carry LMO2 breakpoints in the far upstream region (at minus 80–150 Kbp), and appear to result in upregulation of LMO2 by juxtaposition rather than via covert deletion. STAG2 is a component of the chromosomal cohesin complex which acts as a transcriptional coactivator, and which has been recently identified as a potential driver of oncogene transcription in acute myeloid leukemia (Walter et al., Proc Natl Acad Sci U S A. 2009;106:1295). MBNL1 controls RNA splicing and is a rare BCL6 partner gene in B-cell lymphoma, but this is the first report of its involvement in T-ALL. Conclusion: Given their frequency and variety in a small sample, we propose that cryptic chromosome rearrangements targeting LMO2 upregulation may be significantly more frequent than hitherto appreciated in T-ALL. Unlike canonical LMO2 rearrangements, both t(X;11) and t(3;11) would appear to function positively by upregulation of LMO2 via juxtaposition with noncoding driver elements within these novel partner genes. Perspectives: Future work will address the regulatory potential of candidate enhancer sequences embedded within conserved noncoding intronic sequences of MBNL1 and STAG2. Cytogenetically inconspicuous cell lines displaying LMO2 upregulation will be subjected to more detailed scrutiny using high density genomic SNP arrays. Disclosures: No relevant conflicts of interest to declare.

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Obinutuzumab (GA101) Significantly Enhances Cell Death and ADCC Compared to Rituximab Against CD20+ sensitive and Rituximab Resistant B-Cell Non-Hodgkin Lymphoma (NHL) and Lymphoblastic Leukemia (BLL)

Blood ◽

10.1182/blood.v120.21.4865.4865 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4865-4865 ◽

Cited By ~ 1

Author(s):

Aradhana Awasthi Tiwari ◽

Janet Ayello ◽

Carmella van de Ven ◽

Danielle Glassman ◽

Anthony Sabulski ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Nk Cells ◽

Cell Lines ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Dismal Prognosis

Abstract Abstract 4865 Background: Patients who relapse with CD20+ B-NHL and B cell lymphoblastic leukemia (B-LL) have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012,Mils/Cairo et al. BJH,2012) and often require alternative therapeutic strategies. Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Cairo M.S. et al. ASCO, 2010). Resistance to RTX, however, may predispose patients with CD20+ NHL to an increase risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, mediates enhanced cell death vs RTX and has a glycoengineered Fc region that induces significantly enhanced ADCC (Mössner et al. Bld, 2010; Niederfellner G. et al. Bld, 2011; Bologna L et al. JI, 2012). Objective: To evaluate the in-vitro efficacy of GA101 compared to RTX against RTX sensitive and resistant CD20+ B-NHL and B-LL cell lines. Methods: Raji (CD20+,ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany), Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) and Raji-2R and Raji-4RH (generously supplied by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS and incubated with GA101 and/or RTX at 100 μg/ml for 24 hrs (n=6), 48 and 72 hrs (n=5). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. Loucy cells (CD20−) were used as the negative control. The caspase 3/7 activity was measured by FAM caspase 3/7 assay kit by FLICA™ methodology. RSCL, RRCL, U698-M and Loucy were incubated with GA101 and RTX treatment for 24, 48 and 72 hrs, and caspase3/7 activity was detected by FACS using 488 nm excitation and emission filter (n=3). ADCC were performed with K562-IL-15–41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) as well as IL-2 expanded NK cells, at 20:1 effector: target ratio (E: T, n=3) using europium release assay (Perkin-Elmer). Results: GA101 induced significantly more cell death compared to RTX in B-NHL and BLL cell lines. (Table-1) GA101 vs RTX shows a significantly increase in caspase 3/7 activity in Raji 16.92±0.84% vs 11.76±0.08% compared to Raji2R 6.7±0.62% vs 2.8±0.7%, Raji4RH 5.8±0.35% vs 2.0±0.3% and U698-M 12.54±0.44% vs 9.6±0.95% compared to Loucy 3.22±0.45% vs 2.59±0.05%, respectively, at 24 hrs of treatment (p<0.0001). GA101 vs RTX also elicited a significant increase a ADCC with K562-IL15–41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% compared to Raji-2R 38.0±2.0% vs 21.6±1.2%, Raji-4RH 40.0±1.6% vs 0.5±1.1% and U698-M 70.0±1.6% vs 45.56±0.1%, compared to Loucy 21.67±0.48% vs 15.92±0.52%, respectively (p<0.001) at day 7.The IL-2 alone expanded Hu-NK cells demonstrated a reduction of 10–20% cytotoxicity compared to K562-IL15–41BBL Hu-NK cells at day 7 against BLL, RSCL and RRCL, in-vitro. Conclusion: Obinutuzumab compared to RTX significantly enhanced cell death, caspase3/7 activity and NK mediated ADCC in sensitive and RTX resistant B-NHL and B-LL. Obinutuzumab represents a promising candidate for treating RTX sensitive and resistant CD20+ B-Cell Lymphomas and lymphoblastic leukemia. Further studies will investigate the combination of activated NK cells or chemotherapy that may enhance or synergize with the efficacy of GA101 (Obinutuzumab) both in -vitro and in-vivo in xenografted NOD/SCID mice. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of InsP3R with Xestospongin B Reduces Mitochondrial Respiration and Induces Selective Cell Death in T Cell Acute Lymphoblastic Leukemia Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22020651 ◽

2021 ◽

Vol 22 (2) ◽

pp. 651

Author(s):

Pablo Cruz ◽

Ulises Ahumada-Castro ◽

Galdo Bustos ◽

Jordi Molgó ◽

Daniela Sauma ◽

...

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Lines ◽

Mitochondrial Respiration ◽

Lymphoblastic Leukemia ◽

Specific Inhibitor ◽

Prolonged Treatment ◽

Cell Acute Lymphoblastic Leukemia ◽

Trisphosphate Receptor

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy whose chemoresistance and relapse persist as a problem despite significant advances in its chemotherapeutic treatments. Mitochondrial metabolism has emerged as an interesting therapeutic target given its essential role in maintaining bioenergetic and metabolic homeostasis. T-ALL cells are characterized by high levels of mitochondrial respiration, making them suitable for this type of intervention. Mitochondrial function is sustained by a constitutive transfer of calcium from the endoplasmic reticulum to mitochondria through the inositol 1,4,5-trisphosphate receptor (InsP3R), making T-ALL cells vulnerable to its inhibition. Here, we determine the bioenergetic profile of the T-ALL cell lines CCRF-CEM and Jurkat and evaluate their sensitivity to InsP3R inhibition with the specific inhibitor, Xestospongin B (XeB). Our results show that T-ALL cell lines exhibit higher mitochondrial respiration than non-malignant cells, which is blunted by the inhibition of the InsP3R. Prolonged treatment with XeB causes T-ALL cell death without affecting the normal counterpart. Moreover, the combination of XeB and glucocorticoids significantly enhanced cell death in the CCRF-CEM cells. The inhibition of InsP3R with XeB rises as a potential therapeutic alternative for the treatment of T-ALL.

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Genome-Wide Analysis Reveals Conserved and Divergent Features of Notch1/RBPJ Binding in Human and Murine T Lymphoblastic Leukemia Cells

Blood ◽

10.1182/blood.v118.21.5236.5236 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5236-5236

Author(s):

Jon C Aster ◽

Hongfang Wang ◽

James Zou ◽

Yumi Yashiro-Ohtani ◽

Bo Zhao ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Cell Lines ◽

Binding Sites ◽

Target Genes ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Human T Cell ◽

Two Factors

Abstract Abstract 5236 Activated Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is an important oncoprotein in murine and human T cell acute lymphoblastic leukemia/lymphoma (T-ALL), yet the interplay between Notch1 and other factors that regulate the transcriptional output of T-ALL cells is poorly understood. Using ChIP-Seq and starting with Notch1-dependent human and murine T-ALL cell lines, we find that Notch1 binds preferentially to promoters, to RBPJ binding sites, and near sites for ZNF143, as well as Ets and Runx factors. By ChIP-Seq, ZNF143 binds to ∼40% of Notch1 sites, whereas Ets1 binding is observed within 100 basepairs of ∼70% of genomic Notch1 binding sites. Notch1/ZNF143 “co-sites” have high Notch1 and ZNF143 signals, frequent co-binding of RBPJ to sites embedded within ZNF143 motifs, strong promoter bias, and low mean levels of “activated” chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in T-ALL cell lines. In contrast, Ets1 binding sites flank RBPJ/Notch1 binding sites and are associated with high levels of activated chromatin marks, whereas Runx sites are predominantly intergenic. Although Notch1 predominantly binds promoters, ∼75% of direct Notch1 target genes lack promoter binding and appear to be regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R and the GIMAP gene cluster. Both Ets1 and Notch1 binding to an intronic enhancer located in DTX1 were required for expression of this well characterized Notch1 target gene, suggesting that these two factors coordinately regulate DTX1 expression. Although the association of Notch1 binding with ZNF143, Ets, and Runx sites was highly conserved, binding near certain important genes showed substantial divergence. For example, in human T-ALL lines Notch1/RBPJ bind a 3' enhancer near the IL7R gene, whereas in murine T-ALL lines no binding was observed near Il7r. Similarly, in human T-ALL lines Notch1/RBPJ bound an enhancer located ∼565 kb 5' of MYC, whereas in murine T-ALL cells Notch1/RBPJ bound an enhancer located ∼1 Mb 3' of Myc. Human and murine T-ALL genomes also have many sites that bind only RBPJ. Murine RBPJ “only” sites are highly enriched for imputed sites for the corepressor REST, whereas human RPBJ “only” sites lack REST motifs and are more highly enriched for imputed CREB binding sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in T-ALL cell lines, as well as novel classes of divergent RBPJ “only” sites that also likely regulate transcription. To extend these findings to normal and pathophysiologic tissues, ChIP-Seq was used to identify RBPJ/Notch1 binding sites in primary murine thymocytes and primary murine T-ALL associated with Notch1 gain-of-function mutations. Early findings appear to indicate that primary T-ALLs closely resemble normal DN3a thymocytes in terms of the distribution of Notch1 binding sites and associated chromatin marks. These data suggest that Notch1-driven T-ALLs epigenetically resemble the DN3a stage of T cell development, during which Notch1 signaling is high and cells are rapidly proliferating. Disclosures: No relevant conflicts of interest to declare.

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YB-1 Is Activated by IL-7 and Is Overexpressed in Pediatric Pre-B Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.1468.1468 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1468-1468

Author(s):

Amina Kariminia ◽

Sabine M Ivison ◽

Vivian Leung ◽

Sandra E. Dunn ◽

Aru Narendran ◽

...

Keyword(s):

Flow Cytometry ◽

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

Signaling Pathways ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

B Cell Lymphoma ◽

Fluorescent Intensity

Abstract Abstract 1468 Background: Increased YB-1 expression correlates with poor prognosis, drug resistance and metastasis in several different cancers including B cell lymphoma. Phosphorylation and nuclear localization of YB-1 in response to growth factors leads to increased survival through expression of proteins such as survivin and multidrug resistance protein 1. Until now, its role in leukemia has not been investigated. We hypothesized that YB-1 expression is aberrantly regulated in pediatric pre-B acute lymphoblastic leukemia (pre-B ALL), and that YB-1 may be activated downstream of IL-7. This cytokine facilitates the differentiation and survival of pre-B cells and has been implicated in the drug resistance of pre-B ALL. Methods: YB-1 and IL-7Ra protein expression was investigated by flow cytometry in normal pre-B cells (CD19+CD10+CD20−), and mature B cells (CD19+CD10−CD20+) as well as diagnostic and relapsed pre-B ALL (CD19+CD10+/−). Cell surface and cytoplasmic expression was quantified by mean fluorescent intensity (MFI). Bone marrow from healthy donors was used as a source of normal pre-B cells, while mature B cells were derived from PBMCs; leukemic cells at presentation and relapse were obtained following local IRB approval and informed consent. Activation of YB-1 downstream of IL-7 stimulation (25 ng/ml) was examined in pre-B ALL cell lines or NSG (NOD scid gamma) mice-expanded pre-B ALL by Western blotting using anti-phosphoYB-1(S012). Pre-B ALL cell lines used in these experiments were 697, 380, RCH and RS-4;11. Signaling pathways were investigated by pre-treatment of cells with pharmacological inhibitors followed by Western analyses. For the transient overexpression of YB-1, pEGFP or a pEGFP/YB-1 fusion protein was electroporated into freshly isolated mature B cells (which have a low basal expression of YB-1) and YB-1 and IL-7Ra expression was assessed by flow cytometry after 24 h. Results: While intracellular YB-1 expression was significantly higher in leukemia samples at presentation compared to normal pre-B cells, the highest YB-1 levels were found in relapsed pre-B ALL (see figure, part A). All examined pre-B ALL cell lines had levels comparable to those of the relapse samples. Similarly, surface IL-7Ra (CD127) levels (MFI medians; upper-lower range) were increased in onset (221; 150–286), and relapsed (1840; 651–2030) ALL compared to normal pre-B cells (528; 333–2673). (normal pre-B vs. leukemia at presentation, p<0.001, Mann-Whitney). Overexpression of YB-1-GFP in normal mature B cells resulted in increased expression of IL-7Ra (see figure, part B), suggesting an link between the YB-1 and IL-7 signaling pathways. Activated YB-1 is phosphorylated on S102 and relocated to the nucleus. Addition of IL-7 to pre-B ALL cell lines led to phosphorylation of YB-1 within 30 min. Similar results were shown for patient-derived, NSG mice-expanded pre-B ALL samples. Intracellular immunostaining using Imagestream technology (Amnis) showed that IL-7 treatment of pre-B ALL cell lines increased nuclear YB-1 levels 4-fold. As PI3K and MEK1 are involved in signaling downstream of IL-7, we investigated their role in YB-1 signaling in both pre-B ALL cell lines and NSG-mouse expanded pre-B ALL using pharmacological inhibitors. Western analyses showed that inhibition of PI3K using LY294002 did not prevent IL-7-mediated phosphorylation of YB-1 but the MEK1 inhibitor U0126 did, indicating the involvement of MAPK (see figure, part C). Conclusion: We show that YB-1, which is highly expressed in pediatric pre-B ALL compared to normal pre-B cells, is expressed at even higher levels after relapse. We demonstrate a link between the YB-1 and IL-7 signaling pathways which could offer a novel target for the treatment of refractory leukemia. Disclosures: No relevant conflicts of interest to declare.

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