scholarly journals The Transcription Factor TAp63 Exerts Pro-Survival Effects in Chronic Lymphocytic Leukemia Acting through the BCL2 Pathway

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3110-3110
Author(s):  
Stamatia Laidou ◽  
Stavroula Ntoufa ◽  
Sofia Papanikolaou ◽  
Konstantia Kotta ◽  
Maria Koutroumani ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
Cell Survival ◽  
Mrna Expression ◽  
Cell Line ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Mrna Levels

Abstract Recent evidence indicates that TAp63, the prevalent isoform of TP63 in Chronic Lymphocytic Leukemia (CLL), is implicated in disease pathogenesis. In CLL, TAp63 expression, modulated by both immune signaling and epigenetic modifications, promotes leukemic cell survival and homing to the bone marrow. In activated normal B cells, the TAp63 transcription factor binds the BCL2 gene, participating in an anti-apoptotic pathway (axis NF-κB/TAp63/BCL2) augmenting cell survival. In this study, we investigated the expression of TAp63 in a large cohort of CLL cases and its potential fluctuation during disease progression. Additionally, in order to further understand the pro-survival role of TAp63 in CLL, we interrogated at the molecular level the interplay betweenΤAp63 and BCL2. Initially, using RT-qPCR we quantified TAp63 mRNA expression in 166 CLL patients, consisting of 89 with unmutated IGHV genes (U-CLL) and 77 with mutated IGHV genes (M-CLL), prior to administration of treatment. Significantly higher TAp63 mRNA levels were observed in U-CLL vs M-CLL (FD=13.83, p<0.0001). However, outliers were identified in both subgroups, prompting us to re-classify all cases into TAp63high and TAp63low subgroups using ROC curve and Youden index statistical procedures. TAp63high patients displayed significantly shorter time-to-first-treatment (TTFT) (TAp63highmedian TTFT: 1.58 years; TAp63lowmedian TTFT: 4.07 years; p=0.03) and shorter overall survival (OS) (TAp63highmedian OS: 7.825 years; TAp63lowmedian OS: not yet reached; p=0.046). Next, we analyzed TAp63 mRNA expression in longitudinal samples of 25 U-CLL cases treated with either FCR or rituximab-chlorambucil. In each case, samples were collected at three 'landmarks'; diagnosis, first progressionand first relapse. Expression analysis by RT-qPCR showed that TAp63 levels significantly increased at disease relapse compared to diagnosis (FD=3.47, p=0.02). We subsequently investigated links between TAp63 and BCL2 by measuring BCL2 mRNA levels in 56 U-CLL cases from the present cohort and found statistically significant correlation with the corresponding TAp63 mRNA levels (spearman rho=0.31, p=0.01). To validate this observation, we undertook functional studies in the MEC1 CLL cell line. Considering that MEC1 cells express high TAp63 mRNA levels, we generated a stable MEC1 cell line to inducibly downregulate TAp63, using CRISPR/dCas9-KRAB upon treatment with doxycycline (Dox). We used 2 different guide RNAs (sgRNAs; sgRNA1, sgRNA2) targeting 2 distinct regions of the endogenous TAp63 promoter. After 5 days of induction, the expression levels of both TAp63 and BCL2 were quantified by one step RT-qPCR in Tet-on-dCas9-KRAB-sgRNA-TAp63 MEC1 cells. Inducible downregulation of TAp63 expression (gRNA1: FD=1.7, gRNA2: FD=1.53) resulted in downregulation of BCL2 expression (gRNA1: FD=1.34, gRNA2: FD=1.12) with strong correlation (rho=0.97, p<0.0001) between TP63 and BCL2 mRNA levels. Furthermore, we also observed correlation between TAp63 and BCL2 protein expression in primary cells of one representative TP63high CLL case (rho=0.94, p=0.01), in which TAp63 was silenced by RNA interference (RNAi) with 3 different siRNAs. Prompted by these results, we additionally assessed ex vivo the effect of the BCL2 inhibitor Venetoclax in primary CLL cells of both TAp63high (n=8) and TAp63low (n=6) cases. Cell viability was measured by flow cytometry at 24 and 48 hours after treatment. TAp63high cases were more resistant to treatment with Venetoclax as they showed no statistically significant reduction in cell viability compared to the respective (DMSO-treated) controls, in contrast to TAp63low cases (24h: FD=3.63, p=0.004; 48h: FD=7.17, p=0.005). In conclusion, we provide evidence suggesting that up-regulated TAp63 expression represents a novel resistance mechanism to chemoimmunotherapy in CLL. The pro-survival role of TAp63 is supported by its strong association with BCL2. Indeed, based on the present findings, TAp63 appears to act as a positive modulator of BCL2 in CLL cells, rendering them less responsive to apoptosis induction with the BCL2 inhibitor Venetoclax. Disclosures Hadzidimitriou: Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Chronic Lymphocytic Leukemia (CLL) Cells Require Microenvironmental Stimuli to Resist Apoptosis through Activation of STAT3 Mediated by VEGF.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1069-1069
Author(s):  
Iris Gehrke ◽  
Julian Paesler ◽  
Rajesh Kumar Gandhirajan ◽  
Regina Razavi ◽  
Alexandra Filipovich ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Lymphocytic Leukemia ◽  
Vegf Receptor ◽  
Mrna Levels ◽  
Activated State ◽  
Survival Effect

Abstract B-cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, but incompetent B-cells due to a decrease of apoptosis rather than an increase in proliferation. Vascular endothelial growth factor (VEGF) has been suggested to play an important role in this so called apoptotic block. However, so far little is understood whether VEGF is acting mainly as a microenvironmental stimulus and/or whether CLL cells themselves contribute to the enhanced apoptotic resistance by maintaining an autocrine VEGF loop. Moreover, it is unknown by which mechanisms VEGF prevents apoptosis and whether this can be circumvented by inhibition of VEGF signaling. By quantitative real time PCR we found no significant difference in mRNA VEGF levels in B-cells from CLL patients and healthy donors after isolation from blood. In contrast, ELISA revealed clearly increased levels of secreted VEGF in plasma of CLL patients and in the supernatant under culture conditions compared to healthy individuals. In addition, we found the VEGF receptor 2 (VEGFR2), which is existent in CLL and healthy B-cells, in a phosphorylated, hence activated state, to a significantly higher extent in CLL cells as assessed by intracellular phospho flow cytometry. In conclusion, despite its expression in healthy B-cells VEGF does not seem to be secreted and therefore, no VEGF receptor phosphorylation takes place. Whereas CLL cells exhibit a long life span in vivo, they die rapidly in vitro, suggesting major survival factors being existent in the CLL cells microenvironment. We found levels of secreted VEGF in supernatant decreasing with time in culture, going along with decreasing levels of phosphorylated VEGFR2 and increasing cell death as assessed by Annexin V-FITC/PI staining. This further supports the role of VEGF in CLL cell survival. Coculturing primary CLL cells with the bone marrow stromal derived cell line HS5 dramatically increased VEGF transcription and secretion and improved cell survival. Hence, VEGF expression in CLL cells is not only mediated by autocrine, but also paracrine stimuli involving bone marrow stromal. Knocking down VEGF in HS5 cells and subsequent coculture with CLL cells might prove the major role of VEGF in this survival supporting coculture setting. Besides coculturing also supplement of culture medium with recombinant human VEGF (rhVEGF) increased survival, but to a lesser extent than coculture, indicating a direct cell-cell interaction as advantageous. Furthermore, we found a downregulation of anti apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP), myeloid cell leukemia 1 (MCL1) and BclXL upon VEGF stimulation. Also cyclinD1 was upregulated as seen by immunoblotting. We further tried to discover the underlying mechanism of how VEGF mediates its pro survival effect and found STAT3 to become phosphorylated on tyrosine 705 upon VEGF stimulation. In CLL STAT3 is known to be constitutively phosphorylated on serine 727. This phosphorylation is not sufficient to induce target gene expression though. We could show that Y705 phosphorylation of STAT3 is responsible for upregulation of anti apoptotic BCLXL and cyclinD1. A PCR array detecting mRNA levels of 84 transcription factors in untreated and VEGF stimulated CLL cells shall provide more information about mechanistical details how VEGF mediates it pro survival effect. Since VEGF seems to be a major player in CLL cell survival it might be a suitable target to overcome the apoptotic block. In first experiments we found an induction of apoptosis after neutralization of VEGF or inhibition of the VEGF receptor. This additionally highlights the severe importance of VEGF in the apoptotic block in CLL cells. Therefore, VEGF might serve as an excellent therapeutic target in CLL.

scholarly journals Intraclonal Diversification Occurs in Chronic Lymphocytic Leukemia Expressing B Cell Receptors Belonging to the IGHV4 Gene Family

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 944-944
Author(s):  
Filippo Vit ◽  
Francesca Maria Rossi ◽  
Tiziana D'Agaro ◽  
Tamara Bittolo ◽  
Antonella Zucchetto ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Excision Repair ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Direct Role ◽  
Region Gene ◽  
Mutational Status ◽  
Ighv Gene

Abstract Background. The pivotal role of the Immunoglobulin (Ig) receptor and antigenic stimulation have been proven to be landmarks for the understanding of the ontogeny and evolution of chronic lymphocytic leukemia (CLL). In addition, the mutational status of the Immunoglobulin Heavy-Chain Variable region gene (IGHV) was confirmed to be a reliable prognostic factor, supporting an antigen-driven model of CLL development. To clarify aspects regarding an antigenic involvement in CLL evolution, studies focusing on intraclonal diversification (ID) of Ig genes have provided relevant information, although mainly conducted in a pre-Next Generation Sequencing (NGS) era. Aim. To apply a NGS approach to investigate ID in CLL. Methods. IGHV genes from 530 CLL patients with Royal Masden Hospital score 4-5 (Fig. 1A) was sequenced using NGS (Lymphotrack). The bio-informatic pipeline was based on the pRESTO/ChangeO packages. Specific pathological clones were selected based on the presence of same IGHV, junction genes and with similar HCDR3 sequence according to Hamming's distance. Through the R-Alakazam package, we generated rarefaction curves to evaluate the clonal diversity inside the pathological clone (Fig. 1B). Focusing on the Simpson index (represented by the Hill number of order q=2), which gives more weight to larger clones minimizing the smaller ones (Fig. 1B), we selected a Diversity Score (DS) of 4 for the definition of cases without ID (clonal; DS <4) and cases with ID (intraclonal; DS ≥4) (Fig. 1B). Results. Using the reported threshold we identified 469 (88.5%) clonal cases, expressing a single clone (Fig. 1C), and 61 (11.5%) cases with ID (median DS 9.2, range 4.4-66.0) characterized by the presence of two or multiple pathological clones expressing the same IGHV gene and HCDR3 (Fig. 1C). Notably, cases with ID expressed both a mutated (M) (39/61, 63.9%) and an unmutated (UM) (22/61, 36.1%; p=0.066) IGHV gene configuration (Fig. 1C). Of note, we observed a significant skewing toward the usage of VH4-family genes when comparing cases with ID (38/61, 62.3%) vs. cases without ID (78/469, 16.6%; p<0.0001, Fig 1D). Moreover, the IGHV4-39 and IGHV4-34 genes were the most used genes in the context of cases with ID (Fig. 1E), although none of them belonging to known stereotyped subsets. By focusing on VH4-family only cases, we observed that cases with ID and UM IGHV genes displayed higher mutation frequencies in WA/TW motifs, a mutational signature which suggests an involvement of both Activation-Induced (Cytidine) Deaminase (AID) and error-prone polymerase eta (Fig. 1F), a pattern not observed in its counterpart with UM IGHV genes but without ID (Fig. 1F). Conversely, in cases with ID and M IGHV genes, mutations preferentially clustered in AID hotspots (WRC/GYW motifs), suggesting a direct role of AID and the Base Excision Repair machinery in the mutational overload (Fig. 1F). Consistently, M IGHV cases with ID expressed significantly higher AID mRNA levels than M IGHV cases without ID (p=0.0024; Fig. 1G). These expression levels were overall comparable with those found in UM IGHV cases, irrespective to the evidence of ID (Fig. 1G), which however were not associated with an increased number of mutations in AID-specific hotspots (Fig. 1F). Conclusions. By taking advantage of a new method for ID assessment in CLL, we demonstrated that ID prevalently affects VH4-family cases which display different mutational patterns dependent to the IGHV gene status. This data are in keeping with previous reports indicating the IGHV4 genes as particularly prone to generate immunoglobulin subjected to continuous/persistent stimulation by external/auto-antigens, hence particularly prone to generate features of ID. Further experiments in selected cases with ID through a non-random barcode strategy are needed. Disclosures Zaja: Sandoz: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Abbvie: Honoraria.

scholarly journals RPS15 mutations Repress mRNA Translation in Chronic Lymphocytic Leukemia Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1843-1843
Author(s):  
Stavroula Ntoufa ◽  
Stamatia Laidou ◽  
Fotis Psomopoulos ◽  
Marina Gerousi ◽  
Larry Mansouri ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Transcription Level ◽  
Triplet Periodicity

Abstract We and others recently reported mutations within the RPS15 gene, encoding a component of the 40S ribosomal subunit, in clinically aggressive chronic lymphocytic leukemia (CLL). RPS15 mutations resided within an evolutionary conserved region, alluding to an oncogenic rather than a tumor-suppressor role. Our pilot functional analysis revealed that, similar to other ribosomal proteins (RPs), RPS15 also binds MDM2 and may impact on the p53 response. Here, we performed ribosome profiling in order to gain global insight into changes in translation induced by RPS15 mutations in CLL cells. This technique involves measuring translational efficiency (TE), by comparing the levels of ribosome-associated mRNA footprints against the total mRNA for each gene. For 6 CLL cases bearing mutant (mut, n=3) or wildtype (wt, n=3) RPS15, we obtained both ribosome-protected footprints (RPFs) and matching mRNA sequencing data. In parallel, we created stable MEC1 CLL cell lines expressing an additional copy of wt or mut RPS15 (131S) by lentiviral transduction; validation of the transgene expression was performed by Sanger sequencing of amplified cDNAs. Ribosome footprinting and subsequent library preparation of RPFs and total mRNA for all samples was performed with the Illumina Truseq Ribo Profile Kit and all libraries were sequenced on a NextSeq500 instrument. Reads were aligned to the human hg19 genome using Bowtie2. SystemPipeR was used to determine the percentage of reads mapping to 5' UTRs, CDS, and 3' UTRs and triplet periodicity was assessed using RibORF. The RPFs were of high quality, as assessed by expected RPF size (28-30nt), CDS enrichment, and triplet periodicity. To determine differentially expressed genes between RPS15-mut vs RPS15-wt cases we used DESeq2 while, for differentially translated genes we used Xtail. Changes in transcription and translation between PRS15-wt vs RPS15-mut cases showed limited overlap in both primary CLL cells and cell lines (12.8% and 12.9%, respectively), indicating the potential of ribosome profiling to reveal additional information compared to RNA sequencing alone. In primary CLL cells, 474 genes showed differences only at the transcription level (log2FC mRNA>I1I, p<0.05), while 742 genes were modulated only at the translation level (log2FC RPF>I1I, p<0.05). We identified 322 genes with differential TE (log2FC TE<I1I, p<0.05) between PRS15-wt vs RPS15-mut CLL cases; 262/322 (81%) showed reduced TE in RPS15-mut versus RPS15-wt cases. Similar analysis for the stable MEC1 cell lines revealed 749 genes displaying differences only at the transcription level (log2FC mRNA>I1I), while 1859 genes were regulated only at the translation level (log2FC RPF>I1I). Overall, 771 genes displayed differential TE (log2FC TE<I1I, adjusted p<0.1) between PRS15-wt vs RPS15-mut MEC1 cell lines; 48% of the genes showed reduced TE in mut vs wt cell lines and the remaining 52% augmented TE. The slightly different results compared to those obtained from primary CLL cells, may be attributed to the following reasons: (i) MEC1 cells are TP53-aberrant; (ii) the PRS15-wt cell line overexpresses the RPS15 gene compared to primary CLL cells; and,( iii) the RPS15-mut cell line expresses both the wt and mut RPS15 mRNAs (22% of the mapped reads correspond to the mut RPS15 and 78% to the wt gene, respectively). Gene ontology analysis (Enrichr) of the genes showing differential TE, revealed that in both primary CLL cells and MEC1 cell lines a large fraction of the deregulated transcripts is implicated in RNA binding processes (adj-p=0.0001; adj-p=1.98X10^-13, respectively) which are known to induce translational repression. Interestingly, in primary CLL cells, amongst genes with reduced TE we identified genes implicated in tRNA biosynthesis, protein processing in the endoplasmatic reticulum and the Hippo signaling pathway (p<0.01). Additionally, enrichment analysis revealed that a proportion of genes with reduced TE were targets of the MYC transcription factor (adj-p=0.0005). RP genes, despite unchanged mRNA levels, showed changes in RPF levels and differential TE, suggesting that RPs are also deregulated at the translational level. In conclusion, we show that RPS15 mutations rewire the translation program of CLL cells by reducing the TE of critical molecules, including translation initiation factors and other regulatory elements. Disclosures Hadzidimitriou: Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.

HIF-1α Upregulation in TP53 Disrupted Chronic Lymphocytic Leukemia Cells and Its Potential Role As a Therapeutic Target

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 305-305
Author(s):  
Valentina Griggio ◽  
Candida Vitale ◽  
Maria Todaro ◽  
Chiara Riganti ◽  
Joanna Kopecka ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
Signaling Pathways ◽  
Research Funding ◽  
Transcriptional Activity ◽  
Advisory Board ◽  
Lymphocytic Leukemia ◽  
Kinase Signaling ◽  
Rhoa Kinase

Abstract Background : In chronic lymphocytic leukemia (CLL), disease aggressiveness and drug responsiveness can be ascribed to intrinsic genetic features of the tumor cells, such as TP53 disruption, and to interactions of CLL cells with stromal cells (SC) of the tumor microenvironment. The transcription factor HIF-1α is critically involved in the regulation of genes implicated in key cellular processes, such as cell survival and tumor progression, and also modulates the interactions of CLL cells with SC. HIF-1α expression and transcriptional activity depend on genetic alterations of tumor suppressor genes (e.g. TP53), and on extrinsic signals such as oxygen deprivation and soluble factors. In CLL cells, HIF-1α is active even in normoxia, and its expression is rapidly elevated during hypoxia. We have already reported that HIF-1α activity in CLL cells is upregulated by SC, via activation of Akt, and Ras/ERK1-2 and RhoA/RhoA kinase signaling pathways. SC are well known pro-survival factors, which protect CLL cells from spontaneous apoptosis and fludarabine-induced cytotoxicity. The cytotoxic effects of HIF-1α inhibition, and the ability of HIF-1α targeting agents to reverse constitutive or SC-induced fludarabine resistance, need to be investigated. Aim : The aim of this study was twofold: 1) to unravel the HIF-1α regulatory pathways in CLL cells, also discriminating between TP53 wild type (TP53wt)and disrupted (TP53dis), and 2) to evaluate the ability of HIF-1α inhibition to exert a direct cytotoxic effect and potentiate fludarabine cytotoxicity. Methods: CLL cells were considered TP53dis when TP53 mutation or 17p deletion (>10%) were present (n=33). Otherwise they were considered TP53wt(n=49). HIF-1α gene and protein expression were evaluated by RT-PCR and Western Blot. HIF-1α transcriptional activity was evaluated by ELISA in nuclear extracts. CLL cells were cultured alone or with the M2-10B4 SC line under normoxic or hypoxic conditions. CLL cell cultures were exposed to fludarabine (F-ara-A, 10 μM), BAY 87-2243 (BAY, 1 μM), simvastatin (1 μM) or idelalisib (10 μM), used as single agents or in combination. Cell viability was analyzed by AnnexinV/propidium Iodide immunostaining and flow cytometry. Samples were considered resistant to fludarabine when the relative viability of CLL cells exposed to F-ara-A compared to untreated controls was >0.43 (median value for the whole cohort). Results: TP53dis CLL cells expressed a constitutively higher amount of HIF-1α protein compared to TP53wt CLL cells. This upregulation was not due to a higher HIF-1α gene expression, and was associated to a significantly higher transcriptional activity of HIF-1α (p=0.009). We also evaluated the effect of extrinsic factors on the regulation of HIF-1α. We observed a further increase in HIF-1α expression when both TP53dis and TP53wt CLL cells were cultured under hypoxia. Similarly, the co-culture of CLL cells with SC further upregulated HIF-1α, via activation of Akt, Ras/ERK1-2 and RhoA/RhoA kinase signaling cascades, in both subsets. These inducing effects were particularly evident in TP53dis CLL cells. The specific inhibition of HIF-1α with BAY induced a significant reduction in viability of TP53dis and TP53wt CLL cells (p<0.001), confirming the role of HIF-1α in maintaining leukemic cells survival. We also evaluated the ability of BAY to sensitize constitutively resistant TP53dis CLL cells to F-ara-A. Results from these experiments showed a significant impairment of cell viability after dual treatment with BAY and F-ara-A compared to single agents and untreated control (mean viability: 21.8% BAY + F-ara-A, 34.1% BAY, 47.6% F-ara-A and 59.6% controls; p<0.01). In the last set of experiments, we investigated the role of HIF-1α in the microenvironment-mediated resistance to fludarabine. We found that the direct HIF-1α inhibition with BAY, and the targeting of Ras/ERK1-2 and PI3K/Akt signaling pathways using simvastatin or idelalisib were all able to counteract the protection exerted by SC toward F-ara-A-induced cell death, in both TP53dis and TP53wtCLL cells (p<0.03). Conclusions:Our data demonstrate that HIF-1α is constitutively overexpressed by TP53dis CLL cells compared to TP53wt, and is positively regulated by hypoxia and SC in both subsets. HIF-1α targeted inhibition results in high cytotoxicity of CLL cells, and is able to circumvent constitutive p53-related and SC-induced resistance to fludarabine. Disclosures Rossi: Gilead: Honoraria, Research Funding; Janssen: Honoraria; AbbVie: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Massaia:Janssen: Other: advisory board; Roche: Other: advisory board, research support; Gilead: Other: advisory board. Boccadoro:Mundipharma: Research Funding; Abbivie: Honoraria; SANOFI: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BMS: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Coscia:Gilead: Honoraria; Janssen: Honoraria; ROCHE: Honoraria, Other: Advisory board; Mundipharma: Honoraria; Karyopharm: Research Funding.

scholarly journals Pharmacologic Inhibition with Duvelisib (IPI-145) and Genetic Inhibition of PI3K p110δ Antagonizes Intrinsic and Extrinsic Survival Signals in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3324-3324 ◽  
Author(s):  
Shuai Dong ◽  
Daphne Guinn ◽  
Jason A Dubovsky ◽  
Yiming Zhong ◽  
Amy Lehman ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Nk Cells ◽  
Cell Line ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Pi3k Signaling

Abstract Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen, and bone marrow. CLL cells display up-regulated B-cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton’s tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. Both the PI3K p110δ inhibitor, idelalisib, and the BTK inhibitor, ibrutinib, have received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies. Methods/Results: Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, duvelisib (IPI-145), which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of duvelisib in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM duvelisib for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to duvelisib, displaying about 20% decrease in viability at concentrations greater than 0.5μM; however, the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM duvelisib for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, duvelisib is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in the leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose-dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, duvelisib. Disclosures Dubovsky: Principia Inc.: Research Funding. Kutok:Infinity Pharmaceuticals, Inc.: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

Tp63 Contributes to the Apoptosis Resistant Phenotype in Aggressive Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4142-4142
Author(s):  
Stavroula Ntoufa ◽  
Nikos Papakonstantinou ◽  
Despoina Papazoglou ◽  
Maria Tsagiopoulou ◽  
Sarka Pospisilova ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
B Cell ◽  
Cell Survival ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Richter’S Transformation ◽  
Richter's Transformation

Abstract Alterations in the expression of TP63, a TP53 gene family member, are known to critically affect tumorigenesis though the gene is almost never mutated. In cancer, the following two TP63 isoforms have been extensively studied with contrasting/opposite functions: DNp63, known to promote oncogenesis, and TAp63, which instead has been shown to promote apoptosis. Conflicting information is available regarding the role of TP63 in chronic lymphocytic leukemia (CLL), in particular its effects on cell death (pro-apoptotic or anti-apoptotic?). In a previous DNA methylation profiling study of aggressive CLL subgroups with unmutated B-cell receptor immunoglobulin (BcR IG), we identified TP63 among the differentially methylated and expressed genes. In more detail, TP63 was overexpressed and hypomethylated in stereotyped subset #8 (IGHV4-39/IGKV1(D)-39; IgG-switched), while the opposite was seen for subset #6 (IGHV1-69/IGKV3-20); a finding that might be clinically relevant since subset #8 displays the highest risk for Richter's transformation among all CLL. Given recent evidence that the increased propensity for RT in subset #8 might be associated with intense B-cell receptor (BcR) signaling capacity, here we investigated potential links between BcR stimulation, TP63 expression and CLL cell survival. To this end, we studied primary leukemic cells negatively isolated prior to treatment from 14 cases assigned to subset #6 (n=5), subset #8 (n=4) or non-subset CLL cases with unmutated BcR IG (U-CLL, n=5). RT-PCR analysis detected mRNA of the TAp63 isoform only, whereas the DNp63 isoform was not expressed in any case. As revealed by Western blotting analysis, BcR stimulation for 21 hours with anti-IgM (in subset #6 and non-subset cases) or anti-IgG (exclusively in subset #8) had differential effects on TP63 expression in the different CLL subgroups analyzed. More specifically, whereas TP63 levels remained unaltered in subset #6, a significant up-regulation (Fold=2.8; p<0.05) was seen in subset #8 cases and, in contrast, downregulation was observed in non-subset cases (Fold=-1.6; p<0.05). Next, we investigated the functional impact of TP63 expression on CLL cell viability at 48 hours after BcR stimulation by flow cytometry analysis, using Annexin V and Propidium iodide staining. As expected, no changes were seen in stimulated versus unstimulated (control) subset #6 CLL cells. In contrast, TP63 upregulation after BcR stimulation of subset #8 cases (n=4) was followed by significantly augmented CLL cell survival (Fold=1.6; p<0.05), whereas the opposite was seen in non-subset U-CLL cases (Fold=-1.5; p<0.05), where p63 downregulation was associated with reduced CLL cell survival (p<0.001). In order to investigate whether the observed effects on cell viability were regulated by TP63, we downregulated TP63 expression in primary cells from 5 CLL cases using TP63 -specific siRNA. This treatment led to a significant (Fold=-1.8; p<0.05) reduction in cell viability, further supporting a pro-survival function of TP63. In conclusion, we show that TP63 expression can be regulated by BcR stimulation, but the observed effect differs and can be opposite in CLL subgroups with distinct immunogenetic features. In particular, we highlight p63 as a novel pro-survival factor in CLL subset #8, thus identifying another player in the complex pathophysiology of this very aggressive subset with a high risk for Richter's transformation. Disclosures Ghia: Janssen Pharmaceuticals: Research Funding. Stamatopoulos:Janssen Pharmaceuticals: Research Funding; Gilead Sciences: Research Funding.

Heme-Regulated Inhibitor (HRI) Activates Transcription Factor ATF4 to Promote BCL11A Transcription and Fetal Hemoglobin Silencing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 814-814
Author(s):  
Peng Huang ◽  
Scott A. Peslak ◽  
Xianjiang Lan ◽  
Eugene Khandros ◽  
Malini Sharma ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Research Funding ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Major Pathway ◽  
Specific Regulation

Reactivation of fetal hemoglobin in adult red blood cells benefits patients with sickle cell disease and β-thalassemia. BCL11A is one of the predominant repressors of fetal γ-globin transcription and stands as an appealing target for therapeutic genome manipulation. However, pharmacologic perturbation of BCL11A function or its co-regulators remains an unmet challenge. Previously, we reported the discovery of the erythroid-enriched protein kinase HRI as a novel regulator of γ-globin transcription and found that HRI functions in large part via controlling the levels of BCL11A transcription (Grevet et al., Science, 2018). However, the specific mechanisms underlying HRI-mediated modulation of BCL11A levels remain unknown. To identify potential HRI-controlled transcription factors that regulate BCL11A, we performed a domain-focused CRISPR screen that targeted the DNA binding domains of 1,447 genes in the human erythroid cell line HUDEP2. Activating transcription factor 4 (ATF4) emerged as a novel γ-globin repressor. Prior studies reported that ATF4 production is under positive influence of HRI. Specifically, HRI phosphorylates translation factor EIF2α which in turn augments translation of ATF4 mRNA. As expected, HRI deficiency reduced ATF4 protein amounts in HUDEP2 and primary erythroid cells. We further found that the degree of γ-globin reactivation was similar in ATF4 and HRI-depleted cells. ATF4 ChIP-seq in both HUDEP2 and primary erythroblast identified 4,547 and 3,614 high confidence binding sites, respectively. Notably, we did not observe significant enrichment of ATF4 binding or even the presence of an ATF4 consensus motif at the γ-globin promoters, suggesting that ATF4 regulates the γ-globin genes indirectly. However, ATF4 specifically bound to one of the three major BCL11A erythroid enhancers (+55) in both cell types. This was the sole binding site within the ~0.5Mb topologically associating domain that contains the BCL11A gene. Eliminating this ATF4 motif via CRISPR guided genome editing lowered BCL11A mRNA levels and increased γ-globin transcription. Capture-C showed that ATF4 knock-out or removal of the ATF4 site at the BCL11A (+55) enhancer decreased chromatin contacts with the BCL11A promoter. Forced expression of BCL11A largely restored γ-globin silencing in cells deficient for ATF4 or lacking the ATF4 motif in the BCL11A (+55) enhancer. An unexplained observation from our prior study was that HRI loss did not significantly lower Bcl11a levels in murine erythroid cells. Therefore, we mutated the analogous ATF4 motif in the Bcl11a enhancer in the murine erythroid cell line G1E. Unlike in human cells, Bcl11a mRNA synthesis was decreased only very modestly, and there was no effect on the murine embryonic globin genes whose silencing requires Bcl11a. This suggests that the species specific regulation of BCL11A by HRI results from divergent functional roles of ATF4 binding at the BCL11A (+55) enhancer. In sum, our studies uncover a major pathway that extends linearly from HRI to ATF4 to BCL11A to γ-globin. Moreover, these results further support HRI as a pharmacologic target for the selective regulation of BCL11A and γ-globin. Disclosures Blobel: Pfizer: Research Funding; Bioverativ: 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.

Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2328-2334 ◽  
Author(s):  
Mı́riam Molina-Arcas ◽  
Beatriz Bellosillo ◽  
F. Javier Casado ◽  
Emili Montserrat ◽  
Joan Gil ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chronic Lymphocytic Leukemia ◽  
Expression Patterns ◽  
Individual Variability ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Nucleoside Transporter ◽  
Drug Bioavailability

Nucleoside derivatives are currently used in the treatment of hematologic malignancies. Although intracellular events involved in the pharmacologic action of these compounds have been extensively studied, the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in leukemia cells has not been comprehensively addressed. We have monitored the amounts of mRNA for the 5 nucleoside transporter isoforms cloned so far (CNT1, CNT2, CNT3, ENT1, and ENT2) in several human cell types and in normal human leukocytes. We then examined the expression patterns of these plasma membrane proteins in patients with chronic lymphocytic leukemia (CLL) and correlated them with in vitro fludarabine cytotoxicity. Despite a huge individual variability in the mRNA amounts for every transporter gene expressed in CLL cells (CNT2, CNT3, ENT1, and ENT2), no relationship between mRNA levels and in vitro fludarabine cytotoxicity was observed. Fludarabine accumulation in CLL cells was mostly, if not exclusively, mediated by ENT-type transporters whose biologic activity was clearly correlated with fludarabine cytotoxicity, which reveals a role of ENT-mediated uptake in drug responsiveness in patients with CLL.

scholarly journals A Study of Ribonucleotide Reductase mRNA Expression and Its Prognostic Role in Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4678-4678
Author(s):  
Sevastianos Chatzidavid ◽  
Christina-Nefeli Kontandreopoulou ◽  
Panagiotis T Diamantopoulos ◽  
Nefeli Giannakopoulou ◽  
Panagiota Katsiampoura ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Mrna Expression ◽  
Board Of Directors ◽  
Ribonucleotide Reductase ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Line Treatment ◽  
Prognostic Role ◽  
Advisory Committees ◽  
Trisomy 12

Abstract Background Ribonucleotide Reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides required for DNA replication and repair. RNR consists of two subunits, termed subunit 1 (RRM1) and 2 (RRM2). Imbalance in the regulation of RNR activity and control of dNTPs' pool leads to genomic instability and increases mutation rate. RNR expression has been associated with prognosis in pancreatic, non-small-cell lung, breast, and biliary tract cancer. However, RNR expression in chronic lymphocytic leukemia (CLL) and its possible prognostic role have not been investigated yet. Aim In this study we evaluate the possible prognostic role of RNR expression in CLL. Method The study comprised patients with immunophenotypically confirmed disease at the time of sample collection. Peripheral whole blood samples were collected from 84, 27, 15, and 9 patients before treatment, after one, two, and three lines of treatment respectively. RNA extraction and reverse transcription were carried out using standard protocols. A Taqman based real-time PCR was performed on a CFX96 RT-PCR system (Bio-Rad Laboratories, Hercules, CA, USA). For both the housekeeping and target genes, a Taqman primer/probe mix was used according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). RRM1 and RRM2 mRNA levels were expressed as an RRM1-2/GAPDH ratio. Western blot analysis was performed to quantify the RRM1 protein levels in a random sample of 41 patients. Antibodies used were: RRM1 #3388, β-actin #4967 and anti-rabbit IgG HRP-conjugated #7074 (Cell Signaling Technology, Danvers, MA, USA). Detection was done using the ECL western blotting reagents. Statistical analysis was conducted to study the possible correlations between the variables. All reported p values are two-tailed. Statistical significance was set at p&lt;0.05 and analyses were conducted using SPSS statistical software (version 22.0). Results From 135 CLL patients included in the study 56.3% were female and the median age at diagnosis was 64 years. Peripheral blood was collected in 84 treatment-naïve patients (62.2%). Median follow up was 6.66 years (3.47 ─ 11.13) and median time from diagnosis until 1st line treatment was 23.1 months (IQR: 5.8 - 56.5 months). Out of 135 patients, 69 (51,1%) received 1 st line treatment and 35 patients (25,9%) 2 nd line treatment with median time between the two treatment lines being 26.5 months (IQR: 7.8 - 40.8 months). Furthermore, 48.5%, 33.8%, 12.3%, 3.1% and 2.3% of the patients had Rai score 0, I, II, III, IV respectively. The median mRNA expression of RRM1 was 0.04 (IQR: 0 - 0.09) and of RRM2 was 0.01 (IQR: 0 - 0.1). RRM1 mRNA expression was significantly higher in patients without anemia (p=.025) and without lymphadenopathy (p=.002). Higher values of ESR (r=-.30; p=.028), LDH (r=-.20; p=.026) and Rai score (r=-.18; p=.037) were associated with lower expression of RRM1 mRNA. In addition, TP53 gene deletion detected by FISH was associated with higher RRM1 mRNA expression (p=.036). Significantly higher RRM2 mRNA expression was reported in patients without lymphadenopathy (p=.021) and Rai score 0 (p=.003). Moreover, higher was the expression of RRM2 mRNA in cases with Trisomy 12 (p=.050). In samples collected before treatment, higher values of RRM1 mRNA expression were statistically significantly associated with lower RAI score (r=-.30; p=.005) and longer time periods between the first two lines of treatment (r=.95; p=.050). Western blot analysis confirmed detection of RRM1 protein but statistical correlation was not carried out due to lack of material from the whole group of patients. Conclusion For the first time, mRNA expression of RRM1 and RRM2 is studied in patients with CLL. These results show RNR involvement in the pathophysiology of CLL. RRM1 and RRM2 mRNA higher expression found in 17p deletion and trisomy 12 cases respectively may be consistent with the existence of a methylation-depended mechanism proposed by other studies. Therefore, these results demonstrate RNR's potential role as a prognostic factor, and make it a probable therapeutic target. A study including a larger number of cases could further confirm our results. Figure 1 Figure 1. Disclosures Kyrtsonis: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/Genesis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria; Sanofi: Membership on an entity's Board of Directors or advisory committees. Panagiotidis: Abbvie: Research Funding; Pfizer: Research Funding; Janssen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Sandoz: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Research Funding; Astellas: Research Funding. Viniou: Sandoz: Research Funding; Takeda: Research Funding; Novartis: Honoraria, Research Funding; Sanofi: Research Funding; Janssen: Honoraria, Research Funding; Pfizer: Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Roche: Research Funding; Astellas: Research Funding; Celgene: Research Funding.

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