scholarly journals Functional Calcitriol/Vitamin D Receptor Signaling in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3019-3019
Author(s):  
Marina Gerousi ◽  
Fotis Psomopoulos ◽  
Kostantia Kotta ◽  
Niki Stavroyianni ◽  
Achilles Anagnostopoulos ◽  
...  
Keyword(s):  
Vitamin D ◽  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathways ◽  
Cell Line ◽  
Vitamin D Receptor ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Biologically Active ◽  
Differentially Expressed ◽  
Rna Seq

Calcitriol, the biologically active form of vitamin D, modulates a plethora of cellular processes following its receptor ligation, namely the vitamin D receptor (VDR), a nuclear transcription factor that regulates the transcription of diverse genes. It has been proposed that vitamin D may play a role in prevention and treatment of cancer while epidemiological studies have linked vitamin D insufficiency to adverse disease outcome in chronic lymphocytic leukemia (CLL). Recently, we reported that VDR is functional in CLL cells after calcitriol supplementation, as well as after stimulation through both the calcitriol/VDR signaling system and other prosurvival pathways triggered from the tumor microenvironment. In this study, we aimed at investigating key molecules and signaling pathways that are altered after calcitriol treatment and are known to play a relevant role in CLL pathophysiology. CD19+ primary CLL cells were negatively selected from peripheral blood samples of patients that were treatment naïve at the time of sample collection. CLL cells were cultured in vitro with calcitriol or co-cultured with the HS-5 mesenchymal cell line for 24 hours. VDR+, CYP24A1+, phospho-ERK+ and phospho-NF-κB p65+ cells were determined by Flow Cytometry (FC). Total RNA was extracted from calcitriol-treated and non-treated CLL cells, while mRNA selection was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module. Library preparation for RNA-Sequencing (RNA-Seq) analysis was conducted with the NEBNext Ultra II Directional RNA Library Prep Kit. The libraries were paired-end sequenced on the NextSeq 500 Illumina platform. Differential expression analysis was performed using DESeq2; genes with log2FC>|1| and P≤0.05 were considered as differentially expressed. RNA-Seq analysis (n=6) confirmed our previous findings that the CYP24A1 gene is significantly upregulated by calcitriol, being the top upregulated gene, whereas the VDR gene remains unaffected by this treatment. Overall, 85 genes were differentially expressed in unstimulated versus calcitriol-treated cells, of which 28 were overexpressed in the latter thus contrasting the remaining 57 which showed the opposite pattern. Pathway enrichment and gene ontology (GO) analysis of the differentially expressed genes revealed significant enrichment in PI3K-Akt pathway and Toll-like receptor cascades, as well as in vitamin D metabolism and inflammatory response pathways. Additionally, flow cytometric analysis showed that calcitriol-treated CLL cells displayed increased pERKlevels (FD=1.3, p<0.05) and, in contrast decreased pNF-κBlevels (FD=2.7, p<0.05), highlighting active VDR signaling in CLL. Aiming at placing our findings in a more physiological context, we co-cultured CLL cells with the HS-5 cell line. Based on our previous finding that co-cultured CLL cells showed induced CYP24A1 levels, we evaluated pNF-κB expression. pNF-κB levels were found to be increased in co-cultured CLL cells (FD=4.2, p<0.05), while the addition of calcitriol downregulated pNF-κB (FD=1.5, p<0.05). Moreover, ex vivo calcitriol exposure of CLL cells from patients under ibrutinib treatment (at baseline, +1 and +3-6 months, n=7) resulted in significant upregulation of pERK (FD=1.6, p<0.01; FD=1.4, p<0.01; FD=1.9, p<0.01; for each timepoint respectively) but, significant downregulation of pNF-κΒ (FD=3.4, p<0.01; FD=3, p<0.05; FD=2.3, p<0.05; for each timepoint respectively), indicating preserved calcitriol/VDR signaling capacity. In conclusion, we provide evidence that the calcitriol/VDR system is active in CLL, modulating NF-κB and MAPK signaling as well as the expression of the CYP24A1 target gene. This observation is further supported by RNA-Seq analysis that confirms CYP24A1 upregulation and highlights new signaling pathways that need to be validated. Interestingly, the calcitriol/VDR system appears relatively unaffected by either stimulation or inhibition (ibrutinib) of microenvironmental signals that promote CLL cell survival and/or proliferation, indicating context-independent signaling capacity. Disclosures Kotsianidis: Celgene: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

scholarly journals The Calcitriol/Vitamin D Receptor System Regulates Key Immune Signaling Pathways in Chronic Lymphocytic Leukemia

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 285
Author(s):  
Marina Gerousi ◽  
Fotis Psomopoulos ◽  
Konstantia Kotta ◽  
Maria Tsagiopoulou ◽  
Niki Stavroyianni ◽  
...  
Keyword(s):  
Vitamin D ◽  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathways ◽  
Ex Vivo ◽  
Active Form ◽  
Lymphocytic Leukemia ◽  
Biologically Active ◽  
Sequencing Analysis ◽  
Receptor System

It has been proposed that vitamin D may play a role in prevention and treatment of cancer while epidemiological studies have linked vitamin D insufficiency to adverse disease outcomes in various B cell malignancies, including chronic lymphocytic leukemia (CLL). In this study, we sought to obtain deeper biological insight into the role of vitamin D and its receptor (VDR) in the pathophysiology of CLL. To this end, we performed expression analysis of the vitamin D pathway molecules; complemented by RNA-Sequencing analysis in primary CLL cells that were treated in vitro with calcitriol, the biologically active form of vitamin D. In addition, we examined calcitriol effects ex vivo in CLL cells cultured in the presence of microenvironmental signals, namely anti-IgM/CD40L, or co-cultured with the supportive HS-5 cells; and, CLL cells from patients under ibrutinib treatment. Our study reports that the calcitriol/VDR system is functional in CLL regulating signaling pathways critical for cell survival and proliferation, including the TLR and PI3K/AKT pathways. Moreover, calcitriol action is likely independent of the microenvironmental signals in CLL, since it was not significantly affected when combined with anti-IgM/CD40L or in the context of the co-culture system. This finding was also supported by our finding of preserved calcitriol signaling capacity in CLL patients under ibrutinib treatment. Overall, our results indicate a relevant biological role for vitamin D in CLL pathophysiology and allude to the potential clinical utility of vitamin D supplementation in patients with CLL.

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 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.

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.

scholarly journals Spatial Genomic Heterogeneity in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3017-3017
Author(s):  
Clare Sun ◽  
Yun-Ching Chen ◽  
Aina Zurita Martinez ◽  
Delong Liu ◽  
Daniel Rosebrock ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Clonal Evolution ◽  
Lymphocytic Leukemia ◽  
Checkpoint Blockade ◽  
Rna Seq ◽  
Stable Group ◽  
Single Nucleotide Variants ◽  
Activation Markers

Activation and proliferation of chronic lymphocytic leukemia (CLL) cells depend on signals from the lymph node (LN) tumor microenvironment (TME). Separately, the genetic makeup of CLL has been closely linked to disease aggressiveness and its capacity to evolve under the selective pressures of treatment. Here, we investigated the intersection between the TME and molecular events in CLL pathogenesis. Whole exome and RNA sequencing (RNA-seq) were performed on CD19+ cells of paired peripheral blood (PB) and LN samples and matched germline DNA from 14 patients with treatment-naïve CLL. RNA-seq was also done on unsorted LN samples containing tumor and non-tumor cells from the same patients. A median of 27 (range 11-69) somatic single nucleotide variants (sSNVs) and 3 (0-10) insertions and deletions (sIndels) were detected per exome. All but one patient had copy number alterations (CNAs), most commonly del 11q and del 13q. Cancer cell fractions (CCFs) of sSNVs, sIndels, and CNAs were inferred from variant allele frequencies then clustered over the two anatomic compartments for each patient. Genetic compartmentalization (ΔCCF > 0.25, false discovery rate [FDR] < 0.1) was observed in 7 patients (50%), of whom 6 demonstrated subclonal expansion in LN. To understand factors contributing to spatial heterogeneity, we compared the tumor transcriptome based on the presence (shifted group) or absence (stable group) of an expanded subclone in LN. Most differentially expressed genes between PB and LN were shared by all patients and reflected the activation of CLL cells in the LN TME as previously shown. However, cell cycle genes (e.g. E2F2, CDC25A) were more upregulated (log2FC > 0.5, FDR < 0.05) in LN of the shifted group, while lymphocyte activation markers (e.g. CD83, CD69) were more upregulated in LN of the stable group. We hypothesized the latter finding could indicate immune-mediated control of clonal outgrowth. We therefore evaluated the expression of an 18-gene T-cell associated inflammatory signature in unsorted LN samples. This signature was originally developed as a predictive biomarker for response to immune checkpoint blockade in multiple cancer types. Unsupervised hierarchical clustering of signature genes revealed an inflamed TME in the stable group relative to the shifted group. In summary, genetic compartmentalization is a common phenomenon in CLL. Clonal equilibrium is maintained by a T-cell inflamed TME. When immune surveillance is inactivated, subclones with a competitive advantage may expand in response to support signals provided by the TME. An immunotherapy-based clinical study using checkpoint blockade to restrict clonal evolution is currently in progress (NCT03204188). This research was supported by the Intramural Research Program of the NIH, NHLBI. Disclosures Getz: Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding. Wiestner:Merck: Research Funding; Pharmayclics: Research Funding; Acerta: Research Funding; Nurix: Research Funding.

scholarly journals Comparative transcriptomics analysis revealing flower trichome development during flower development in two Lonicera Japonica Thunb. cultivars using RNA-seq

2020 ◽  
Author(s):  
Jianjun Li ◽  
Chenglin Ye ◽  
Cuifang Chang
Keyword(s):  
Signaling Pathways ◽  
Plant Hormones ◽  
Flower Development ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Molecular Networks ◽  
Lonicera Japonica ◽  
Rna Seq ◽  
Trichome Development ◽  
A Genome

Abstract Background: Trichomes comprise specialized multicellular structures that have the capacity to synthesize and secrete secondary metabolites and protect plants from biotic and abiotic stresses. However, little is known about the trichome formation mechanism during flower development in Lonicera Japonica Thunb.Results: Here, we present a genome-wide comparative transcriptome analysis between two L. japonica cultivars, toward the identification of biological processes and functional gene activities that occur during flowering stage trichome development. In this study, the density and average lengths of flower trichomes were at their highest during three green periods. Using the Illumina RNA-Seq method, we obtained 134,304 unigenes, 33,733 of which were differentially expressed. In an analysis of 40 differentially expressed unigenes (DEGs) involved in trichome development, 29 of these were transcription factors. The DEGs analysis of plant hormone signal transduction indicated that plant growth and development may be independent of GA and CTK signaling pathways, and plant stress may be independent of JA and ET signaling pathways. We successfully isolated key genes involved in the floral biosynthesis of odors, tastes, colors, and plant hormones, and proposed biosynthetic pathways for sesquiterpenoid, triterpenoid, monoterpenoid, flavonoid, and plant hormones. Furthermore, 82 DEGs were assigned to cell cycles and 2,616 were predicted as plant resistance genes (PRGs).Conclusions: This study provides a comprehensive characterization of the expression profiles of flower development during the seven developmental stages of L. japonica, thereby offering valuable insights into the molecular networks that underly flower development in L. japonica.

scholarly journals Mechanisms of Adaptation to Ibrutinib in High Risk Chronic Lymphocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 585-585 ◽  
Author(s):  
Valeria Spina ◽  
Gabriela Forestieri ◽  
Antonella Zucchetto ◽  
Alessio Bruscaggin ◽  
Tamara Bittolo ◽  
...  
Keyword(s):  
High Risk ◽  
Chronic Lymphocytic Leukemia ◽  
Nuclear Localization ◽  
Peripheral Blood ◽  
Research Funding ◽  
Clonal Evolution ◽  
Lymphocytic Leukemia ◽  
Adaptation Process ◽  
Over Time ◽  
Stimulation Of

Abstract Introduction. Ibrutinib inhibits the BTK molecule downstream the B-cell receptor (BCR). Though highly active in high risk chronic lymphocytic leukemia (CLL), the most typical response achievable in patients is a minimal residual disease (MRD) positive partial remission (PR) which is maintained until the development of genetically driven resistance caused by the acquisition of mutations in the BTK or PLCG2 genes. The study aims at characterizing the adaptation process allowing residual CLL cells to persist despite BTK inhibition. Methods. The IOSI-EMA-001 study (NCT02827617) is an observational study consisting in the prospective and longitudinal collection of peripheral blood samples and clinical data from high risk CLL patients treated with ibrutinib. Peripheral blood CLL cells longitudinally drawn from patients before treatment start and at fixed timepoints under ibrutinib were monitored by: i) next generation flow cytometry approaches for changes in proliferation rate, surfaceome, and pathway activation; and ii) CAPP-seq targeted deep next generation (sensitivity ~10-3) for clonal evolution. Results. The study cohort comprised 31 high risk CLL patients, including 15 treatment naïve, 16 relapsed, 80% IGHV unmutated, 42% 17p deleted and 55% TP53 mutated. Median duration of ibrutinib treatment was 45 weeks (24-72 weeks). All patients obtained a MRD positive PR that was maintained in all but one who progressed with a PLCG2 mutation (VAF 3%). Compared to baseline, under ibrutinib therapy CLL cells slowed down their proliferation, as suggested by the decreased expression of Ki-67, the reduction of the proliferating fraction (CXCR4dimCD5bright), and the increase of the resting fraction (CXCR4brightCD5dim). Compared to baseline, under ibrutinib therapy CLL cells also upregulated BCR and adhesion/homing proteins, and decreased the expression of BCR inhibitor proteins. Upon stimulation of the BCR with anti-IgM, the downstream path through pBTK and pPLCG2 was inhibited by ibrutinib, while conversely the downstream path through pAKT and pERK was still inducible throughout all the assessed timepoints. The proportion of CLL cells harboring nuclear localization of NF-kB progressively increased over time under ibrutinib. NF-kB nuclear localization was inducible throughout all the assessed timepoints by CD40L stimulation of the non-canonical NF-kB pathway, but not by anti-IgM stimulation of the BCR/canonical NF-kB pathway. Overall, 880 individual mutations were longitudinally discovered and monitored across a total of 121 sequential timepoints collected during ibrutinib treatment. Clonal evolution was observed in (67.7%) cases, a proportion rate previously documented in CLL treated with chemoimmunotherapy. Clonal evolution appeared to be heterogeneous involving different genes without a stereotypic targeting. Consistently, none of the main driver gene mutations was homogeneously selected or suppressed by ibrutinib suggesting that the biological adaptation of CLL cells under ibrutinib is not genetically driven. Clonal evolution propensity was not associated with any of the biomarkers of the disease, and it did not decrease over time under ibrutinib. Conclusions. Taken together these results suggest that residual CLL cells persisting under ibrutinib therapy adapt their phenotype by upregulating adhesion molecules, chemokine receptors and BCR molecules, and by maintaining a competence of BCR signaling through the PI3K/AKT/ERK pathway. The progressive selection of CLL cells having NF-kB in the nucleus, likely due to the BTK independent non-canonical NF-kB pathway, might explain their survival despite ibrutinib therapy. Finally, clonal evolution is not suppressed by ibrutinib chemotherapy, and despite does not seem to be directly involved in such adaptation process, may ultimately favor the acquisition of BTK and PLCG2 ibrutinib resistance mutations. Disclosures Zucca: Celltrion: Consultancy; AstraZeneca: Consultancy. Ghia:Sunesis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding. Montillo:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding. Tedeschi:Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy; AbbVie: Consultancy. Gaidano:AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Morphosys: Honoraria; Roche: Consultancy, Honoraria.

scholarly journals Lenalidomide and Rituximab Maintenance Therapy after Front-Line Induction Chemoimmunotherapy in Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5470-5470
Author(s):  
Julie E Chang ◽  
Vaishalee P. Kenkre ◽  
Christopher D. Fletcher ◽  
Aric C. Hall ◽  
Natalie Scott Callander ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Maintenance Therapy ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Second Malignancies ◽  
Front Line ◽  
First Line ◽  
11Q Deletion ◽  
Line Chemotherapy

Introduction: Chronic lymphocytic leukemia (CLL) is incurable with standard therapy. With first-line chemotherapy, some patients (pts) may achieve durable remissions of many months/years. Lenalidomide (LEN) has improved progression-free survival (PFS) when given as maintenance (MNT) therapy after front-line chemotherapy (CALGB10404, CLLM1). The combination of LEN + rituximab (LR) has activity in relapsed CLL, hypothesizing benefit as MNT therapy after first-line chemotherapy. Methods: Adult pts ≥18 years with previously untreated CLL received induction bendamustine (B) 90 mg/m2 IV days 1 & 2 and rituximab (R) IV day 1 (375 mg/m2 cycle 1, then 500 mg/m2 cycles 2-6) for 6 treatment cycles (as few as 4 cycles allowed). MNT therapy with LR was initiated within 12 weeks after cycle 6, day 1 of BR. Criteria to start LR MNT included: neutrophils ≥1000/microliter (uL), platelets ≥75 K/uL, and creatinine clearance ≥40 mL/min. LEN was administered in 28-day cycles for 24 cycles, initially 5-10 mg daily continuous dosing, later modified to 5-10 mg on days 1-21 of each 28-day cycle in 6/2018 due to neutropenia and second malignancy risk. LEN was reduced to 5 mg every other day for toxicities at 5 mg/day. R 375 mg/m2 IV was given every odd cycle (total of 12 doses). Patients discontinuing LEN for any reason were allowed to continue R MNT per protocol. The primary endpoint is PFS with LR MNT therapy, calculated from the first day of MNT therapy until progressive disease (PD), death, or start of a new therapy. Secondary endpoints are response rate and overall survival. Results: Thirty-four pts have enrolled beginning 11/2013, with follow-up through 6/2019. Median age is 64 years, with 8 pts ≥70 years; 8 women and 26 men. CLL FISH panel is available on all pts: 14 with 13q (as sole abnormality), 9 with 11q deletion, 6 with trisomy 12, 4 with normal FISH panel and 1 with 17p deletion. Heavy chain mutation analysis is available on 11 pts: 8 unmutated, 2 mutated, 1 indeterminate. Thirty-one pts completed 4 (n=2) or 6 cycles of induction BR; 3 pts are receiving induction BR. Twenty-four pts have received MNT LR; 7 did not receive LR for reasons of PD during induction (n=2), infection (n=1), pt preference (n=2), renal insufficiency (n=1), and new carcinoma (n=1). MNT LR was completed in 7 pts; 9 pts are still receiving LR. Fourteen subjects have discontinued protocol therapy, 3 during induction due to PD (n=2) and infection (n=1), and 8 during MNT. Toxicities that led to discontinuation of LR were recurrent infections in 7 pts, including 2 events of PJP pneumonia; 4 pts had recurrent neutropenia with infections; 1 pt had neutropenia without infections. Response is assessable in 31 patients using the International Working Group Consensus Criteria. Best responses to treatment were: partial response 65% (22/34), complete response (CR)/unconfirmed CR 24% (8/34). The median number of MNT cycles received is 16. The dose intensity of LEN across total cycles received (n=278): 5 mg every other day (52.5%), 5 mg/day (43.9%), and 10 mg/day (3.6%). The most common reason for dose reduction or dose holding was neutropenia. Most common Gr 3/4 toxicities (reported as events Gr3/Gr4) during MNT therapy were: neutropenia (20/20), leukopenia (19/4), febrile neutropenia (3/1), and infections (11/-). The majority of Gr3 infections were pneumonia/respiratory (n=5). One event of disseminated herpes zoster occurred. Second malignancies during MNT included: basal cell CA (n=1), squamous cell carcinoma (n=5), and colon cancer (n=1). No unexpected second malignancies were observed in pts receiving LR. Two-year PFS (defined from day 1 of MNT therapy) is 90% (95% confidence interval [CI] 0.78-1), and the median follow-up for 24 patient who started maintenance therapy is 1.79 years (95% CI 1.53-2.7). There have been no deaths. Conclusion: The combination of LR is effective in sustaining remissions after a BR induction in previously untreated CLL, but with frequent neutropenia and infections even at low doses of LEN. Most patients discontinuing MNT did so due to neutropenia and/or infections. A shorter planned interval of MNT LR (i.e., 6-12 months) may confer similar benefit to extended dosing that is more tolerable. Pts at high risk for short remissions after front-line chemotherapy (e.g., unmutated heavy chain status, 11q deletion and/or failure to achieve minimal residual disease after induction) may be the populations for which LR MNT therapy is most appropriate. Disclosures Chang: Genentech: Research Funding; Adaptive Biotechnologies: Research Funding; Celgene: Research Funding. OffLabel Disclosure: Lenalidomide administered as maintenance therapy for first treatment of CLL/SLL.

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