Apoptosis In Primary Chronic Lymphocytic Leukemia Cells Research Articles

Abstract 518: The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation

Abstract Richter transformation (RT), characterized by the transformation of chronic lymphocytic leukemia (CLL) to an aggressive large-cell lymphoma, remains a challenge for the therapy. Genetic aberrations, such as Myc activation, TP53 abnormality, NOTCH1 gene mutation or loss of CDKN2A are associated with uncontrolled RT cell proliferation and escaping of apoptosis. Among those changes, gain of Myc activity, found in around 70% of RT, plays a nexus role in the pathogenesis of RT. As a transcription factor, Myc activation has a profound effect on cell survival, proliferation, adhesion and metabolism. Because of its essential role in lymphoma transformation and aggressiveness, Myc is a vital target for therapy toward RT. Fadraciclib (CYC065) is a second-generation CDK inhibitor with selective potency toward CDK2 and CDK9. We have shown previously that by inhibiting CDK9-mediated transcription, fadraciclib reduced expression of the short-lived anti-apoptotic protein Mcl-1 to initiate apoptosis in primary CLL cells. Like Mcl-1, Myc is a quintessential example of an oncogene with rapid turnover in both its mRNA and protein, therefore a promising target of CDK9 inhibition. Thus, we proposed that fadraciclib would target both the survival and proliferation pathways of RT pathogenesis through collaborative inhibition of Mcl-1, Myc and CDK2. First, our data showed that fadraciclib effectively induced apoptosis in the RT cell line HPRT1, as well as the lymphoma cell lines with Myc amplification, including Raji, Ramos and SU-DHL-2. The IC50 for apoptosis was 0.3 µM at 24h in the HPRT1 cells, which is less than the average IC50 for the primary CLL cells (0.8 µM). The IC50 for the lymphoma lines ranged from 0.8 µM to 1.3 µM. Immunoblots showed reduction of RNA Pol2 phosphorylation by fadraciclib, consistent with the inhibition of CDK9. As a result of loss of RNA Pol2 activity and pausing of transcription, there was a clear reduction of both Mcl-1 and Myc mRNA, measured by real-time RT-PCR, that was associated with the reduction of their protein levels. CDK2 was also inhibited by fadraciclib, shown by the reduction of the phosphorylation of both RB and NPM. Second, Fadraciclib inhibited cell proliferation of the HPRT1 cells at an IC50 of 0.25 µM measured by the CellTiter-Glo assay. The IC50 for growth inhibition in the lymphoma lines ranged from 0.5 to 0.7 µM. CFSE tracing demonstrated halting of cell division upon fadraciclib incubation. Cell cycle analysis by Click-iT EdU showed reduction of S-phase population and arresting of cells in both G1 and G2. We are currently working on dissecting the contributions of inhibiting Myc, Mcl-1 and CDK2 individually and together, in apoptosis induction and proliferation blockade. Taken together, these data suggest that fadraciclib is active in RT cells and Myc-dependent lymphoma cell lines and support expansion of its clinical testing in patients with RT. Citation Format: Rong Chen, Yuling Chen, Ping Xiong, William Plunkett. The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 518.

Prognostic value and therapeutic targeting of XPO1 in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a subtype of B-cell malignancy with high heterogeneity. XPO1 is highly expressed in many hematological malignancies, which predicts poor prognosis. In the study, we aimed to explore the prognostic role of XPO1 and the therapeutic effect of Selinexor, a selective inhibitor of nuclear export, which targets XPO1. We collected 200 CLL samples in our center to confirm XPO1 mRNA expression and analyzed the correlation between XPO1 expression and prognosis. Then, we decreased XPO1 expression with Selinexor to explore the effect of proliferation inhibition, cell cycle arrest, and apoptosis in CLL cell lines. RNA-Seq was performed to explore potential mechanisms. We analyzed XPO1 expression in a cohort of 150 treatment naive patients and another cohort of 50 relapsed and refractory (R/R) patients and found that XPO1 expression was upregulated in 76% of CLL patients compared with healthy donors. Survival analysis suggested that patients with increased XPO1 expression had inferior treatment-free survival (P = 0.022) and overall survival (P = 0.032). The inhibitor of XPO1, Selinexor, induced apoptosis in primary CLL cells. We showed the effects of Selinexor on proliferation inhibition, cell cycle arrest, and apoptosis in CLL cell lines with JVM3, MEC1, and ibrutinib-resistant (MR) cells via nuclear retention of cargo proteins of IκBα, p65, p50, and FOXO3a. Moreover, downregulation of the NF-κB and FOXO pathways was a common feature of the three CLL cell lines responding to Selinexor, indicating the potential application of XPO1 inhibitor even in the high-risk CLL cells. We identified XPO1 as an unfavorable prognostic factor for CLL patients and provided a rationale for further investigation of the clinically XPO1 targeted therapeutic strategy against CLL.

CDK7 Inhibitor THZ1 Inhibits Glycolytic Metabolism By Suppressing HIF1α Activity in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL), the most common type of leukemia in Western adults, is characterized by the progressive accumulation of CD19+ malignant B cells in blood, secondary lymphoid tissues and bone marrow. Currently, CLL remains incurable in spite of the explosive development of novel therapeutics in the past decade. Cyclin-dependent kinases (CDKs) have been suggested as potential therapeutic targets for CLL. Flavopiridol, a pan-CDK inhibitor, has demonstrated safety and efficacy in patients with relapsed CLL, including patients with high-risk cytogenetic features. In this study, we assessed the therapeutic potential of a covalent CDK7 inhibitor, THZ1, in preclinical CLL models. We observed that THZ1 had a significantly lower IC50 in CLL cell lines MEC1 and MEC2 (7.23nM and 7.35nM, respectively), which was 10-fold more potent than flavopiridol and CR8, two other CDK inhibitors that were previously studied in CLL. THZ1 inhibited cell growth and induced apoptosis in MEC1, MEC2 and primary CLL cells in both a dose-dependent and time-dependent manner. The induction of apoptosis by THZ1 in MEC1, MEC2 and primary CLL cells was associated with PARP1 cleavage and down-regulation of anti-apoptotic protein MCL-1. Particularly, THZ1 was able to overcome protection signals provided by the tumor microenvironments and induce apoptosis in primary CLL cells in stromal-coculture conditions. To elucidate the mechanism of action of THZ1, we performed a time course RNA-seq expression analysis in both MEC1 and MEC2 treated with 50nM THZ1. Based on the incremental reduction of transcript levels over a 12-hour period, we identified 728 and 647 THZ1-sensitive transcripts (~5.2% and 4.5% of all active transcripts) in MEC1 and MEC2 cell lines, respectively. Pathway analysis demonstrated that THZ1 down-regulated transcripts are significantly enriched in regulation of cellular metabolism including hypoxia, mTORC1 signaling, and glycolysis pathways. In particular, when analyzing the entire THZ1 down-regulated expression signature for both MEC1 and MEC2 at 8 hours using Gene Set Enrichment Analysis (GSEA), two of the most significantly enriched gene sets contain gene expression signatures from hypoxia induction and HIF1α target genes. Several of the metabolic transcripts sensitive to THZ1 treatment including SLC2A1 , SLC2A3 , HK2 , ENO2 , and PKFKB4 were validated using quantitative RT-PCR. Immunoblot analysis demonstrated that mTORC1 substrates 4E-BP1 and p70S6K were hypophosphorylated in MEC1 and MEC2 when treated with increasing concentrations of THZ1 over time, indicating the inhibition of mTORC1 activity by THZ1. We also observed that the metabolically sensitive AKT Ser473 phosphorylation had essentially been fully repressed by THZ1 treatment in both MEC1 and MEC2. Furthermore, THZ1 was able to mitigate glucose uptake and reduce lactate production in MEC1 and MEC2 cells. THZ1 also prevented the induction of glycolytic transcripts by hypoxia and hypoxia-mimetic CoCl2 (200μM) treatment in MEC1, MEC2 and primary CLL cells. Analysis of CDK7 ChIP-seq data in Jurkat cells revealed that CDK7 directly binds to the promoter and -24kb, -35kb distal enhancers of SLC2A3 that are known to directly interact with HIF1α, suggesting that CDK7 is likely involved in transcriptional regulation of HIF1α target genes via direct enhancer binding. Experiments are currently underway to determine if CDK7 physically interacts with HIF1α under hypoxia. Overall, we observed that THZ1 could effectively disrupt CLL cell viability and overcome the protective signals present in the leukemia microenvironment by inhibiting the transcriptional activity of HIF1α, which is a key regulator of the interaction of CLL cells with the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of the CDK7 inhibitor as novel therapies for CLL. DisclosuresNo relevant conflicts of interest to declare.

The Clinical BET Inhibitor, GS-5829, Is Active Against Chronic Lymphocytic Leukemia As Single Agent and in Combination with B-Cell Receptor Signaling Inhibitors

The Clinical BET Inhibitor, GS-5829, Is Active Against Chronic Lymphocytic Leukemia As Single Agent and in Combination with B-Cell Receptor Signaling Inhibitors

Inhibition of AKT By MK2206 but Not PI3-Kinase δ By Idelalisib Is Effective Against CLL Cells in an In Vitro Model of the Hypoxic Microenvironment

Treatment of chronic lymphocytic leukemia (CLL) relies on targeting the leukemic cells that populate the bone marrow and lymph nodes. Recent studies (Shachar, Cohen et al. 2012, Muz, de la Puente et al. 2014, Valsecchi, Coltella et al. 2016) suggest that the ability of CLL cells to adapt to low oxygen tensions, particularly within the bone marrow, may play an important role in their survival, proliferation and resistance to current therapies. Understanding the mechanisms underlying hypoxia-induced drug resistance may help to identify novel treatment strategies.In this study, we compared the effects of the AKT inhibitor MK2206 and the PI3-kinase δ inhibitor idelalisib under in vitro conditions that mimic the hypoxic tumour microenvironment. MK2206 induced apoptosis of primary CLL cells in co-culture with stromal cells and caused a G0/G1 phase cell cycle arrest of OSU-CLL cells under both normoxic (21 % O2) and hypoxic (1 % O2) conditions. Consistent with other reports, no cytotoxic or cytostatic effects of idelalisib at clinically relevant doses were observed under these in vitro conditions. Treatment of both primary and OSU-CLL cells with MK2206 induced an accumulation of reactive oxygen species in the mitochondria and downregulated expression of P70-S6-kinase and Mcl-1, effects which were not observed following treatment with idelalisib.Collectively these data suggest that the anti-apoptotic and cell cycle-regulatory roles of AKT in CLL cells may not be dependent on activity of the d-isoform of PI3-kinase within the hypoxic tumour microenvironment. The efficacy of MK2206 in vitro and the toxicities associated with idelalisib suggest further studies of MK2206 may be warranted, particularly for CLL patients with bulky disease. DisclosuresNo relevant conflicts of interest to declare.

Modulation of the Tumor Suppressor Protein PP2A Using a Small Molecule Agonist Overcomes Multi-Drug Resistance through Mitochondrial Permeability Transition Pore (MPTP) Dependent Induction of Apoptosis in Chronic Lymphocytic Leukemia

The advent of molecularly targeted therapies has revolutionized the treatment of Non-Hodgkin's Lymphoma (NHL), including Chronic Lymphocytic Leukemia (CLL). Venetoclax (VEN, an inhibitor of Bcl-2) and ibrutinib (IBR, an inhibitor of BTK) generated excellent clinical responses in CLL patients singly and even more effectively in combination (Portell et al. Blood, 2019; Tam et al.NEJM, 2018). Despite its efficacy, the majority of VEN or VEN+IBR responses are partial and resistance usually develops. We previously reported that treatment with microenvironmental agonists ex vivo can generate anti-apoptotic resistance to the VEN+IBR combination in CLL cells via NF-kB-dependent upregulation of multiple anti-apoptotic proteins (Mcl-1 and Bcl-xL) (Jayappa et al. Blood Adv, 2017). Here, we report that circulating CLL cells of lymph node origin (CD69positive) exhibit resistance to several BH-domain antagonists (inhibitors of Bcl-2, Mcl-1, and Bcl-xL) when used as single agents. Apoptosis resistance was shown to occur at a pre-mitochondrial level by insufficient activation of Bax/Bak proteins (Fig. 1A-B). Supportive of these findings, ex vivo treatment of primary CLL cells with agonists (sCD40L, TLR9 agonist CpG-ODN, and IL10) known to be expressed in the cancer microenvironment in vivoalso resulted in apoptosis restriction due to defective Bax/Bak activation. Our molecular studies suggest that this resistance is driven by the upregulation of anti-apoptotic proteins, which generates an intracellular environment in which single-drug treatments allow pro-apoptotic proteins (e.g. Bim) to swap between upregulated anti-apoptotic proteins (Mcl-1/Bcl-xL/Bcl-2), leading to defective Bax/Bak activation. Hence, therapies aimed at depleting this reservoir must block multiple anti-apoptotic proteins simultaneously or bypass Bax/Bak-dependent apoptosis. Several cancers, including NHL, have evolved mechanisms to suppress the activity of Protein Phosphatase 2A (PP2A), a serine/threonine phosphatase known to regulate cell survival/proliferation. In our current study involving various cancer cell lines (~250), a small molecule agonist of PP2A (SMAP, TRC-382) showed broad activity across blood cancer cell lines. A further pharmacologically optimized SMAP compound (DT061), also known to be safe in animal models (Tohmé et al. JCI Insight, 2019), was effective even in blood cancer cell lines and agonist treated CLL samples resistant to several BH-domain antagonists (inhibitors on Mcl-1, Bcl-xL, and Bcl-2), suggesting that PP2A activation could overcome pre-mitochondrial apoptosis restriction. DT061 was able to overcome drug resistance in patient-derived CD69positive CLL cells through the induction of Bax/Bak-independent apoptosis (Fig. 1C). Supportive of this finding, DT061 was also able to induce apoptosis in Bax/Bak double knockout CLL cell line (MEC1) (Fig. 1D). To determine the mechanisms underlying SMAP-induced apoptosis, we examined additional pathways capable of triggering apoptosis such as mitochondrial permeability transition pore (MPTP), calcium channels, and VDAC1 using selected small molecule inhibitors. Only inhibitors blocking MPTP (NIM811 or cyclopsorin-A/CspA) significantly impaired the DT061- but not VEN-induced apoptosis in primary CLL cells (Fig. 2A-B). Additionally, our analysis using the CalceinAM/CoCl2 method revealed that DT061 was able to induce MPTP opening in primary CLL cells, which was inhibited in the presence of NIM811 or CspA, suggesting that DT061 induces apoptosis through MPTP activation (Fig. 2C). In summary, we identify, in the circulation of treatment-naïve and treated CLL patients, microenvironmentally-activated leukemic B cells that exhibit apoptosis resistance at the level of Bax/Bak activation. Therapies aimed at depleting this reservoir of drug-resistant leukemic B cells must trigger a process of Bax/Bak independent apoptosis. We demonstrate that PP2A activation using the SMAP (DT061) induces apoptosis in drug resistant CLL cells bypassing the Bax/Bak pathway. The apoptosis induction was dependent on MPTP activation. Collectively, this work highlights the existence of an anti-apoptotic multi-drug resistant pool of CLL cells in patients, and validates a novel pharmaceutically tractable pathway to deplete this reservoir. Disclosures Williams: Janssen: Research Funding; Pharmacyclics: Research Funding; TG Therapuetics: Research Funding; Celgene: Honoraria; Gilead: Honoraria; TG Therapeutics: Honoraria; Abbvie: Honoraria; Kite: Honoraria; Xian Janssen: Honoraria. Portell:Xencor: Research Funding; Roche/Genentech: Consultancy, Research Funding; Infinity: Research Funding; TG Therapeutics: Research Funding; AbbVie: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Bayer: Consultancy; BeiGene: Consultancy, Research Funding; Kite: Consultancy, Research Funding; Acerta/AstraZeneca: Research Funding. Narla:The Icahn School of Medicine at Mount Sinai: Patents & Royalties: nternational Application Numbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358 B2; Mount Sinai: Other: Mount Sinai is actively seeking commercial partners for the further development of the technology. G.N. has a financial interest in the commercialization of the technology.; RAPPTA Therapeutics: Consultancy, Current equity holder in private company.

Abstract 5230: Modulation of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia

Abstract Splicing factor SF3B1 is frequently mutated in chronic lymphocytic leukemia (CLL) patients and has been suggested as a potential therapeutic target. In this study, we demonstrated that SF3B1 modulator sudemycin D6 (SD6) effectively inhibits cell growth and induces apoptosis in CLL cells. RNA sequencing analysis revealed significant increases in global intron retention in SD6-treated CLL cells. Pathway analysis of the genes associated with increased intron retention suggested that B-cell receptor (BCR) and PI3K signaling pathways were among the most important pathways being affected by SD6. The increases in intron retention were inversely correlated with decreases in mRNA and protein levels of the affected BCR/PI3K pathway molecules including BLNK, BTK, AKT, PLCγ2, and PI3Kδ. SD6 treatment also induced a time-dependent exon-skipping event in MCL-1 mRNA and resulted in significant down-regulation of another anti-apoptotic gene TRAF1, thus collectively contributing to the SD6-induced apoptosis. Furthermore, SD6 treatment can overcome the pro-survival and pro-growth signals and synergize with established CLL therapies ibrutinib, idelalisib, and venetoclax to induce apoptosis in primary CLL cells co-cultured with bone marrow stromal cells and T-cell-derived cytokines. Finally, in vivo treatment with SD6 at 10mg/kg/day for 7 days significantly inhibited the growth of xenograft tumors that were established by subcutaneous inoculation of 5×106 MEC1 CLL cells into NOD mice. Collectively, these results provide a strong rationale for the future clinical development of spliceosome modulators and potential combination therapies for the treatment of CLL. Citation Format: Qimei Han, Jianbo Wang, Austin Y. Shull, Fang Shi, Libin Deng, Jeong-Hyeon Choi, Eun-Jeong Park, Shuo Tu, Lirong Pei, Farrukh T. Awan, Roni Bollag, Locke J. Bryan, Hong-bo Xin, Chandraiah Lagisetti, Thomas R. Webb, Victor Jin, Huidong Shi. Modulation of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5230.

Targeting CD38 Enhances the Antileukemic Activity of Ibrutinib in Chronic Lymphocytic Leukemia.

CD38 has emerged as a high-impact therapeutic target in multiple myeloma, with the approval of daratumumab (anti-CD38 mAb). The clinical importance of CD38 in patients with chronic lymphocytic leukemia (CLL) has been known for over 2 decades, although it's relevance as a therapeutic target in CLL remains understudied. We investigated the biological effects and antitumor mechanisms engaged by daratumumab in primary CLL cells. Besides its known immune-effector mechanisms (antibody-dependent cell-mediated cytotoxicity, complement-dependent death, and antibody-dependent cellular phagocytosis), we also measured direct apoptotic effects of daratumumab alone or in combination with ibrutinib. In vivo antileukemic activity was assessed in a partially humanized xenograft model. The influence of CD38 on B-cell receptor (BCR) signaling was measured via immunoblotting of Lyn, Syk, BTK, PLCγ2, ERK1/2, and AKT. In addition to immune-effector mechanisms; daratumumab also induced direct apoptosis of primary CLL cells, which was partially dependent on FcγR cross-linking. For the first time, we demonstrated the influence of CD38 on BCR signaling where interference of CD38 downregulated Syk, BTK, PLCγ2, ERK1/2, and AKT; effects that were further enhanced by addition of ibrutinib. In comparison to single-agent treatment, the combination of ibrutinib and daratumumab resulted in significantly enhanced anti-CLL activity in vitro and significantly decreased tumor growth and prolonged survival in the in vivo CLL xenograft model. Overall, our data demonstrate the antitumor mechanisms of daratumumab in CLL; furthermore, we show how cotargeting BTK and CD38 lead to a robust anti-CLL effect, which has clinical implications.

AKT/mTORC2 Inhibition Activates FOXO1 Function in CLL Cells Reducing B-Cell Receptor-Mediated Survival.

To determine whether inhibition of mTOR kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL). Stratification of mTOR activity was carried out in patients with primary CLL samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B-cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib. Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT-dependent manner, to promote FOXO1 nuclear localization, activity, and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib. Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib.

Dual inhibition of MEK1/2 and AKT by binimetinib and MK2206 induces apoptosis of chronic lymphocytic leukemia cells under conditions that mimic the tumor microenvironment

Several key pathways mediate signaling via the B-cell receptor, including the mitogen-activated protein kinase-ERK1/2 pathway. However, inhibition of MEK1/2, a key component of the MAPK-ERK1/2 signaling cascade, results in paradoxical activation of AKT in chronic lymphocytic leukemia (CLL) cells. In the current study we demonstrate synergy between the MEK1/2 inhibitor binimetinib and the AKT inhibitor MK2206, which combined induce apoptosis of primary CLL cells and restrict the cell cycle progression and proliferation of the OSU-CLL cell line. The mechanisms of action of the drug combination involve dual inhibition of MAPK-ERK1/2 and AKT signaling and down-regulation of Mcl-1 expression. Collectively, these data suggest that dual inhibition of MEK1/2 and AKT may represent a therapeutic option for CLL, capable of overcoming the pro-survival effects of the lymph node and bone marrow microenvironments.

Expression and Effects of Ligand-activated Estrogen Receptors in Chronic Lymphocytic Leukemia.

Recent epidemiological data indicate that lymphoid tumors may be influenced by estrogens. The effects of estrogens are mediated via nuclear estrogen receptors α (ERα) and β (ERβ). This study investigated the potential functions of ligand-activated ERs in chronic lymphocytic leukemia (CLL). The ER mRNA expression in B lymphocytes isolated from patients with CLL was analyzed by quantitative real-time polymerase chain reaction. To evaluate the effects of ERβ signaling, primary CLL cells and CLL-derived MEC1 cells were treated with selective ERβ agonists. The mRNA expression of ERα, ERβ1 and its splice variant ERβ2 was detected in CLL cells. Selective ERβ agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile induced apoptosis in primary CLL cells and suppressed the growth of CLL-derived MEC1 cells. A suppressive effect of ERβ agonists on the growth of ERβ-expressing CLL cells was found, indicating that ERβ may be considered as a potential therapeutic target in CLL.

NFATC1 activation by DNA hypomethylation in chronic lymphocytic leukemia correlates with clinical staging and can be inhibited by ibrutinib.

B cell receptor (BCR) signaling is a key for survival of chronic lymphocytic leukemia (CLL) cells, and BCR signaling inhibitors are clinically active. However, relapse and resistance to treatment require novel treatment options. To detect novel candidate therapeutic targets, we performed a genome-wide DNA methylation screen with custom arrays and identified aberrant promoter DNA methylation in 2,192 genes. The transcription factor NFATC1 that is a downstream effector of BCR signaling was among the top hypomethylated genes and was concomitantly transcriptionally upregulated in CLL. Intriguingly, NFATC1 promoter DNA hypomethylation levels were significantly variant in clinical trial cohorts from different disease progression stages and furthermore correlated with Binet disease staging and thymidine kinase levels, strongly suggesting a central role of NFATC1 in CLL development. Functionally, DNA hypomethylation at NFATC1 promoter inversely correlated with RNA levels of NFATC1 and dysregulation correlated with expression of target genes BCL-2, CCND1 and CCR7. The inhibition of the NFAT regulator calcineurin with tacrolimus and cyclosporin A and the BCR signaling inhibitor ibrutinib significantly reduced NFAT activity in leukemic cell lines, and NFAT inhibition resulted in increased apoptosis of primary CLL cells. In summary, our results indicate that the aberrant activation of NFATC1 by DNA hypomethylation and BCR signaling plays a major role in the pathomechanism of CLL.

TP53-induced glycolysis and apoptosis regulator protects from spontaneous apoptosis and predicts poor prognosis in chronic lymphocytic leukemia

ObjectivesCirculating chronic lymphocytic leukemia (CLL) cells appear not to be overly utilizing aerobic glycolysis. However, recurrent contact with CLL cells in a stromal microenvironment leads to increased aerobic glycolysis and the cells’ overall glycolytic capacity, which promotes cell survival and proliferation. TP53-induced glycolysis and apoptosis regulator (TIGAR) has been directly implicated in cellular metabolism in the control of glycolysis. TIGAR inhibits glycolysis and protects cells from intracellular reactive oxygen species (ROS)-associated apoptosis. MethodsTIGAR mRNA expression was investigated by quantitative PCR in 102 newly diagnosed CLL patients. Furthermore, the relationship between the expression of TIGAR and its clinical characteristics and prognosis were investigated. Moreover, we also investigated the correlation between TIGAR expression and apoptosis in primary CLL cells. ResultsOur data revealed that TIGAR overexpression was correlated with the protection from spontaneous apoptosis in CLL cells, and is strongly associated with advanced Binet stage, unmutated immunoglobulin heavy-chain variable region (IGHV) status, CD38 positivity, β2-microglobulin and p53 aberrations. Higher expression of TIGAR was associated with shorter treatment-free survival (median: three months vs. 51 months, P=0.0108), worse overall survival (median: 74 months vs. not reached, P=0.0242), and the diverse responses to fludarabine-based chemotherapy. TIGAR expression in patients resistant to chemotherapy was significantly higher than in patients sensitive to chemotherapy (mean: 0.3859±0.1710 vs. 0.0974±0.0291, P=0.0290). ConclusionTaken together, our findings revealed that high TIGAR expression is closely correlated with worse clinical outcome in CLL patients, and depicted how bioenergetic characteristics could be therapeutically exploited in CLL.

Idelalisib for the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma.

Idelalisib is a first-in-class selective oral PI3Kδ inhibitor for the treatment of patients with relapsed chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma, a predominantly elderly population with high comorbidity. The drug promotes apoptosis in primary CLL cells ex vivo, independent of common prognostic markers and inhibits CLL cell homing, migration and adhesion to cells in the microenvironment. Idelalisib has shown efficacy with acceptable safety as monotherapy and combination therapy in relapsed/refractory CLL. Idelalisib has clinical activity in patients with CLL with del(17p). The development of other novel B-cell-targeted agents provides the opportunity to evaluate additional idelalisib treatment combinations for their potential to further improve outcomes in CLL/small lymphocytic lymphoma.

Reactivation of Smac-mediated apoptosis in chronic lymphocytic leukemia cells: mechanistic studies of Smac mimetic.

Dysfunctional apoptotic machinery is a hallmark feature of chronic lymphocytic leukemia (CLL). Accordingly, targeting apoptosis regulators has been proven a rational approach for CLL treatment. We show that CLL lymphocytes express high levels of XIAP, cIAP1, and cIAP2 compared to normal lymphocytes. Smac mimetic, Smac066, designed to bind to BIR3-domain of IAPs, induce apoptosis in primary CLL cells (n=71; p<0.0001), irrespective of prognostic markers. Apoptosis was mediated by diminished levels of IAPs (XIAP-p=0.02; cIAP-p<0.0001) and increased activation of caspases-8, −9, −3. The caspase-cleavage was in direct association with the levels of apoptosis (r2=0.8 for caspases-8, −9, −3). Correlative analysis revealed a direct relationship between reduction in IAPs and degree of apoptosis (r2=0.6 (XIAP); 0.5 (cIAP2)). There was a strong association between apoptosis, IAP-degradation, and concurrent caspase-activation. Pan-caspase inhibitor Z-Vad-fmk reversed the degradation of Mcl-1, but not IAPs suggesting that smac066 is selective to IAPs, however, Mcl-1 degradation is through caspase-mediated cleavage. Immunoprecipitation experiments revealed physical interaction between caspase-3 and XIAP that was disrupted by smac066. Importantly, XIAP and cIAP2 were markedly induced in bone-marrow and lymph-node microenvironments, providing a basis for IAP antagonists as anti-tumor agents in CLL. Smac066 synergized with ABT-737, revealing a mechanistic rationale to jointly target BH3 and BIR3 domains.

Functional characterization of NAD dependent de-acetylases SIRT1 and SIRT2 in B-Cell Chronic Lymphocytic Leukemia (CLL)

ABSTRACTSirtuins (SIRT) are nicotinamide adenine dinucleotide (NAD+) dependent deacetylases or ADP- ribosyl transferases (ARTs) that deacetylate lysine residues on various proteins regulating a variety of cellular and metabolic processes. These enzymes regulate metabolism, cell survival, differentiation and DNA repair. SIRT proteins play an important role in the survival and drug resistance of cancer cells. The purpose of the present study was to investigate the expression and role of SIRT in chronic lymphocytic leukemia (CLL). We analyzed the expression of SIRT1 and SIRT2 in CLL and normal B cells using the Oncomine database as well as by Western blotting of fresh CLL cells from patients and pro-lymphocytic leukemia (PLL) cell lines, JVM-3 and MEC-2. We showed that both primary CLL cells and JVM-3 and MEC-2 cell lines overexpress high levels of functional SIRT1 and SIRT2. SIRT inhibitors EX-527 and sirtinol impair cell growth, induce ROS production, loss of mitochondrial membrane potential and apoptosis in primary CLL cells and cell lines. Using shRNA knock down of SIRT1 and SIRT2 in JVM-3 and MEC-2 cell lines, we showed that expression of both proteins is crucial for the survival of these cells. Furthermore, studies in nutrient deprived conditions suggest a role of SIRT in metabolism in CLL. These results demonstrate that the inhibition of SIRT1 and SIRT2 activity may be a new therapeutic approach for CLL.

Combination Effects of B Cell Receptor Pathway Inhibitors (Entospletinib, ONO/GS-4059, and Idelalisib) and a BCL-2 Inhibitor in Primary CLL Cells

BackgroundCompared to single-agent therapy, combination therapy in chronic lymphocytic leukemia (CLL) has the goal to achieve complete responses with greater durability and ultimately to achieve minimal residual disease negativity and cure with finite therapy. The investigational agents entospletinib, ONO/GS-4059, and ABT-199 are selective, oral, small molecule inhibitors of SYK, BTK, and BCL-2, respectively, and currently are each being developed for the treatment of CLL. Idelalisib (Zydelig™), a first-in-class, selective, oral inhibitor of PI3Kδ, is approved for the treatment of relapsed chronic lymphocytic leukemia (CLL) in combination with rituximab. Though significant clinical efficacy has been achieved in CLL with currently approved agents, improvement in outcome could be enhanced with combinations of novel agents. We investigated the activity of combinations of the B cell receptor pathway inhibitors, entospletinib, ONO/GS-4059 and idelalisib, and the BCL-2 inhibitor, ABT-199, in vitro in primary CLL cells to define the potential utility of these combinations for clinical investigation in patients.MethodsPeripheral blood mononuclear cells (PBMCs) prepared from CLL patients were stimulated with αIgM/αIgG and αCD40 with or without co-culture with the human bone marrow stromal cell line, HS-5. Entospletinib, ONO/GS-4059, idelalisib, or ABT-199 were combined and incubated with cells for 66-72 hours at clinically achievable concentrations of each. Apoptosis was assessed in CD5+ CD19+ CLL cells by Annexin V/Live-Deadassay (Promega). Combinations of the compounds were assessed using a range of concentrations in an 8 × 3 or 3 × 3 matrix format and assayed for apoptosis using a minimum of 8 individual CLL donors after 66-72 hours. Synergy was evaluated using the Bliss model of independence (Meletiadis, J., et al., Med Mycol, 2005).ResultsStimulation of CLL cells by αIgM/αIgG/αCD40 led to a variable, but statistically significant, protection of primary CLL cells in vitro (p=0001), and co-culture with the stromal cell line, HS-5, in conjunction with αIgM/αIgG/αCD40 stimulation further reduced the observed level of apoptosis (p=0.0051). Entospletinib, ONO/GS-4059, idelalisib, and ABT-199, were able to induce apoptosis in primary αIgM/αIgG/αCD40 stimulated CLL cells with an average maximal proportion of apoptotic cells of 50.2%, 26.1%, 23%, and 91.4%, respectively. Addition of ONO/GS-4059 + entospletinib or idelalisib, had an additive effect on induction of apoptosis in primary CLL cells from 5/9 and 6/8 patients, respectively (Figure 1). The addition of ABT-199 to entospletinib, ONO/GS-4059, or idelalisib showed additive to synergistic effects on induction of apoptosis in primary CLL cells from all patients tested, and increased the maximal level of apoptosis (Figure 1).Primary CLL cells were exposed to pairwise combinations of ONO/GS-4059 with entospletinib or idelalisib (A) or ONO/GS-4059 entospletinib, or idelalisib with ABT-199 (B) using a range of concentrations in an 8 x 3 or 3 x 3 matrix format for 66-72 h. Each point represents the mean result for each pairwise combination for each compound at each concentration tested. The percent apoptosis was assessed by AnnexinV/Live-Dead cell staining. The plots show the measured apoptosis versus the calculated, Bliss predicted score for each combination at each concentration tested. Observed apoptosis greater than the bliss predicted score indicates a synergistic relationship. Area within the dashed lines, indicate additive responses.SummaryCombination treatment with the B cell receptor pathway inhibitors resulted in additive increases in apoptosis in primary CLL cells compared to treatment with the individual agents. The combination of the BCL2 inhibitor ABT-199 with entospletinib, ONO/GS-4059 or idelalisib resulted in an additive to synergistic increase in apoptosis in these primary CLL cells and achieved the greatest maximal levels of apoptosis. These in vitro data support clinical investigation of these combinations in patients with CLL. [Display omitted] [Display omitted] DisclosuresJones:Gilead Sciences: Employment, Equity Ownership. Axelrod:Gilead Sciences: Employment, Equity Ownership. Tumas:Gilead Sciences: Employment, Equity Ownership. Quéva:Gilead Sciences: Employment, Equity Ownership. Di Paolo:Gilead Sciences: Employment, Equity Ownership.

Abstract 2657: Elevated level of BCL-2 is the primary target for inhibition during duvelisib (IPI-145) therapy: ABT-199 neutralizes the resistance mechanism in chronic lymphocytic leukemia

Abstract Inhibition of BTK by ibrutinib, PI3K-delta with idelalisib, or PI3K-delta and gamma with duvelisib (IPI-145), all components of B-cell receptor (BCR) pathway, has significantly improved the treatment outcome in chronic lymphocytic leukemia (CLL) via disrupting the interactions with microenvironment. Though BCR network inhibitors induce durable remission in the majority of CLL patients, a proportion of patients that initially respond to treatment develop resistance or some show maintained lymphocytosis in the blood. Identifying and overcoming resistance mechanisms will be crucial for the most effective combinatorial use of these agents. Duvelisib is an orally bioavailable, highly potent small molecule inhibitor of p110δ and p110γ with KD values of 0.023 nM and 0.24 nM, respectively. Preclinical investigations with duvelisib overcame signals from PI3K/AKT/S6 pathway and promoted apoptosis in primary CLL cells (Balakrishnan et al, ASH 2013). Duvelisib is currently in a phase III trial in CLL. During first phase I study, we performed molecular investigations. PBMCs collected from CLL patients from Phase I study of duvelisib treatment (Pre/Day 0 and Post/Day 28) were subjected to RPPA analysis (n = 7). Interestingly, of the 141 proteins analyzed, Bcl-2 was maximally and significantly elevated in Day 28 samples (mean fold + SEM: 1.7 + 0.2; p = 0.015), suggesting that this could be in part the mechanism involved in resistance during therapy. Further analysis of protein expression by immunoblotting confirmed that Bcl-2 protein was elevated in post-treatment samples (1.3 + 0.1; p = 0.086; n = 7). This was in conjunction with elevated levels of Bcl-2 transcripts analyzed by mRNA array (TaqMan Human Apoptosis - 93 genes) assay (3.0 + 0.4; p = 0.002) and RT-PCR (1.9 + 0.2; p = 0.003), while other anti-apoptotic genes (Bfl-1, Mcl-1, Bcl-w Bcl-g, Bcl-b, Bcl-xL) were unchanged. Importantly, ex-vivo incubations of pre- and post- duvelisib samples with 3 nM ABT-199, a highly selective clinically promising Bcl-2 protein antagonist, induced significantly greater apoptosis in post-therapy samples (79%) in comparison to pre-treatment (58%) samples suggesting that Bcl-2 is the primary target for inhibition during duvelisib intake (n = 5; p = 0.041). In addition, duvelisib induced sensitivity on ex-vivo post- duvelisib clinical samples (n = 15) was specifically towards ABT-199 and produced significantly more apoptosis in the presence of ABT-199 (45%, p&amp;lt;0.0001; 3 nM) compared to other clinically-relevant agents such as ibrutinib (1%, p = 0.10; 10 μM), idelalisib (4% p = 0.05; 10 μM) or ABT-737 (16%, p = 0.0002; 10 nM). We report that elevated Bcl-2 level is the primary target for inhibition during duvelisib therapy and thereby combination with ABT-199 could be a rational approach to overcome the resistance mechanism. Citation Format: Viralkumar M. Patel, Kumudha Balakrishnan, Renato Guerrieri, William Wierda, Susan O'Brien, Varsha Gandhi. Elevated level of BCL-2 is the primary target for inhibition during duvelisib (IPI-145) therapy: ABT-199 neutralizes the resistance mechanism in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2657. doi:10.1158/1538-7445.AM2015-2657

NFATc1 Is Transcriptionally Activated in Chronic Lymphocytic Leukemia (CLL) By Promotor DNA-Hypomethylation Which Correlates with in-Vitro Vulnerability to Calcineurin Inhibitors

Chronic lymphocytic leukemia (CLL), the most frequent adult leukemia in Western countries, is characterized by progressive accumulation of mature, monoclonal B lymphocytes in blood, bone marrow, and lymphoid tissues. In the pathogenesis and treatment of CLL, B cell receptor (BCR) signaling plays a crucial role, and aberrations in downstream pathways that become activated in CLL need to be better defined. One downstream target of BCR signaling is NFATc1, a transcription factor with a high oncogenic and transforming potential. Employing a genome-wide comparative DNA methylation analysis the NFATc1 5’ region was identified to be DNA hypomethylated in CLL patient samples. The pilot series comprised CD19+ B-cells from 13 CLL patients without previous treatment (9 with mutated IGHV using a 98% similarity cut-off) and CD19+ B-cells from 6 age-matched healthy donors. DNA methylation was relatively quantified using methyl-CpG immunoprecipitation (MCIp) on custom oligonucleotide promoter arrays covering -8kbp to +2.5kbp of all human transcription start sites. In total 1944 genes showed significant DNA hypomethylation in CLL cells compared to non-malignant B-cells (p<0.05), with NFATc1 ranking at position 36. DNA hypomethylation of the NFATc1 promoter was validated in peripheral blood CD19+ B-cells from 76 CLL patients without previous treatment compared to 14 additional age-matched healthy donors using mass spectrometry based quantitative high resolution measurement of DNA methylation (MASSARRAY). These findings suggest activation of the NFATc1 gene in CLL cells. In fact, NFATc1 mRNA was significantly upregulated in CLL samples compared to non-malignant B-cells, and expression correlated highly significantly with DNA methylation (r=-0.66, p<0.001). Inhibition of NFATc1 in primary CLL cells from patients (n=5) using calcineurin inhibitors like CsA and FK-506 induced significant apoptosis in primary CLL cells in-vitro even when protected by co-culture with bone-marrow stromal cells, suggesting a dependence of CLL cells on the activity of NFATc1. For a mechanistic insight into NFATc1 signal transduction in primary CLL cells and to allow modeling of the signaling network, a panel of phospho-specific antibodies is currently used in flow cytometry. This allows to monitor multiple components of signaling pathways in parallel with high time resolution to understand the impact of NFATc1 upregulation on the cellular signaling networks. The approach will shed further light on how CLL cell survival is regulated how this pathway can be targeted in CLL. DisclosuresNo relevant conflicts of interest to declare.

Syk Inhibition By TAK-659 Overcomes Proliferative, Survival and Migratory Signals from the Microenvironment in Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation and proliferation of monoclonal CD5+ mature B cells in peripheral blood, lymph nodes (LN), and bone marrow (BM). The microenvironment found in BM and LN induces proliferation of CLL cells and protects them from spontaneous and chemotherapy-induced apoptosis. Syk protein is a tyrosine kinase essential for the BCR signaling pathway that also participates in signaling from chemokine receptors and has been shown to be deregulated in CLL. Therefore Syk has been hypothesized to be a rational candidate for targeted therapy in CLL and its inhibition has been tested with the non-specific Syk inhibitor fostamatinib (R406). Against this background we tested the effectiveness of the highly specific Syk inhibitor TAK-659 in suppressing the induction of survival, proliferation and migration of CLL cells by the microenvironment.To mimic the microenvironment of the proliferative centers ex vivo, we co-cultured primary CLL cells with the BM stromal cells (BMSC), CD40L, CpG ODN and anti-IgM (BCR stimulation). This co-culture system protected CLL cells from apoptosis (mean % of viable cells relative to suspension: 137.52±26.17, P<0.05); proliferative responses were significantly observed after 72 hours (mean % of Ki-67-positive cells: 0.91±0.22 in suspension vs. 7.00±1.49 in co-culture, P<0.001); CLL cells activation according to CD69, CD38, and CD86 expression was markedly induced (mean MFI of CD69: 137.2±26.3 in suspension vs. 339.1±41.4 in co-culture, P<0.01; mean MFI of CD38: 14.1±1.67 in suspension vs. 29.8±6.26 in co-culture, P<0.01; mean MFI of CD86: 27.3±2.99 in suspension vs. 80.8±15.1 in co-culture, P<0.01). Moreover, in this system CLL cells became chemoresistant to fludarabine and bendamustine.TAK-659 inhibited SykTyr525, Btk, Akt and ERK1/2 phosphorylation after BCR cross-linking with anti-IgM in the B cell line Ramos and in primary CLL cells, as assessed by western blot. Syk inhibition by TAK-659 translated into an induction of apoptosis in primary CLL cells, obtaining a LD50 for CLL cells in suspension of 40.39μM (95%CI 21.7-75.2μM) vs. 16.99μΜ (95%CI 7.67-37.67μM) for CLL cells in co-culture. Interestingly, TAK-659 displayed stronger capacity to induce apoptosis than R406 especially in co-cultured CLL cells (LD50 TAK-659 16.99μΜ vs. LD50 R406 not achieved). In addition, combination of TAK-659 with fludarabine, ibrutinib or idelalisib showed a synergistic effect in inducing apoptosis especially in co-cultured CLL cells (Cooperative index for TAK-659 0.1μΜ combined with fludarabine 1μM: 0.62, with ibrutinib 0.1μM: 0.68, and with idelalisib 0.1μM: 0.18). Treatment with TAK-659 also resulted in almost complete abrogation of co-culture-induced proliferation in a dose-dependent manner (mean % of Ki-67-positive cells: 7.00±1.49 in untreated controls vs. 3.39±0.76 after 0.1μM TAK-659 vs. 1.72±0.20 after 1μM TAK-659 vs. 1.27±0.18 after 10μM TAK-659, P<0.01), and CLL cells activation (mean MFI of CD38: 29.7±6.26 in untreated controls vs. 23.9±4.39 after 0.1μM TAK-659, P<0.01; mean MFI of CD86: 80.8±15.1 in untreated controls vs. 58.7±8.99 after 0.1Μm TAK-659, P<0.05). Since BCR signaling also promotes CLL cell chemotaxis toward BMSCs and the chemokines CXCL12 and CXCL13, we next evaluated the effect of treatment with TAK-659 on the migratory capacity of primary CLL cells and we observed that TAK-659 markedly decreased chemotaxis of CLL cells toward CXCL12, CXCL13 and BMSCs. Given the significant homology between Syk and ZAP-70 protein and the critical role of the latter in T cell signaling we aimed to assess the effects of TAK-659 on Jurkat T cells. Surprisingly, we observed that although TAK-659 inhibited ZAP-70Tyr493 phosphorylation, this inhibition did not translate into inhibition of downstream signaling elements, such as Itk, Akt or ERK. We next analyzed the effects of TAK-659 in viability and activation of primary T cells and we observed that TAK-659 did not induce significant apoptosis neither inhibition of activation in terms of CD69 and CD38 expression.In conclusion, these findings demonstrate that, in this ex vivo system, the specific inhibition of Syk by TAK-659 effectively overcomes the microenvironment signals that promote proliferation, activation, survival and chemoresistance of primary CLL cells. Altogether, this study provides a rationale for the clinical development of TAK-659 in CLL. DisclosuresNo relevant conflicts of interest to declare.