Co-culture Of Chronic Lymphocytic Leukemia Cells Research Articles

Abstract 297: Role of purine metabolism in CLL cell pathobiology and CLL disease progression

Abstract The treatment of Chronic lymphocytic leukemia (CLL) has been revolutionized in recent years, however CLL is still incurable, and the leukemic cells often develop drug resistance. Previous studies show that the interaction of CLL cells with the bone marrow (BM) microenvironment promotes spontaneous and drug induced survival. But the nature of this interaction is still in need of a full understanding. To pursue this we discerned RNA profiles using RNA-seq in paired CLL cells isolated from untreated blood and BM (n=6) and in untreated patients CLL cells (n=4) cultured alone or cocultured with BM stromal cells (BMSCs). CLL cells from blood/BM and CLL cells cultured alone or with BMSCs were examined by Western blot (WB), untargeted metabolomics (LCMS+GCMS) and preclinical drug sensitivity assays. We found upregulation of 232 genes in CLL cells from BM vs the paired blood and 917 genes in cocultured CLL cells compared to CLL cells cultured alone (p&lt0.05, fold change &gt1.5). Here we detected only 13 genes that overlap between BM and cocultured CLL cells. When we analyzed the expression of these 13 genes in blood CLL cells from a cohort of 162 untreated patients by RNA-seq we found a positive relationship between 4 (PNP, C16orf54, MOB3A, CDK2AP2) with CLL patient clinical outcome including overall survival (OS), progression free survival (PFS), and time to first treatment (TTFT) [multivariable Cox proportional hazards models, p&lt0.05]. Out of the 4 genes, purine nucleoside phosphorylase (PNP), an enzyme in the purine salvage pathway, showed the most significant association for patient’s OS, PFS, and TTFT. Metabolomic profiling indicated an increased level of purine salvage pathway metabolites adenosine, inosine, and hypoxanthine in cocultured CLL cells. WB analysis using blood CLL cells from untreated patients showed a variable PNP protein expression (high, low/no PNP) and induction in PNP protein levels were found in blood CLL cells expressing low/no PNP only when in contact with BM/BMSCs. But PNP inhibitor forodesine did not show any significant killing in high vs low/no PNP group. When we treated CLL cells from untreated patients with Bcl2 inhibitors venetoclax, S55746 and LP-118 (Bcl-2/Bcl-xl inhibitor, Newave), CLL cells showed increasing sensitivity to the Bcl2 inhibitors associated with their PNP expression (high&gtlow&gtno PNP). But a covalent and non-covalent BTKi LP-168 (Newave) cultured with CLL cells showed no difference in drug sensitivity associated with CLL PNP levels. In contrast CLL cells when treated with both venetoclax and LP-168, showed more drug sensitivity in the high PNP expressing CLL cells vs low/no PNP. These results indicate that active purine metabolism in CLL cells can contribute to differential novel agent drug responses of CLL patients. Future studies to understand the exact mechanism of how PNP relates to this differential drug sensitivity should be instructive in regard to alternate maneuvers to treat CLL. Citation Format: Sutapa Sinha, Weiguo Han, Zhiquan Wang, Kari G. Rabe, Susan L. Slager, Chantal E. McCabe, Daniel R. O'Brien, Sameer A. Parikh, Esteban Braggio, Yi Chen, Fenlai Tan, Stephen P. Anthony, Yu Chen, Bing Dai, Yue Shen, Neil E. Kay. Role of purine metabolism in CLL cell pathobiology and CLL disease progression [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 297.

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.

Nurse-like Cells Express High-Levels of Wnt5a, Which Induces ROR1-Dependent Signaling That Promotes Migration and Survival of Neoplastic Cells in Chronic Lymphocytic Leukemia

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen expressed on chronic lymphocytic leukemia (CLL) cells, but not on normal postpartum tissues. The ligand that binds to ROR1 is Wnt5a, which we find present at high levels in the plasma of patients with CLL relative to that of age-matched healthy adults. Wnt5a can induce CLL-cell activation of Rho-GTPases in a ROR1-dependent manner, thereby enhancing leukemia-cell proliferation, migration, and survival. Currently, the cell-source(s) of Wnt5a in CLL is not known. Good candidates include Nurse-Like cells (NLCs), which are monocyte-derived accessory cells that develop in the context of CLL cells and that are found within the leukemia-cell microenvironment in lymphoid tissues. Already NLCs have been shown to elaborate factors that can promote leukemia-cell activation/survival, including chemokines (e.g. CXCL12, CXCL13) and members of the tumor-necrosis-factor family (e.g. BAFF and APRIL). Because activated monocytes can produce Wnt5a, we hypothesize that NLC also may produce this non-canonical Wnt factor. For this study, we generated NLCs from blood mononuclear cells of CLL patients through co-culture with leukemia B cells using established methods. NLCs, which stained positive for CD163 and CD68, were isolated free of leukemia cells via adherence. We isolated RNA from purified CLL cells or purified NLC of the same patient for real-time PCR to measure the levels of WNT5A transcripts. WNT5A transcripts were abundant in the RNA isolated from NLC, but were negligible in RNA isolated from CLL cells each patient tested (N = 5). We evaluated for Wnt5a via ELISA in the culture-medium of isolated NLCs of five different patients. The cultured medium of NLCs from each patient had detectable Wnt5a that increased in concentration over time. Furthermore, ultra-high resolution confocal fluorescent microscopy of NLC-CLL co-cultures showed Wnt5a was expressed at high-levels in NLCs, but was undetectable in CLL cells of each patient tested (N = 3). We performed co-culture studies and trans-well assays to assess the survival and migration of CLL cells when cultured with or without NLCs, and with or without a neutralizing mAb specific for Wnt5a or cirmtuzumab, which is a humanized anti-ROR1 mAb that blocks Wnt5a-induced, ROR1-dependent signaling. We found that anti-Wnt5a or cirmtuzumab, but not a mAb of irrelevant specificity, each could comparably and significantly inhibit the protective effects of NLCs for CLL cells in vitro (p= 0.008, N = 3 and p= 0.0032, N = 3, respectively by paired student's t-test). Moreover, anti-Wnt5a or cirmtuzumab each could comparably and significantly inhibit the migration of CLL cells enhanced by co-culture with NLCs in vitro (p < 0.0001, N = 3, by paired student's t-test). We also examined for Rho-GTPase activation in CLL cells co-cultured with NLCs. CLL cells co-cultured with NLCs, but not CLL cells cultured alone, had high-level activation of Rac1 and RhoA. The activation of RhoA and Rac1 in co-cultured CLL cells could be blocked by neutralizing anti-Wnt5a or cirmtuzumab, but not by control mAb of irrelevant specificity. We conclude that NLCs express Wnt5a, which can enhance CLL-cell migration and survival via a ROR1-dependent pathway. We speculate that the high-level Wnt5a noted in the plasma of patients with CLL is produced primarily by NLCs, which reside in lymphoid tissues. If so, then the high-level Wnt5a present in the plasma of CLL patients may extend the protective effects of NLCs beyond the CLL microenvironment in which NLCs reside. Treatment with cirmtuzumab to block ROR1-dependent Wnt5a-signaling may mitigate such effects, potentially enhancing the clearance of leukemia cells when used alone or in combination with drugs that target other survival-signaling pathways in CLL. DisclosuresKipps:AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Oncternal: Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Functional Calcitriol/Vitamin D Receptor Signaling in Chronic Lymphocytic Leukemia

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&gt;|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&lt;0.05) and, in contrast decreased pNF-κBlevels (FD=2.7, p&lt;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&lt;0.05), while the addition of calcitriol downregulated pNF-κB (FD=1.5, p&lt;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&lt;0.01; FD=1.4, p&lt;0.01; FD=1.9, p&lt;0.01; for each timepoint respectively) but, significant downregulation of pNF-κΒ (FD=3.4, p&lt;0.01; FD=3, p&lt;0.05; FD=2.3, p&lt;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.

Wnt5a Induces ROR1-Dependent Upregulation of MMP9 and Enhanced Invasiveness in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) cells recirculate between blood and lymphoid tissue compartments, where they receive growth/survival signals from accessory cells within the lymphoid-tissue microenvironment. Such trafficking requires leukemia cells to pass through endothelial barriers and to transverse the extracellular matrix (EM), a process that is facilitated by matrix metallopeptidases (MMP). We co-cultured CLL cells with marrow mesenchymal stromal cells (MSCs) at a physiologic oxygen tension (e.g., 5% O2 in N2). Under such conditions we found that MSCs could enhance leukemia-cell expression of MMP9, a 92 kDa type IV collagenase and key MMP involved in EM degradation. Unexpectedly, however, we found circulating CLL cells with high-level expression of the onco-embryonic protein, ROR1 (designated ROR1Pos), also had high-level expression of MMP9 despite not having immediate contact with such accessory cells. Moreover, we found that circulating ROR1Pos CLL (N = 12) had significantly greater levels of MMP9 than blood CLL cells with low-to-negligible levels of ROR1 (ROR1Neg CLL, N = 10) (P &lt; 0.001). Short-term culture of ROR1Pos CLL in serum-free media significantly reduced their expressed levels of MMP9, unless we added exogenous Wnt5a, a non-canonical Wnt factor and ligand for ROR1 that we found expressed at significantly higher levels in the plasma of patients with CLL than in age-matched adults (Yu et al., J Clin Invest, 2015). Treatment of ROR1Pos CLL cells with Wnt5a enhanced their expression and release of MMP9 and their capacity to invade Matrigel in a Boyden-Chamber Assay; such effects could not be inhibited by inhibitors of B-cell receptor/chemokine signaling (e.g. Ibrutinib), but could be blocked by cirmtuzumab, a humanized mAb specific for ROR1 that can inhibit leukemia-cell ROR1-signaling in patients with CLL (Choi et al., Cell Stem Cell, 2018). Silencing expression of MMP9 with siRNA or treatment with a MMP9-specific inhibitor (CAS 1177749-58-4) could inhibit the capacity of Wnt5a to enhance the invasive capability of CLL cells, indicating that MMP9 plays a major role in facilitating CLL-cell invasiveness. Targeted siRNA-mediated silencing of cytoskeletal proteins HS1/cortactin, which complex with ROR1 in response to Wnt5a (Hasan et al., Leukemia, 2018; Hasan et al., Leukemia, 2017), also impaired the capacity of Wnt5a to enhance expression and release of MMP9 in ROR1Pos CLL. Collectively, these studies indicate that Wnt5a can induce ROR1-signaling leading to enhanced expression of MMP9, which can facilitate invasion of the EM. Moreover, inhibitors of ROR1 signaling, such as cirmtuzumab, can repress Wnt5a-induced CLL-cell expression of MMP9, potentially impairing the homing of ROR1Pos CLL to their protective niches within the lymphoid microenvironment. Disclosures Kipps: AstraZeneca, Inc.: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Velos-Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jannsen Pharmaceutical Companies of Johnson &amp; Johnson: Honoraria, Membership on an entity's Board of Directors or advisory committees.

CG-806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broad Signaling Inhibition in Chronic Lymphocytic Leukemia Cells

CG-806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broad Signaling Inhibition in Chronic Lymphocytic Leukemia Cells

TR57, an Inhibitor of the Integrated Stress Response, Is Synergistic with Venetoclax Against CLL Cells, Independent of Their TP53 Status

Introduction Despite recent advances in the treatment of chronic lymphocytic leukemia (CLL), a significant proportion of patients still relapse with drug-resistant disease. Patients with deletions and/or mutations in TP53 remain a poor prognostic sub-group. There remains a need for on-going research on identifying novel treatment strategies. We have investigated the therapeutic potential of TR57 (Madera Therapeutics, USA), as a single agent and in combination with the Bcl-2 inhibitor venetoclax in CLL. TR57 is an inhibitor of the integrated stress response (ISR) and has shown efficacy in pre-clinical studies of breast cancer. Methods TR57 and venetoclax, as single agents or in combination, were studied against primary CLL cells co-cultured with CD40L-expressing fibroblasts to mimic the tumour microenvironment (TME). The effects of the drugs were also assessed against an OSU-CLL TP53 knock-out cell line generated using CRISPr Cas-9. Cell viability was assessed using the mitochondrial membrane potential dye DiIC1(5), propidium iodide (PI) and flow cytometry. Cytotoxic synergy between TR57 and venetoclax was determined by calculating combination indices (CI) using the CompuSyn software, with CI values of &lt;1 indicative of synergy. Primary CLL cells were stimulated into cycle primary using Dsp30 in combination with IL-2. Cell cycle distribution and proliferation were analysed by flow cytometry using PI and CFSE, respectively. The migratory and adhesive capacities of primary CLL cells was assessed using stroma-derived factor 1a and by assessing expression of CXCR4 and CD49d. The mechanisms of the synergy between TR57 and venetoclax were studied by immunoblotting. Statistical analyses were performed using the students t-test with P-values of &lt; 0.05 considered significant. Results TR57 was cytotoxic towards primary CLL cells in a nanomolar range, with IC50 values of 38 ± 1.38 nM and 287 ± 50.45 nM against cells in medium and stromal cell co-culture, respectively. No significant difference was observed in the sensitivity of samples with ATM or TP53 aberrations (n=12). Synergy between TR57 and venetoclax against CLL cells in stromal cell co-culture was consistent with a significant (P &lt; 0.001) decrease in the IC50 for both drugs (Figure 1A). A CI value of 0.13 was calculated at a fractional effect of 0.5. TR57 was cytotoxic towards OSU-CLL and OSU-CLLTP53ko cells, with IC50 values of 3.98 ± 1.03 nM and 90.7 ± 3.51 nM, respectively. Synergy between TR57 and venetoclax was evident in both lines with a CI = 0.1 at Fa 0.5. Dose-response analyses in the OSU-CLLTP53ko line are shown in Figure 1B. Following stimulation of primary CLL cells with Dsp30/IL-2 we observed a significant (P &lt; 0.01) increase in the proportion of cells in S, G2 and M phases, which was consistent with an increase in cell proliferation. TR57 and venetoclax in combination had a greater effect than individual drug treatments, significantly reduced the proportion of cells in these cell cycle phases and the proliferative fraction of cells. TR57 and venetoclax in combination also had a significantly greater effect on the migratory capacity (P &lt; 0.05) of CLL cells and on the expression of CXCR4 and CD49d (P &lt; 0.001) than either drug as a single agent. Immunoblotting of primary CLL and OSU-CLL cells showed that treatment with TR57 decreased expression of Grp78, which supports the notion that this drug may function through inhibition of the ISR. We also observed that TR57, alone and in combination with venetoclax, down-regulated the expression of the pro-survival Mcl-1 and Bcl-2 proteins, up-regulated expression of the pro-apoptotic Noxa and Bax proteins and inhibited the phosphorylation of AKT and ERK1/2-MAPK. Conclusions The data presented demonstrate that TR57 is cytotoxic towards CLL cells under in vitro conditions that mimic the TME and cells with lesions of the TP53 pathway. The synergy observed between TR57 and venetoclax suggests that the drug combination may be highly effective at limiting the survival and proliferation of CLL cells as well as their ability to migrate to, and be retained within, the TME. The mechanisms of this synergy include a shift towards a pro-apoptotic balance in Bcl-2 family proteins and inhibition of signalling via the AKT and ERK1/2-MAPK pathways. Collectively, these data suggest that TR57 alone and in combination with venetoclax may be a highly effective treatment strategy for high risk CLL. Disclosures Iwanowicz: Madera Therapeutics, LLC: Other: President;Ownership.

Oxidative stress as candidate therapeutic target to overcome microenvironmental protection of CLL.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental non-malignant cells for survival. We compared the transcriptomes of primary CLL cells cocultured or not with protective bone marrow stromal cells (BMSCs) and found that oxidative phosphorylation, mitochondrial function, and hypoxic signaling undergo most significant dysregulation in non-protected CLL cells, with the changes peaking at 6-8 h, directly before induction of apoptosis. A subset of CLL patients displayed a gene expression signature resembling that of cocultured CLL cells and had significantly worse progression-free and overall survival. To identify drugs blocking BMSC-mediated support, we compared the relevant transcriptomic changes to the Connectivity Map database. Correlation was found with the transcriptomic signatures of the cardiac glycoside ouabain and of the ipecac alkaloids emetine and cephaeline. These compounds were highly active against protected primary CLL cells (relative IC50's 287, 190, and 35 nM, respectively) and acted by repressing HIF-1α and disturbing intracellular redox homeostasis. We tested emetine in a murine model of CLL and observed decreased CLL cells in peripheral blood, spleen, and bone marrow, recovery of hematological parameters and doubling of median survival (31.5 vs. 15 days, P = 0.0001). Pathways regulating redox homeostasis are thus therapeutically targetable mediators of microenvironmental support in CLL cells.

Abstract 4408: Metabolic alterations induced by ibrutinib in CLL cells as a basis for drug combinations to enhance ibrutinib therapeutic activity

Abstract The use of ibrutinib, which targets the Bruton tyrosine kinase, has significantly improved the therapeutic outcome for patients with chronic lymphocytic leukemia (CLL), especially in cases with chromosome 17p deletion, which usually respond poorly to conventional chemotherapy. However, ibrutinib is not completely curative, and a small portion of CLL patients exhibit resistance. The main objectives of this study were to investigate the effect of ibrutinib on CLL mitochondrial metabolism and to develop novel strategies to enhance the therapeutic activity of ibrutinib. Ibrutinib-induced changes in mitochondrial functions were analyzed by measuring oxygen consumption rate (OCR) as an indicator of mitochondrial respiration and extracellular acidification rate (ECAR) as a parameter of lactate production from glycolysis using an extracellular flux analyzer. In a primary CLL cell-stromal cell co-culture system (which maintains long-term CLL cell viability), ibrutinib induced little change in OCR and ECAR during the first 48-72 h. However, prolonged drug exposure (up to 7 days) in vitro caused a consistent and significant decrease in OCR compared to that in untreated co-cultured CLL cells. Importantly, CLL cells isolated from patients treated with ibrutinib for 7 days also had significantly decreased OCR compared to pre-treatment samples from the same patients, suggesting that the impact of ibrutinib on mitochondrial functions occurred in vivo and was clinically relevant. On western blot analysis, ibrutinib induced decreases in expression of certain respiratory chain components associated with AMPK activation. On electron microscopy, CLL cells treated with ibrutinib in vitro for 6 days exhibited lower numbers of mitochondria than untreated cells, which was confirmed by flow cytometry analysis of cells stained with Mitotracker Green. Functional analysis showed that ibrutinib caused CLL cells to take up less glutamine but did not affect glucose uptake, suggesting that ibrutinib has a preferential impact on glutamine metabolism. On testing the possibility that the metabolic alterations and mitochondrial dysfunction induced by ibrutinib render CLL cells vulnerable to compounds that affect mitochondria or inhibit glutamine metabolism, we found that combinations of ibrutinib with metformin (inhibitor of mitochondrial OXPHOS) or with C968 (glutaminase inhibitor) resulted in synergistic activity against CLL cells and induced massive leukemia cell death compared to either drug alone. Notably, the combination of ibrutinib and C968 was synergistic in killing CLL cells with 17p deletion. Overall, our results suggest that ibrutinib induces metabolic alterations characterized by decreased mitochondrial respiration and attenuated glutamine metabolism. Such metabolic alterations provide a biochemical basis for mechanism-based drug combination to enhance the therapeutic activity of ibrutinib. Citation Format: Helene Pelicano, Li Feng, William G. Wierda, Michael J. Keating, Peng Huang. Metabolic alterations induced by ibrutinib in CLL cells as a basis for drug combinations to enhance ibrutinib therapeutic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4408. doi:10.1158/1538-7445.AM2017-4408

Microenvironment-Induced Expression of PIM Kinases Supports Chronic Lymphocytic Leukemia Cells Survival and Promotes CXCR4-mTOR Pathway Dependent Migration

Lymph node microenvironment provides chronic lymphocytic leukemia (CLL) cells with pro-survival and protective signals, fostering resistance to conventional chemotherapeutics. CLL cells overexpress oncogenic PIM kinases, which modulate proteins engaged in transcription, translation, apoptosis, cell cycle and adhesion/motility (Mol Cancer Ther 2014, 13: 1231-45). Herein, we searched for the link between tumor microenvironment and PIMs expression, compared the clinical characteristics of CLL patients with high versus low expression of PIM kinases, and investigated the consequences of their inhibition with newly developed pan-PIM inhibitor, SEL24-B489 in primary CLL cells.We first evaluated the expression of PIM kinases in CD19+ cells derived from 88 newly diagnosed CLL cases. Patients with unmutated IGHV status exhibited significantly higher PIM1 transcript levels than patients with mutated IGHV genes. Subjects with advanced CLL (Binet C) exhibited higher PIM2 expression than patients in Binet A/B stage. Significantly higher PIM2 transcript abundance at the time of diagnosis was also observed in patients who relapsed after first line treatment (p=0.005). Expression of PIM2 and PIM3 kinases in lymph nodes was significantly higher than in peripheral blood, suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of microenvironment in the control of PIM expression, peripheral blood CLL cells were incubated with anti-IgM or CD40 ligand. Both stimuli induced PIM1 and PIM3 expression. Co-culture of CLL cells with stromal cell (HS5) monolayers promoted the expression of PIM3 isoform.We next assessed the consequences of PIM inhibition in CLL cells using novel pan-PIM inhibitor, SEL24-B489. Incubation with SEL24-B489 decreased phosphorylation of PIM substrates, p-FOXO1/3a(T24/T32) and p-4EBP1(S65), and induced dose-dependent apoptosis in 27 out of 28 analyzed cases, regardless of the IGHV mutation status and including relapsed patients. Of note, SEL24-B489 induced higher apoptotic response in primary CLL cells than referential pan-PIM inhibitor AZD1208. CLL cells with 17p13 deletion and obtained from chemo-refractory patients were also vulnerable to SEL24-B489, suggesting that functional p53 is not required for execution of SEL24-B489-mediated apoptosis. Importantly, SEL24-B489 was not toxic for cells derived from healthy donors.Since microenvironmental cues increase expression of PIM kinases, we hypothesized that interactions with stromal cells might hinder the in vitro activity of the PIM inhibitor. To explore this possibility, we compared apoptotic response to SEL24-B489 in CLL cells co-cultured on HS5 monolayers and CLL cells grown without the stromal support. In 6 out of 7 tested cases, SEL24-B489 overrode the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells.PIM1 was shown to regulate CLL cells migration through CXCR4(S339) phosphorylation (Mol Cancer Ther 2014, 13: 1231-45). Accordingly, SEL24-B489 decreased phospho-CXCR4(S339), CXCR4 surface expression, and impaired CLL cells migration in the CXCL12 gradient. Surprisingly, decrease in the CXCR4 surface expression after SEL24-B489 was relatively modest when compared to the effect of this inhibitor on CXCL12-directed migration. We found that incubation of CLL cells with CXCL12 led to increase in the phosphorylation of mTOR(S2448) and Akt(S473). SEL24-B489 reduced the levels of p-mTOR(S2448), p-Akt(S473), p-4EBP1(T37/T46) and p-TSC2(S1798), revealing inhibitory effect on mTOR pathway. Pre-incubation of CLL cells with an mTOR inhibitor similarly restrained CXCL12-mediated mTOR activity and led to impaired CLL cells migration, uncovering the key role of mTOR axis in CXCR4-dependent migration. Thus, SEL24-B489 impairs the CLL cell migration by inhibiting CXCR4 surface expression and the CXCR4-triggered mTOR pathway.Taken together, we show that microenvironment signals increase expression of PIM kinases, supporting CLL cell survival and migration. Inhibition of PIM kinases impairs CXCR4-dependent migration and leads to CLL cells death, regardless of the p53 status. Targeting PIM kinases in CLL patients will likely release the cells from microenvironmental niches and might be a rational therapeutic strategy. DisclosuresWarzocha:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Czardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Bcl-2 Members As Drug Target and Biomarkers for Response to Ibrutinib and Venetoclax in CLL

Introduction.BCL-2 family members are crucial determinants for survival in normal and malignant B cells. Venetoclax, the BCL-2 targeting drug, was recently clinically approved for CLL. CLL cells cycle between the lymph node (LN) and peripheral blood, and between those compartments display large changes in BCL-XL and MCL-1, which are not targeted by Venetoclax. However, novel BH3 mimetics specific for BCL-XL and MCL-1 are under preclinical development. Ibrutinib is a clinically successful BTK inhibitor that forces Chronic Lymphocytic Leukemia (CLL) cells out of the lymph node. After prolonged application resistance develops in a fraction of patients, who then show fast disease progression. Thus, both Venetoclax and Ibrutinib have potential drawbacks when applied as single agents. For long-term successful application of new drug combinations, it is crucial to understand BCL-2 member functionality of malignant B cells in relation to their normal counterparts. In parallel, we studied clinical samples of CLL patients under Ibrutinib or Venetoclax treatment for changes in BCL-2 family members.Experimental procedures.FACS sorting of tonsil B subsets, mRNA and protein profiling, co-culture of CLL cells, co-IP of BCL-2 members, cell death assays with BH3 mimetics specific for BCL-2, BCL-XL or MCL-1 (ABT-199, WEHI-539, A-1210477), intracellular FACS for BCL-2, BCL-XL and MCL-1 in CLL (CD19/CD5/CXCR4).Results.We mapped the clearly distinct expression profiles for BCL-2 members in tonsillar naïve, germinal center (GC), plasma (PC) and memory B cells, which translate into different BH3 mimetic sensitivity profiles. In brief, naïve and memory cells rely on BCL-2, GC cells on MCL-1, and PC on BCL-XL. These approaches were extended to primary CLL cells. In a LN model where cells are fully resistant to single agent ABT-199 due to induction of MCL-1, BCL-XL and BFL-1, we find that dual or triple BH3 mimetic combinations restore killing. Moreover, there is differential sensitivity to (combinations of) BH3 mimetics between healthy and malignant B cells, suggesting there is a therapeutic window.Furthermore, intracellular FACS staining for BCL-2, MCL-1, and BCL-XL was performed on patients treated with Ibrutinib or Venetoclax. In untreated patients, recent LN emigrants (CD5hi/CXCR4lo) have higher BCL-XL and MCL-1 expression than 'old' returning cells (CD5lo/CXCR4hi). This distinction collapses under Ibrutinib treatment, demonstrating that Ibrutinib affects pro-survival BCL-2 members in vivo. Interestingly, in patients that develop Ibrutinib resistance the MCL-1 or BCL-XL pre-treatment profile reappears. In contrast, under Venetoclax treatment BCL-2 member levels do not change and can even increase in some patients, underlining the different mode of action of the two drugs. The pattern differed among patients (n=5) and increases were observed for BCL-2, BCL-XL, or MCL-1.Conclusions. BH3 mimetics have not only found their way to the clinic, but can also be used as tools for BCL-2 functionality profiling. Secondly, our ex vivo data underline that combination treatment between (paired) BH3 mimetics and/or Ibrutinib may afford better long-term efficacy in CLL than single agents. Thirdly, BCL-2 members may be useful as biomarkers for response and progression. DisclosuresEldering:Roche: Research Funding; Gilead: Research Funding. Brown:Pharmacyclics: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Genetech: Consultancy. Kater:Celgene: Research Funding.

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

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. DisclosuresRossi: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.

Abstract 3554: Functional disparity among BCL-2 members in tonsillar and leukemic B cell subsets probed by next generation BH3 mimetic profiling

Abstract Introduction and aim. Long-lived humoral immunity, in the form of plasma cells (PC) and memory B cells, as well as most B cell malignancies, originate in the germinal center (GC). For successful therapies against malignant B cells it is crucial to understand their survival requirements in relation to their normal counterparts. It was previously shown that murine GC B cells depend on pro-survival protein MCL-1, but not BCL-XL, although both are expressed. In contrast, naïve and memory B cells express BCL-2, and as a consequence, they are sensitive to the BH3 mimetic ABT-737, which blocks both BCL-2 and BCL-XL but not MCL-1. The divergent roles of MCL-1 and BCL-XL in GC B cells still remain unexplained, and it is unknown if this also holds for human B cells. We here dissect these aspects in human tonsillar B subsets and in chronic lymphocytic leukemia (CLL) cells, a B cell cancer where expression of BCL-2 members is influenced by signals that also occur in the normal GC. Experimental procedures. We used FACS sorting of tonsil B subsets, mRNA and protein profiling, co-culture of CLL cells, co-IP of BCL-2 members, cell death assays with next generation BH3 mimetics specific for BCL-2, BCL-XL or MCL-1 (ABT-199, WEHI-539, A-1210477). Results. Naive and memory B cells are mainly sensitive to specific inhibition of BCL-2 by ABT-199. In contrast, GC B cells and tonsil PC are insensitive to ABT-199, but undergo apoptosis when MCL-1 is inhibited. Tonsil PC display yet another profile and are remarkably sensitive to inhibition of BCL-XL. MCL-1 expression in GC B cells is regulated post-translationally and its physiological importance is highlighted by exclusive binding to pro-apoptotic BIM. In contrast, BCL-XL is transcriptionally induced in the GC-light zone, and is solely bound to the weak BH3-only sensitizer BIK, which may explain why BCL-XL is not required for GC B cell survival. These approaches were extended to primary CLL cells either resting or stimulated via CD40, as model for circulating and lymph node resident cells. We showed recently that CD40 stimulation strongly induces MCL-1, BCL-XL and BFL-1, similar to the pattern in healthy GC B cells, and confers complete resistance to ABT-199, whereas untreated CLL cells are highly sensitive. We now demonstrate that dual or triple BH3 mimetic combinations are effective in killing CD40-stimulated CLL. In contrast to GC B cells, BIK is absent in CLL cells while NOXA is highly expressed and, like BIM, is bound to MCL-1. This may account for differential sensitivity to (combinations of) BH3 mimetics. Conclusion. Using novel BH3 mimetics and interaction profiles, we were able to probe the contribution of individual BCL-2 members in survival of normal and malignant B cells. Our findings provide clues to determine therapeutic windows in novel treatment strategies in B cell and other malignancies. Citation Format: Victor Peperzak, Rachel Thijssen, Hanneke ter Burg, Erik Slinger, Arnon P. Kater, Eric Eldering. Functional disparity among BCL-2 members in tonsillar and leukemic B cell subsets probed by next generation BH3 mimetic profiling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3554.

Identification of B Cell Receptor Antigens in the Chronic Lymphocytic Leukemia Microenvironment

Background: B cell receptor (BCR) signaling is a central pathway in Chronic Lymphocytic Leukemia (CLL) pathogenesis that is activated by interactions between CLL cells and the microenvironment in secondary lymphoid organs. Nurselike cells (NLCs) are an important component of this microenvironment, and co-culture of CLL cells with NLCs activates BCR signaling. CLL BCRs are able to recognize vimentin and calreticulin proteins exposed on the surface of NLCs and these interactions are responsible for stromal-mediated anti-apoptotic effects. However, the exact mechanism of BCR activation and the nature of the BCR ligands expressed by NLCs still remain incompletely defined.Aim: The aim of this project is to identify and validate ligands expressed by NLCs that activate BCRs on CLL cells.Methods: CLL PBMCs from 3 CLL patients were cultured in vitro for 14 days until outgrowth of NLCs. Then, NLCs were harvested and lysed, followed by immunoprecipitation with recombinant monoclonal antibodies obtained from 4 different CLL patients carrying unmutated IGHV genes (U-CLL). Immunoprecipitation of human hTERT mesenchymal stromal cells was used as a negative control. Immunoprecipitated proteins were analyzed by label-free quantitative mass spectrometry followed by bioinformatic data analysis using the softwares MaxQuant and Perseus. The quantitative mass spectrometric data enabled us to distinguish between unspecific background proteins and putative BCR ligands.Results: In all samples, around 2600 proteins were identified and around 2000 of them were quantified using mass spectrometry. Unsupervised hierarchical clustering identified the enrichment patterns of NLC-derived BCR ligands. We identified 6 different protein clusters; among them, one cluster included 11 putative CLL BCR antigens with a fold-change cut-off above 10, which were enriched in all 3 NLC samples, but not in hTERT cells. These BCR ligands included cytoskeletal proteins, ER-associated proteins, and membrane-associated proteins, some of them with known auto-antigenic function in other diseases.Conclusion: Recombinant BCRs from U-CLL patients recognize a large number of proteins expressed by NLCs, identified through immunoprecipitation of NLC lysates with CLL BCRs, followed by label-free mass spectrometry. The identified ligands will be further validated by epitope-mapping and BCR activation functional studies to allow a better characterization of the pathogenic antigens in CLL, and of the mechanisms driving CLL survival in the tissue microenvironment. DisclosuresWierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Estrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.

IgM and IgD Receptors Differentially Contribute to CLL Survival and Chemokine Secretion: Implications for CLL Biology and Treatment

Background: B-cell receptor (BCR) signaling is a central pathway activated in the tissue microenvironment of patients with Chronic Lymphocytic Leukemia (CLL). CLL cells express IgM and IgD receptors, but the contribution of these isoforms to signaling and functional responses remains incompletely defined. The mutational status of CLL BCRs, on the other hand, has clear correlation with clinical outcome: patients with unmutated IGHV genes (U-CLL) characteristically have more aggressive disease and an inferior prognosis when compared to patients with mutated IGHV (M-CLL).Aim: The aim of this present study is to characterize functional differences between IgM and IgD signaling, in the context of the CLL microenvironment and of ibrutinib treatment.Methods: Freshly isolated, purified CLL cells from 80 patients (40 M-CLL and 40 U-CLL) were stimulated with soluble or bead-bound anti-IgM or anti-IgD; IgM, IgD expression, phosphorylation of the BCR-related molecules HS1 and ERK, F-actin polymerization and cell viability were analyzed by flow cytometry; HS1 and ERK phosphorylation, and BCL6 protein expression were also analyzed by Western Blot. CCL3 and CCL4 chemokine secretion was measured by ELISA. BCL6 binding to the CCL3 gene promoter region was assessed by ChIP-PCR. We utilized nurselike cell (NLC) co-cultures to activate BCR signaling and to induce CCL3 and CCL4 production in this model system that resembles the lymph node microenvironment. Effects of ibrutinib treatment on CLL-cell viability and BCR activation were also tested.Results: Surface IgM and IgD levels were significantly higher in U-CLL (P<0.0001), ZAP70 positive (P<0.01), and/or del17p or del11q CLL cells (P<0.05). Early BCR signaling with activation of the cytoskeletal protein HS1 and F-actin polymerization were similar after anti-IgM and anti-IgD stimulation. Downstream activation of ERK kinases was more prolonged after anti-IgM than after anti-IgD stimulation. Transient activation of IgD signaling was paralleled by rapid IgD receptor endocytosis, as compared to IgM. Furthermore, anti-IgM increased CLL viability to a significantly greater extent than anti-IgD, and exclusively induced secretion of CCL3 and CCL4 chemokines. This effect was consistently observed after stimulation with either soluble or bead-bound anti-IgM and anti-IgD. Expression of BCL6, which functions as a transcriptional repressor for CCL3 gene, was down-regulated following anti-IgM, but not after anti-IgD stimulation. In addition, we observed BCL6 binding to CCL3 promoter regions and noted that BCL6 down-regulation coincided with the onset of CCL3 secretion. In all experimental settings, U-CLL cells were more responsive to BCR stimulation than M-CLL. NLC co-culture induced down-modulation of surface IgM and IgD on CLL cells, increased HS1 and ERK phosphorylation and induced CCL3 chemokine secretion, along with BCL6 down-regulation. When CLL cells were removed from NLCs, recovery of surface IgM and IgD, baseline BCL6 expression, and HS1 and ERK phosphorylation was observed after 72 hours. Ibrutinib treatment preferentially interfered with anti-IgM induced survival signals and BCR signaling activation, in particular of U-CLL cells.Conclusion: Compared to IgD, IgM stimulation induces more durable signaling responses, CCL3 and CCL4 chemokine secretion and increases CLL-cell survival, in particular in U-CLL cells. BCL6 binding to CCL3 promoter and its down-regulation concomitant to CCL3 production suggests that BCL6 represses CCL3 gene expression in CLL cells. Co-culture of CLL cells with NLCs triggers BCR signaling, with functional outcomes that mainly recapitulate the features of IgM stimulation in vitro. Ibrutinib treatment preferentially interferes with IgM-mediated signals, and reduces CCL3 and CCL4 levels in CLL-cell supernatants and in the plasma of ibrutinib-treated patients. Taken together, these results support the relevance of IgM signaling in the pathogenesis of CLL, and suggest that IgM is the main receptor isotype targeted in vivo during ibrutinib treatment. DisclosuresEstrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Nurse-like cells mediate ibrutinib resistance in chronic lymphocytic leukemia patients

Nurse-like cells mediate ibrutinib resistance in chronic lymphocytic leukemia patients

Effective elimination of chronic lymphocytic leukemia cells in the stromal microenvironment by a novel drug combination strategy using redox-mediated mechanisms.

Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia, and is currently incurable due to drug resistance. A previous study indicated that the redox interaction between bone marrow stromal cells and leukemia cells profoundly affected CLL cell viability and drug response. The present study aimed to further investigate the effect of the redox interaction on drug resistance of CLL cells in the bone marrow microenvironment, and to assess a novel redox-mediated strategy to eliminate stromal-protected CLL cells, and thus to achieve maximum therapeutic efficacy of antileukemic drugs. Histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) is a potent novel anticancer agent, however, it exerts limited activity in patients with CLL. The results of the present study demonstrated that SAHA facilitated stromal-mediated glutathione upregulation in the CLL cells, contributing to drug resistance. The addition of β-phenylethyl isothiocyanate (PEITC) induced severe depletion of stromal and SAHA-upregulated glutathione, enhanced SAHA-mediated reactive oxygen species accumulation in the CLL cells and caused oxidation of mitochondrial cardilopin, leading to substantial cell death. The results further demonstrated that stromal cells and SAHA markedly upregulated antiapoptotic protein expression levels of myeloid cell leukemia 1 (Mcl1) in CLL the cells. By inducing protein deglutathionylation and degradation, PEITC suppressed the expression of Mcl1 in co-cultured CLL cells, and increased SAHA sensitivity. The combination of SAHA and PEITC enabled the induction of marked apoptosis of CLL cells co-cultured with bone marrow stromal cells. The present study provided a preclinical rationale, which warrants further clinical investigation for the potential use of SAHA/PEITC as a novel combination treatment strategy for CLL.

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.

Inhibition of the Notch1 Pathway in Chronic Lymphocytic Leukemia Promotes Apoptosis and Inhibits Proliferation

In chronic lymphocytic leukemia (CLL), mutations in the NOTCH1 receptor occur in 4-10% of newly diagnosed patients and 15–20% of multiply relapsed patients. Using next-generation sequencing, our group previously reported NOTCH1 p.P2514fs mutations in 15 CLL patients (9.4%) in an initial cohort of 160 CLL patients in which NOTCH1 mutations were associated with IGHV unmutated (UM) CLL (p=0.0001). Further analysis using a three-group comparison (NOTCH1 mut, IGHV UM vs. NOTCH1 wild-type [wt] IGHV UM vs. NOTCH1 wt IGHV mut) showed that NOTCH1 mutations associated with both trisomy 12 (p=0.049) and 17p deletion (p=0.0008) and poor overall survival (HR 2.99, p=0.008).Given that targeting activating mutations has proven an effective therapeutic strategy in many cancers, we explored the therapeutic potential of a Notch1 inhibitor, PF-03084014, in CLL. Previous studies in T-cell acute lymphoblastic leukemia cells harboring NOTCH1 mutations have shown that gamma secretase inhibitors can induce apoptosis by blocking Notch1 receptor activation. When we tested the gamma secretase inhibitor (GSI) PF-03084014 in 18 CLL samples with NOTCH1 mutations, it consistently induced apoptosis of all CLLs after 48 hours in culture across all cytogenetic groups tested (13.3-47.2% death with 5 μM GSI, p<0.0001)(Figure A). The induction of apoptosis was similar (GSI vs. ibrutinib, p=ns) to that of ibrutinib (n=11,11.9-74.4% death with 5 μM ibrutinib, p<0.0001). In contrast, GSI treatment only induced apoptosis in some (n=10), but not all NOTCH1 wt CLLs (n=6) (p=0.0137). We next tested the effect of GSI PF-03084014 in the context of a stromal environment. Co culture of CLL cells with CD40L-expressing fibroblasts partially mimics the lymph node and bone marrow microenvironments, which are known sites of drug-resistance and proliferation of CLL in vivo. We tested whether the interaction with stromal cells protects CLL cells from Notch1 inhibitor-induced apoptosis, as seen with other drugs including ibrutinib. We found that co culture with CD40L-expressing 3T3s decreased GSI-induced apoptosis in NOTCH1 mutant CLLs (p=0.0006) and in the majority of the NOTCH1 wt CLLs that responded to the GSI (Figure A).Since NOTCH1 mutations have been reported to be an independent marker of aggressive disease in CLL, we tested whether CLL cells with NOTCH1 mutations were more proliferative in vitro compared to NOTCH1 wt CLL cells. We showed that CLL cells upregulate Ki67 expression in co culture with 3T3-CD40L cells and in a cohort of 10 NOTCH1 mutants and 11 NOTCH1 wt, we found the NOTCH1 mutants to be more proliferative than the NOTCH1 wt (median of 7.6% vs. 2.3%, p=0.015). To then address whether blocking of the Notch1 pathway decreases proliferation, we treated CLL cells in co culture with 3T3-CD40L cells with 5 μM GSI for 7 days. GSI treatment decreased the percentage of Ki67+ CLL cells in all but one NOTCH1 mutant (median decrease 28.3%, p=0.044) as wells as in the majority of NOTCH1 wt samples (median decrease 38.7%, p=0.037).Having established that inhibition of Notch1 can reduce proliferation and induce apoptosis in CLL cells in vitro, we were interested in determining the downstream genes that may be the effectors of this activity. We therefore compared the gene expression profiles (GEP) of NOTCH1 mut vs. NOTCH1 wt CLLs, and found upregulation of genes involved in the Notch1 pathway, in apoptosis and in chemokine signaling in the NOTCH1 mutants. Furthermore, comparing GEP of high Ki67 vs. low Ki67 expressing CLL cells revealed higher expression of a range of both upstream and downstream Notch1 pathway genes in high Ki67 expressing CLL cells.In conclusion, we show that PF-03084014 induces apoptosis and decreases proliferation in both NOTCH1 mutant and wt CLL cells. We find NOTCH1 mutant CLL cells to be more proliferative than NOTCH1 wt and show upregulation of Notch1 pathway genes in NOTCH1 mutants compared to wt CLL cells and in high Ki67 expressing compared to low Ki67 expressing CLL cells. Taken together, these results emphasize the important role of Notch1 signaling in CLL in general, perhaps particularly in proliferative compartments like lymph nodes, and demonstrate that Notch1 pathway inhibitors are worthy of therapeutic investigation in CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of STAT3 Reverses the Resistance to Histone Deacetylase Inhibitors Induced By Interleukin-6 in Chronic Lymphocytic Leukemia Cells

Introduction: Interleukin-6 (IL-6) is a pleiotropic cytokine produced by a variety of cell types, including bone marrow stromal cells (BMSCs) and chronic lymphocytic leukemia (CLL) cells. Levels of IL-6 are increased in patients with CLL and correlated with adverse clinical features and short survival. In preliminary studies we observed that IL-6 induced resistance to the pan-histone deacetylase (HDAC) inhibitors vorinostat (SAHA) and panobinostat (LBH589) in CLL cells. Activation of STAT3 induced by IL-6 in CLL cells was further enhanced by low concentration of SAHA and LBH589. We hypothesized that exposure of CLL cells to HDAC inhibitors would further expand the activation of STAT3 signaling by IL-6, and thus the ability of HDAC inhibitors to kill CLL cells may be blunted due to upregulation of STAT3 downstream antiapoptotic proteins. This study was designed to illuminate the role of the STAT3 pathway on the resistance to SAHA and LBH589 in CLL cells, and to evaluate the proapoptotic activity of the combination of SAHA and LBH589 with a STAT3-selective inhibitor, WP1066.Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from samples of 32 CLL patients. The characteristics of CLL patients were shown in Table 1. The PBMCs contained more than 90% of CD19+ B lymphocytes, which were detected by flow cytometry and were referred to as primary CLL cells. BMSCs were isolated from bone marrow mononuclear cells of healthy volunteers. Cytotoxicity of drugs was assessed by carrying out triplicate assays with the Cell Counting Kit-8 (CCK-8), after a 48-hour incubation of primary CLL cells in the presence or absence of recombinant human IL-6, and half-maximal inhibitory concentration (IC50) values were calculated. Effects of drugs on apoptosis of CLL cells, in the presence or absence of IL-6 or BMSCs, were evaluated by Annexin V/7AAD assay, morphological assessment of Giemsa-stained cytospin preparations and western blot analysis of the cleavage of poly(ADP ribose) polymerase (PARP). The levels of STAT3 phosphorylation (pSTAT3) and STAT3 downstream proteins were evaluated by western blotting.Results: IL-6-induced pSTAT3 was completely abolished by 4-hour incubation with 20µM of SAHA or 100nM of LBH589 before IL-6 was added. But when CLL cells were incubated with lower concentration of SAHA or LBH589 (e.g., SAHA, 1µM; LBH589 10nM), the level of pSTAT3 induced by IL-6 was further enhanced (Fig. 1A-C). Treatment of CLL cells with 10ng/mL IL-6 for 24 hours resulted in greater than 2-fold upregulation of the antiapoptotic proteins, Mcl-1 and Bcl-xL. The upregulation of Mcl-1 and Bcl-xL induced by IL-6 was further enhanced in the presence of 1µM or 2.5µM of SAHA (Fig. 1D-E). IL-6 decreased the sensitivity of CLL cells to each of the HDAC inhibitors tested, as evidenced by an increase in the mean IC50 values. Co-incubation with 5µM of WP1066 blocked the protective action of IL-6 (Fig. 1F-G). Apoptosis induction by SAHA and LBH589 was reduced in the presence of IL-6 and this reduction could be inhibited by WP1066 (Fig. 2A-B). The antiapoptotic action of IL-6 and the ability of WP1066 to restrain cytoprotection by IL-6 were also confirmed by morphological assessment and PARP cleavage analysis (Fig. 2C-E). WP1066 decreased the pSTAT3 level and inhibited increases of Mcl-1 and Bcl-xL proteins induced by IL-6 in the presence or absence of SAHA (Fig. 1D-E). Co-culture of CLL cells with BMSCs resulted in the activation of STAT3 in CLL cells and protected CLL cells from apoptosis induction by HDAC inhibitors, and this cytoprotection was reversed by WP1066 (Fig. 2F-I).Conclusions: Our studies indicated that CLL cells were protected from HDAC inhibitor-induced apoptosis by IL-6 or co-culture with BMSCs. This protection may be mediated by activation of STAT3 signal pathway. Furthermore, WP1066 reversed the resistances of CLL cells to SAHA and LBH589 induced by IL-6 or co-culture with BMSCs. Our findings suggest that targeting the STAT3 pathway may be a novel way to improve the efficacy of HDAC inhibitor in CLL patients by overcoming antiapoptotic signaling of the microenvironment.Table 1CharacteristicCase (n)Total (n)Age (years)32≥6020<6012Sex32Male25Female7Rai stage3205I9II11III4IV3IgVH mutational status25Unmutated (≥98% homology)8Mutated (<98% homology)17ZAP7022≥20%7<20%15CD3824≥20%8<20%16FISHcytogenetics21Normal karyotype1013q-711q-217p-1Trisomy 122 [Display omitted] [Display omitted] DisclosuresLu:Novartis Pharmaceuticals Inc: LBH589 was kindly provided by Novartis Pharmaceuticals Inc. Other.