Targeting Metabolic Alterations in CLL Microenvironment; Inhibition of Glutamine Import Attenuates Venetoclax Resistance

Abstract Introduction: For chronic lymphocytic leukemia (CLL), especially in the lymph node (LN) setting where cells receive proliferative and pro-survival signals, in-depth studies of altered metabolism and its relationship with therapeutic responses are still lacking. Venetoclax, a BCL-2 inhibitor currently in wide clinical use for CLL, has shown high efficiency yet emerging resistance is a growing clinical problem. In cell line models, induced resistance to Venetoclax was accompanied by profound metabolic changes 1. This is in accordance with our earlier findings on metabolic and apoptotic changes that CLL cells undergo within the LN environment 2. In the current study, we performed RNA sequencing and applied fluxomics with 13C 6-glucose and 13C 5-glutamine to investigate in detail the metabolic routes in LN CLL. This led to studies to manipulate glutamine metabolism in a venetoclax resistance model. Methods: Peripheral blood (PB) samples from CLL patients were in vitro stimulated for 24 hrs by CD40 or B cell receptor (BCR), which are two potential key signals in LN. RNAseq analysis was compared with microarray data of paired PB/LN patient samples 3. For fluxomics, CLL cells were cultured for 2 hrs in medium containing either 5 mM 13C 6-glucose or 1 mM 13C 5-glutamine. Incorporation of 13C in metabolic intermediates was analyzed by LC-MS. For glutamine blockade, CLL cells were stimulated in presence of specific inhibitors of glutamine/glutamate metabolism or amino acid transporters. Cells were then treated with venetoclax, and viability was measured. Results: Gene expression profiles demonstrated that CLL cells obtained from LN tissue as well as after in vitro CD40 or BCR stimulation showed increased expression of gene sets involved in glycolysis, oxidative phosphorylation / citric acid cycle (OXPHOS/TCA) and amino acid metabolism as well as Myc activation. This confirmed that in vitro stimulation can be used to model the CLL LN setting. For unstimulated PB CLL cells, fluxomics data demonstrated low uptake of either glucose or glutamine, with 13C labelling close to zero for most metabolites. In contrast, both CD40 or BCR stimulation increased the uptake and utilization of glucose and glutamine. 13C labelling from glucose was detected in all glycolytic intermediates analyzed in both CD40- and BCR-stimulated CLL cells. Glucose was catalyzed to lactate and also partly converted to acetyl-CoA, which entered the TCA cycle. Additionally, labelling from glucose was also increased in several metabolites of the pentose phosphate pathway (PPP) suggesting it entered nucleotide synthetic routes. Compared to glucose, the contribution of glutamine was much higher in the TCA cycle in both BCR and CD40-stimulated cells. All intermediates of the TCA cycle were highly enriched with 13C from glutamine (Figure 1A). Combined, these data revealed that glutamine is the key metabolite to fuel the TCA cycle in LN CLL cells, and prompted us to study effects of glutamine blockade in conditions of Venetoclax resistance. It was found that venetoclax resistance induced by CD40 or BCR stimulation was clearly attenuated by glutamine uptake inhibition. CLL cells became re-sensitized to Venetoclax in both CD40- or BCR-stimulated samples, with an approximate 100-fold shift in IC50 (Figure 1B). Conclusions: Our study highlights the role of glutamine, in addition to glucose, in the metabolic reprogramming that CLL cells undergo in the LN (Figure 1C). These processes show potential for therapeutic targeting. Inhibition of glutamine import could contribute to dampen tumor microenvironment-induced Venetoclax resistance. References 1. Guièze, R. et al. Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies. Cancer Cell 36, (2019). 2. Chen, Z. et al. Effects of Ibrutinib on Metabolic Alterations and Micro-Environmental Signalling in Chronic Lymphocytic Leukaemia. Blood 136, (2020). 3. Herishanu, Y. et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-κB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 117, 563-574 (2011). Figure 1 Figure 1. Disclosures Forconi: AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria; Novartis: Honoraria; Gilead: Research Funding. van der Windt: Genmab: Current Employment. Kater: Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Abbvie: Honoraria, Other: Ad Board, Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee.

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The Syk\Jak Inhibitor Cerdulatinib (PRT062070) Shows Promising Preclinical Activity in Chronic Lymphocytic Leukemia By Antagonising B Cell Receptor and Microenvironmental Signalling

Blood ◽

10.1182/blood.v126.23.1716.1716 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1716-1716 ◽

Cited By ~ 1

Author(s):

Matthew D Blunt ◽

Jack Parnell ◽

Marta Larrayoz ◽

Lindsay Smith ◽

Rachel Dobson ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

Kinase Inhibitors ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Signalling Pathways ◽

Simultaneous Inhibition ◽

Induced Apoptosis

Abstract The emergence of B cell receptor (BCR) kinase inhibitors has proved effective for the treatment of a number of B-cell malignancies including chronic lymphocytic leukemia (CLL). BTK and PI3K inhibitors have clear efficacy in suppressing tumor progression but have not been curative. A number of patients have developed resistance to these drugs following mutation of the BTK or PLCγ2 gene. Whilst, other patients are unable to tolerate these drugs due to adverse events or progress whilst on therapy for unknown reasons. Thus the development of novel drugs which are still effective once other BCR-kinases inhibitors become ineffective is of paramount importance. Spleen tyrosine kinase (Syk) is essential for B cell receptor signalling pathways as well as a variety of other surface receptors such as MHCII, FC receptors and integrins, all of which have been shown to play a role in CLL biology. Importantly, Syk inhibition has been shown to overcome resistance to ibrutinib, identifying Syk inhibition as a promising strategy to treat these patients. Furthermore, we have previously shown that IL-4 is found in CLL lymph nodes and can promote resistance to ibrutinib and idelalisib by restoring αIgM induced calcium flux and phosphorylated ERK (ASH 2014, abstract #3299). IL-4 signalling is mediated through the JAK/STAT signalling pathways via JAK1 and JAK3, therefore simultaneous inhibition of both Syk and JAK1/3 may be therapeutically beneficial over BCR kinase inhibitors alone. Cerdulatinib (PRT062070) is a dual JAK/Syk inhibitor in a phase I open label dose escalation study and is currently demonstrating clinical activity in patients with relapsed/refractory B cell malignancies including CLL. Our group has now demonstrated in vitro that cerdulatinib, at plasma concentrations achievable in patients, can induce apoptosis of CLL cells in a concentration and time dependent manner with a mean IC50 of 3µM and 1µM at 48 and 72h respectively, defined by annexin V/PI and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). Apoptosis was caspase dependent since treatment with the pan caspase inhibitor ZVAD.fmk significantly inhibited cerdulatinib induced cell death at 24h. Cerdulatinib induced apoptosis coincided with an increase in pro-apoptotic proteins Noxa and Puma and a decrease in the anti-apoptotic protein Mcl-1. Cerdulatinib significantly inhibited IL-4 induced phosphorylation of STAT6 at 300nM (p=.005), BCR induced phosphorylation of AKTS473 with soluble (p=.008) and bead immobilised (BI) (p=.025) αIgM at 30nM and phosphorylation of AKTT308 with BI αIgM at 300nM (p=.008). Furthermore, in patients with CLL, it is thought that CD40L and IL-4 are key factors, which promote survival of CLL cells in proliferation centres within the lymph node microenvironment. Therefore, we cultured CLL cells with a vehicle control or IL-4\CD40L, prior to treatment with cerdulatinib. Cerdulatinib alone induced similar levels of apoptosis irrespective of IL-4/CD40L treatment, suggesting cerdulatinib may be able to overcome microenvironmental signals and target cells within the lymph node. Next we explored the possibility of augmenting cerdulatinib induced apoptosis by simultaneous inhibition with the Bcl-2\Bcl-XL inhibitor ABT-199. In vitro in the presence of IL-4/CD40L, ABT-199 synergised with cerdulatinib to induce significantly greater cell death than with either agent alone. Therefore these data provide in vitro evidence for the use of cerdulatinib in clinical trials for the treatment of CLL as either a single agent or in combination with other therapies such as ABT-199. Disclosures Strefford: Roche: Research Funding. Davies:Seattle Genetics: Research Funding; Takeda: Honoraria. Coffey:Portola Pharmaceuticals Inc: Employment, Equity Ownership, Research Funding. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015.

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Chronic lymphocytic leukemia: revelations from the B-cell receptor

Blood ◽

10.1182/blood-2003-12-4312 ◽

2004 ◽

Vol 103 (12) ◽

pp. 4389-4395 ◽

Cited By ~ 286

Author(s):

Freda K. Stevenson ◽

Federico Caligaris-Cappio

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Cell Of Origin ◽

Regulatory B Cell ◽

V Genes

Abstract The finding that chronic lymphocytic leukemia (CLL) consists of 2 clinical subsets, distinguished by the incidence of somatic mutations in the immunoglobulin (Ig) variable region (V) genes, has clearly linked prognosis to biology. Antigen encounter by the cell of origin is indicated in both subsets by selective but distinct expression of V genes, with evidence for continuing stimulation after transformation. The key to distinctive tumor behavior likely relates to the differential ability of the B-cell receptor (BCR) to respond. Both subsets may be undergoing low-level signaling in vivo, although analysis of blood cells limits knowledge of critical events in the tissue microenvironment. Analysis of signal competence in vitro reveals that unmutated CLL generally continues to respond, whereas mutated CLL is anergized. Differential responsiveness may reflect the increased ability of post-germinal center B cells to be triggered by antigen, leading to long-term anergy. This could minimize cell division in mutated CLL and account for prognostic differences. Unifying features of CLL include low responsiveness, expression of CD25, and production of immunosuppressive cytokines. These properties are reminiscent of regulatory T cells and suggest that the cell of origin of CLL might be a regulatory B cell. Continuing regulatory activity, mediated via autoantigen, could suppress Ig production and lead to disease-associated hypogammaglobulinemia. (Blood. 2004;103:4389-4395)

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The Bruton Tyrosine Kinase Inhibitor, PCI-32765, Inhibits Activation and Proliferation of Human Chronic Lymphocytic Leukemia Cells in the NSG Xenograph Mouse Model of the Tissue Microenvironment

Blood ◽

10.1182/blood.v118.21.596.596 ◽

2011 ◽

Vol 118 (21) ◽

pp. 596-596 ◽

Cited By ~ 1

Author(s):

Sarah E. M. Herman ◽

Xiameng Sun ◽

Joseph J. Buggy ◽

Georg Aue ◽

Patricia Perez-Galan ◽

...

Keyword(s):

T Cell ◽

Peripheral Blood ◽

Cell Receptor ◽

Gene Signature ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

T Cell Proliferation ◽

Tissue Microenvironment

Abstract Abstract 596FN2 PCI-32765, a specific inhibitor of Bruton's tyrosine kinase (Btk), can disrupt several signaling pathways involved in tumor microenvironment interactions. In vitro, PCI-32765 has been demonstrated to induce apoptosis, to varying degrees, in tumor cells and prevent CpG-ODN induced proliferation of cultured chronic lymphocytic leukemia (CLL) cells (Herman et al, Blood 2011). PCI-32765 has been shown to be well tolerated in CLL with preliminary clinical trial data showing that >85% (34/39) of patients remained on therapy at a median follow-up of four months. In addition, a significant shrinkage of lymph nodes has been observed in the majority of patients displaying lymphadenopathy. As with other B-cell receptor (BCR) directed therapies, PCI-32765 results in an initial increase in the absolute lymphocyte count. These observations are not explainable by the available in vitro data, demonstrating the need for in vivo investigation. In order to study the effect of PCI-32765 in vivo we chose to use the recently established NOD scid gamma null (NSG) - human CLL xenograft model with some modifications (Bagnara et al., Blood 2011). NSG mice were conditioned with 25 mg/kg busulfan 24 hours before injection of 1 × 108 CLL peripheral blood mononuclear cells previously labeled with 1μM CFSE. We first demonstrated that xenografted CLL cells isolated from the mouse spleen acquire an activated phenotype and proliferate, mimicking the phenotype of CLL cells isolated from human lymph nodes (Sun et al., abstract submitted). Next we sought to use this model to investigate the effect ot PCI-32765 on CLL cell activation and proliferation. Mice received PCI-32765 or vehicle in their drinking water at 0.16 mg/ml dissolved in 1% HP-beta-CD starting at the time of busulfan treatment. Mice were bled weekly and sacrificed between 3 and 4 weeks post xenografting. We found that PCI-32765 treatment resulted in a significant reduction in proliferation (defined as CFSE low cells) compared to mice that received vehicle water; this was observed in all three biological compartments: peripheral blood (84.5% decrease, p=0.007), spleen (72.4% decrease, p=0.012) and bone marrow (92.5% decrease, p=0.049). In comparison, PCI-32765 treatment did not result in a significant reduction in T-cell proliferation in any of the compartments (p>0.4). Although peripheral blood CLL counts were comparable between treated and untreated mice, we found that there were substantially more CLL cells in the spleens of the vehicle treated mice than in those of the PCI-32765 treated mice. In contrast, no differences in T-cell number or localization were observed between treated and untreated mice. Lastly, we sought to determine whether activation of CLL cells in the microenvironment could be blocked by PCI-32765. As we have previously shown, CLL cells in the human lymph node display a gene signature indicating B-cell receptor (BCR) and NF-kB activation compared to CLL cells in the peripheral blood (Herishanu et al., Blood 2011). We used quantitative RT-PCR (pre-designed Taqman Gene Expression assays) to measure expression of representative BCR and NF-kB target genes. PCI-32765 significantly reduced expression of EGR1 (p=0.049), EGR3 (p=0.023) and GFI1 (p=0.023) (BCR signature) and CCL3 (p=0.013) and CCND2 (p=0.046) (NF-kB signature) compared to vehicle treated mice. In addition, we also observed decreases in the proliferation gene signature (CDT1, PCNA and RRM2) (signature score, p=0.035) in the CLL cells from mice treated with PCI-32765; consistent with the assessed CFSE proliferation measurements. Taken together, our results show that PCI-32765 inhibits CLL activation and proliferation in the tissue microenvironment in vivo without affecting T-cell proliferation. These results demonstrate that targeting Btk is sufficient to block key interactions between tumor cells and the microenvironment and thus warrants the use of PCI-32765 as a targeted agent in CLL. Disclosures: Buggy: Pharmacyclics, Inc.: Employment.

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Anergic Response to B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia: The Differing Roles of IgD and IgM in the Microenvironment and Peripheral Blood.

Blood ◽

10.1182/blood.v120.21.2889.2889 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2889-2889

Author(s):

Tom Butler ◽

Alexander Montoya ◽

Andrew James Clear ◽

Rita Coutinho ◽

David C Taussig ◽

...

Keyword(s):

Mass Spectrometry ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Bcr Signaling ◽

B Cell Receptor Signaling ◽

Cell Receptor Signaling

Abstract Abstract 2889 Chronic Lymphocytic Leukemia (CLL) cells depend on B cell receptor signaling as well as other microenvironmental survival signals (1). Drugs targeting the BCR signaling pathways are showing exciting results in CLL clinical trials. A peculiarity of CLL is that IgD signaling is generally preserved, whilst IgM signaling is decreased and it has been suggested that this pattern mimics anergic B-cells, and might be consistent with chronic autoantigen exposure. We examined the differing roles of IgM and IgD signaling in CLL using a theoretical framework of anergy. Peripheral blood (PB) CLL cells exhibited higher IgD expression, as compared to IgM (n=204, p<0.0001), but this did not have prognostic impact. When we examined IgM and IgD expression in LN biopsies compared to paired PB (n=10) expression, IgM expression was lower in LN (p=0.002) whilst IgD expression was unchanged. Although the number of these paired samples is small, cases with lower LN IgM levels had poorer prognosis, and we are investigating this further with a larger cohort. We hypothesize that reduced LN IgM expression reflects antigen engagement and an anergic response in the microenvironment. We sought to replicate Mockridge et al' s model of reversible anergy (2) by monitoring the dynamic changes in IgM/D expression after in vitro incubation. Most (18/20) PB CLL samples underwent calcium (Ca) flux after IgD crosslinking, whereas only 13/20 cases underwent IgM Ca flux, and the level of Ca flux was less than with IgD, a well recognized anergic pattern. Incubation for 24h in vitro led to partial restoration of IgM Ca flux and some improvement in IgD Ca flux. This was impaired by treatment with anti-IgD or IgM F(ab)2 fragments, mimicking antigen exposure, and in keeping with a model of CLL cells engaging autoantigen in vivo. Further support for the pro-survival role of the BCR in CLL was demonstrated by the finding that both IgD and IgM ligation was associated with reduced apoptosis in vitro, with a significant decrease in apoptosis with IgD ligation as compared to IgM. To examine the mechanistic differences of signaling via IgM and IgD further, we used high-throughput mass-spectrometry based phosphoproteomics. This allows analysis of multiple active signaling pathways without a priori knowledge of which pathways to investigate. 6 CLL samples were compared to 5 tonsil controls. 4,575 unique phosphopeptides were identified using MASCOT proteomics software and quantified using a label-free technique based on extracted ion currents. 174 phosphoproteins (p<0.001, fold change up to >4000-fold) were over-expressed in CLL relative to healthy B-cells. These included components of RNA processing complexes, cytoskeletal regulators and MAPK signaling pathway components. Kinase prediction based on phosphoprotein substrates confirmed activation of kinases known to be active in CLL (such as AKT1, ERK1/2, CK2), but several novel kinases (such as CaMK1, CRIK, ROCK1 and BCKDK) were also active in CLL relative to healthy controls. Evaluation of differentially expressed phosphoproteins after BCR ligation included components of the spliceosome, regulators of the cytoskeleton, as well as known BCR signaling components. BCR-induced kinase activities included mTOR, CDK family members, MAPKs, BCKDK and others. There was much overlap between kinases active after IgM and IgD ligation, but also marked differences in CLL and tonsil BCR signaling. CONCLUSIONS Anergic IgM signaling is contrasted with IgD as a dynamic and plastic process that appears different in the LN and PB compartments in CLL. Mass-spectrometry based phosphoproteomics offers a powerful tool for interrogating intracellular signaling, with networks of phosphorylation characterizing the topology of pathways. BCR signaling in healthy B-cells has not previously been studied using this approach and comparisons with CLL highlight known pathways as well as suggesting novel treatment targets. The ultimate goal is to identify kinases active in CLL that will provide rational and effective drug combinations. Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

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High TCL1 levels are a marker of B-cell receptor pathway responsiveness and adverse outcome in chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2009-03-208256 ◽

2009 ◽

Vol 114 (21) ◽

pp. 4675-4686 ◽

Cited By ~ 61

Author(s):

Marco Herling ◽

Kaushali A. Patel ◽

Nicole Weit ◽

Nils Lilienthal ◽

Michael Hallek ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Clinical Stage ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Advanced Clinical Stage ◽

Therapy Regimen ◽

Cell Counts

Abstract Although activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL), its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1), the AKT modulator and proto-oncogene, is differentially expressed in CLL and linked to its pathogenesis based on CD5+ B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes, which further included the kinases LYN, SYK, ZAP70, and PKC. High TCL1 levels were also strongly associated with aggressive disease features, such as advanced clinical stage, higher white blood cell counts, and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie, shorter progression-free survival, P < .001), regardless of therapy regimen, especially for ZAP70+ tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful.

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MEK1/2 Inhibition By MEK162 Is Effective Against Chronic Lymphocytic Leukaemia Cells Under Conditions That Mimic Stimulation of B-Cell Receptor-Mediated Signaling

Blood ◽

10.1182/blood.v124.21.3330.3330 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3330-3330 ◽

Cited By ~ 1

Author(s):

Kyle Crassini ◽

William S Stevenson ◽

Stephen P. Mulligan ◽

Oliver Giles Best

Keyword(s):

Protein Kinase ◽

B Cell ◽

Cell Survival ◽

Research Funding ◽

Cell Receptor ◽

B Cell Receptor ◽

Spontaneous Apoptosis ◽

Viable Cells ◽

Cells Cultured

Abstract BACKGROUND Chronic Lymphocytic Leukemia (CLL) is characterised by the clonal expansion of apoptosis resistant B-lymphocytes. However, in vitro and in the absence of pro-survival factors primary CLL cells undergo spontaneous apoptosis. B-cell receptor (BCR) signalling plays a major role in the survival and proliferation of CLL cells, which is highlighted by the clinical efficacy of the Btk and PI3-kinase inhibitors, ibrutinib and idelalisib. Mitogen activated protein kinase (MAPK) is an important mediator of signals downstream of both Btk and PI3-kinase but few studies have shown that inhibitors of MEK1/2, a critical component in the MAPK pathway, have any potential benefit for therapy of CLL. METHODS We sought to investigate the potential of the MEK1/2 inhibitor MEK162 against CLL cells in vitro. To mimic the tonic BCR stimulation experienced in vivo, primary CLL cells were stimulated using an immobilised antibody to IgM or were treated with PMA, a less specific B-cell activator which promotes protein kinase C-dependent MAPK-ERK1/2 signaling. Sensitivity to MEK162 and effects on MAPK-ERK1/2 pathway activity were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and western blot analyses respectively. RESULTS MEK162 treatment of CLL cells cultured in media alone resulted in a modest but significant (P < 0.01) reduction in cell viability; 20µM MEK162 reduced the proportion of viable cells remaining after 48 h to 77.40 +/- 7.81 % relative to vehicle-treated controls. In contrast, BCR stimulation through IgM ligation promoted cell survival 3.2 +/- 1.01 fold and sensitised CLL cells to MEK162; 20µM MEK162 reduced the proportion of viable cells by 56.28 +/- 2.37 % (P < 0.001 relative to control). A similar effect was observed in response to PMA stimulation; cell viability increased 1.78 +/- 0.15 fold and was reduced by 59.55 +/- 10.33 % (P < 0.001 relative to control) following treatment with 20µM MEK162 (Figure 1). At concentrations > 0.05 µM MEK162 was significantly (P < 0.05) more effective against CLL cells stimulated with either anti-IgM or PMA than against cells cultured in media alone. By western blotting we observed low levels of MAPK-ERK1/2 activity in cells cultured in media alone, which we suggest may contribute to the spontaneous apoptosis of these cells and the low degree of sensitivity to MEK162 under these conditions. We confirmed that stimulation with either IgM or PMA results in activation of MAPK-ERK1/2 and show that this response can be effectively blocked using MEK162. The effects of anti-IgM and PMA on cell survival and response to MEK162 were independent of ZAP-70 expression or ATM/TP53 functional status. CONCLUSIONS Our data illustrate the important role of MAPK-ERK1/2 activity in BCR-mediated CLL cell survival and suggest that MEK162 may have potential for CLL therapy. These data highlight the importance of employing appropriate culture conditions in order to make accurate assessments concerning the efficacy of novel agents for the treatment of CLL. Figure 1. Stimulation with IgM or PMA sensitises B-CLL cells to MEK1/2 inhibition by MEK162. Figure 1. Stimulation with IgM or PMA sensitises B-CLL cells to MEK1/2 inhibition by MEK162. Disclosures Mulligan: Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria.

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TAK-243, a Small Molecule Inhibitor of Ubiquitin-Activating Enzyme (UAE), Induces ER Stress and Apoptosis in CLL Cells

Blood ◽

10.1182/blood-2018-99-114964 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1867-1867

Author(s):

Scott R Best ◽

Taylor Rowland ◽

Cody Paiva ◽

Nur Bruss ◽

Stephen E Spurgeon ◽

...

Keyword(s):

B Cells ◽

Er Stress ◽

B Cell ◽

Cell Lines ◽

Small Molecule ◽

Research Funding ◽

Cell Receptor ◽

B Cell Receptor ◽

Ubiquitin Conjugation

Abstract Introduction: Despite the promise of B-cell receptor-associated kinase inhibitors (BCRi) in CLL, resistance to these agents is inevitable. Ubiquitin-proteasome systems are altered in cancer, leading to destabilization of tumor suppressors, overexpression of proto-oncogenes (e.g., MYC), and impaired DNA repair. Neoplastic B cells exhibit a state of heightened cellular stress and are thereby susceptible to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The UPR is activated in CLL cells upon sIgM signaling and inhibited by ibrutinib. Proteasome inhibitors demonstrate clinical activity in certain types of B-cell neoplasia but are inactive in CLL. Here, we investigated an alternative approach to harness the pro-apoptotic UPR in CLL by using TAK-243, a first-in-class small molecule UAE inhibitor. Methods: Peripheral blood cells were obtained from patients with CLL (N=20) and isolated using Ficoll-Hypaque techniques. TAK-243 was obtained from Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA). TAK-243 efficacy was assessed in CLL cells and 10 DLBCL (diffuse large B-cell lymphoma) cell lines. Results: TAK-243 induced ER stress and the UPR in CLL cells, followed by rapid apoptosis within 2 hours. Following 24-hour incubation, we established an IC50 of ~50 nM (Annexin V+ cells) in CLL cells. By contrast, primary B cells and T cells were less sensitive to TAK-243. Given the importance of tumor microenvironment in CLL cell survival, we evaluated the effect of TAK-243 in a CD40L-expressing stromal co-culture model. Whereas CD40L-stimulated CLL cells were resistant to BCRi, they were fully sensitive to UAE inhibition. TAK-243 had a similar IC50 (~50 nM) across DLBCL cell lines, independent of cell of origin. Treatment with TAK-243 rapidly disrupted ubiquitin conjugation and degradation of proteins controlled by the UPS in CLL cells and DLBCL cell lines. UPR induction occurred within 2 hours, as shown by activation of eIF2α (in both CLL and DLBCL cells) and oligomerization and autophosphorylation of PERK (in DLBCL cells). After 4 hours, neoplastic B cells exhibited late apoptotic phase of the UPR: transcriptional induction of CHOP, GADD34, and NOXA. These events were accompanied by upregulation of pro-apoptotic BH3-only proteins, stabilization of Mcl-1 and, ultimately, cleavage of caspase-3 and PARP. TAK-243 inhibited NFκB pathway, as shown by accumulation of IκBα, a negative pathway regulator. The extent of the UPR in CLL cells varies depending on the initiating signal. For example, B-cell receptor crosslinking induced expression of CHOP and GRP78 in CLL cells, but only weak activation of PERK and no IRE1-dependent processing of XBP1 (Krysov S, et al. Blood. 2014). Targeting UAE in CLL cells induced robust activation of eIF2α, upregulation of CHOP, GADD34 as well as NOXA mRNA, indicating high sensitivity to this pathway. TAK-243 induced a more rapid UPR and exhibited lower IC50 compared with the proteasome inhibitor bortezomib in CLL and DLBCL cells. While both drugs induced autophagy as shown by LC3 processing, only bortezomib treatment led to p62 degradation, suggesting that autophagy was inefficient in response to TAK-243 due to lack of ubiquitin conjugation. Our findings were confirmed in a mouse lymphoma xenograft model. OCI-LY3 cells were inoculated subcutaneously in the right flank of NSG mice and treatment with TAK-243 (10 or 20 mg/kg IV twice weekly) or vehicle control began when tumors reached 10 mm in size. Treatment led to reduced tumor progression, induction of ER stress, and decreased cell proliferation and survival. Conclusions: The UAE inhibitor TAK-243 induces ER stress and promotes apoptosis in CLL cells in vitro and restricts lymphoma growth in vivo. TAK-243 exhibited greater in-vitro cytotoxicity in lymphoma cells compared to bortezomib. Targeting UAE is a novel approach to disrupt the UPS which may hold promise in therapy of CLL and other B-cell malignancies. Disclosures Spurgeon: Bristol Myers Squibb: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Oncternal: Research Funding; Acerta: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding; MEI Pharma: Consultancy. Berger:Takeda Pharmaceuticals International Co.: Employment. Danilov:TG Therapeutics: Consultancy; Aptose Biosciences: Research Funding; Astra Zeneca: Consultancy; Genentech: Consultancy, Research Funding; Bayer Oncology: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Takeda Oncology: Research Funding.

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B Cell Receptor-ERK1/2 Signal Cancels PAX5-Dependent Repression of BLIMP1 Probably Through PAX5 Phosphorylation: A Mechanism of Antigen-Triggering Plasma Cell Differentiation

Blood ◽

10.1182/blood.v118.21.1109.1109 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1109-1109

Author(s):

Takahiko Yasuda ◽

Fumihiko Hayakawa ◽

Shingo Kurahashi ◽

Tomoki Naoe

Keyword(s):

Cell Differentiation ◽

B Cell ◽

Plasma Cell ◽

Transcriptional Repression ◽

Research Funding ◽

Cell Receptor ◽

B Cell Receptor ◽

Luciferase Reporter ◽

Plasma Cell Differentiation

Abstract Abstract 1109 Plasma cell differentiation is initiated by antigen stimulation of B cell receptor (BCR). Until BCR stimulation, BLIMP1, a master regulator of plasma cell differentiation, is suppressed by PAX5, a key transcriptional repressor to maintain B cell identity. After BCR stimulation, upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed and thought to be the trigger of plasma cell differentiation; however, the trigger that derepresses BLIMP1 expression is yet to be revealed. Here, we demonstrated PAX5 phosphorylation by ERK1/2, the main component of BCR signal, in vitro and in vivo. The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In luciferase reporter assays, transcriptional repression on BLIMP1 promoter by PAX5 was canceled by PAX5 phosphorylation. Furthermore, transcriptional repression by phosphorylation-defective mutant of PAX5 was attenuated by CA-MEK1 co-expression to a significantly lesser extent than that by wild-type PAX5, indicating its resistance to ERK1/2 signal-dependent cancelation of the transcriptional repression (Figure A). Finally, BCR stimulation induced strong ERK1/2 activation, phosphorylation of endogenous PAX5 (Figure B), and upregulation of BLIMP1 mRNA expression in B cells. These phenomena were inhibited by U0126, MEK1 inhibitor. These data imply that PAX5 phosphorylation by BCR signal is the initial event in plasma cell differentiation (Figure C). Disclosures: Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

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Allosteric Akt Inhibitor MK2206 Synergizes with Bendamustine in Promoting the Apoptosis of Chronic Lymphocytic Leukemia Cells and Selectively Targets B-Cell Receptor Mediated Cytokine Production

Blood ◽

10.1182/blood.v120.21.3928.3928 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3928-3928

Author(s):

Wei Ding ◽

Tait D. Shanafelt ◽

Connie Lesnick ◽

Traci Sassoon ◽

Charla Secreto ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Research Funding ◽

Cytokine Production ◽

Cell Receptor ◽

B Cell Receptor ◽

Leukemic Cells ◽

Akt Inhibitor ◽

Akt Activation

Abstract Abstract 3928 Background: Accumulating data support the critical role of PI3K/Akt in CLL B cell receptor (BCR) mediated signal transduction, cell proliferation and survival. In addition recent preclinical and clinical studies indicate that specific PI3K blockade results in robust preclinical and clinical efficacy in CLL. In our model system of CLL B cell-stromal cultures which feature their interaction, platelet derived growth factor (PDGF) present in CLL culture medium drives VEGF production through PI3K/Akt activation in stromal cells (Blood. 2010. 116:2984). Indeed Akt was found to be activated in leukemic cells during the CLL-stroma interaction (Leuk Res. 2008. 32:1565). Therefore, we hypothesized that Akt inhibition should promote CLL B cell apoptosis and abrogate BCR mediated cytokine production. MK2206 is an orally bioavailable highly specific allosteric Akt inhibitor. It has been tested in patients with refractory solid tumors and was demonstrated to be safely administered in a phase I trial. Therefore the goal of this study was to test the preclinical efficacy of MK2206 on both the survival and the BCR mediated cytokine production of CLL leukemic B cells. Methods: Peripheral blood mononuclear cells isolated from CLL patients (n=37) were treated with escalating concentrations of MK2206 (1–16 μM) for 24 hours, 48 hours or 72 hours. The levels of leukemic B cell viability were tested using an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The potential impact (antagonistic/additive/synergistic) of Bendamustine in combination with MK2206 was also tested by using the MTT assay. We used the Calcusyn system to calculate the effect of drug interactions. The combination index (CI) as calculated by the program usually indicates synergy when ≤ 0.8 and indicates additive outcomes when between 0.8–1.2. A CI >1.2 indicates antagonism. Downstream signals of Akt activation in CLL B cells were evaluated by testing their expression of Mcl-1, 4EBP1 and p70S6K using immunoblot. The impact of Akt inhibition by MK2206 on cytokine production in response to B-cell receptor ligation with anti-IgM was also tested using a multiplex cytokine analysis (Invitrogen) in a time-course experiment. Results: MK2206 treatment induced concentration- and time-dependent apoptosis in CLL leukemic cells. At 72 hours, the IC50 of MK2206 in the experiments using CLL leukemic cells in vitro is ∼8 mM. MK2206 incubation at 1 or 5 mM cultured with CLL B cells over a 48-hour period abolished of Akt and p70S6K phosphorylation while native PARP was cleaved into the 85 kD polypeptide fragment. However, the expression level of the upstream signal molecule, PI3K, was not changed. Among the CLL patients tested (n = 37), we did not find any difference in sensitivity to MK2206 induced apoptosis based on critical prognostic factors of CD38, ZAP-70, IGHV and del(17p) status. Importantly, we detected synergistic or additive activity between MK2206 and Bendamustine in 11 tested CLL samples when these combinations were used to treat CLL cells in vitro for 72hrs. Thus the median CI value for this group of patients was 0.8 (0.1 – 1.1). Six were found to have CI ≤ 0.8 and five fell within the additive CI values (0.8 – 1.2). Production of immune or chemotactic cytokines (e.g. CCL3, CCL4, MCP-1, IL-1Ra, IL-8 and IL-2R) at 24 hour incubation increased significantly above baseline when CLL cells were stimulated anti-IgM. Akt inhibition with MK2206 selectively abrogated upregulation of CCL3, CCL4, MCP-1 and IL-2R production, but not for IL-8 or IL-1Ra secretion. MK2206 also abolished BCR mediated Akt activation and decreased Erk activation. Conclusion: MK2206, a robust and selective Akt inhibitor, induced significant in vitro apoptosis of CLL B-cells in vitro. Preclinical evidence of a synergistic effect between MK2206 and Bendamustine was also observed independent of prognostic risk. MK2206 abolished BCR mediated Akt activation and selectively abrogates BCR mediated production of cytokines that may promote apoptotic resistance. These findings support the use of MK2206 in treating CLL and indeed we have initiated a phase I/II trial of MK2206 in combination with Bendamustine and Rituximab for relapsed CLL patients(N1087, October 2011). Acknowledgments: This study was funded by the NCI-K23, NCCTG and CLL Global Foundation. Disclosures: Shanafelt: Cephalon: Research Funding; Genentech: Research Funding. Kumar:Genzyme: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Honoraria. Kay:Celgene: Research Funding.

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

Blood ◽

10.1182/blood.v126.23.2922.2922 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2922-2922

Author(s):

Elisa ten Hacken ◽

Thomas Oellerich ◽

Maria Gounari ◽

Julia Hoellenriegel ◽

Kuan-Ting Pan ◽

...

Keyword(s):

Mass Spectrometry ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Research Funding ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Label Free ◽

Bcr Signaling ◽

Associated Proteins

Abstract 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. Disclosures Wierda: Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Estrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.

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