Overactive STAT3 drives accumulation of disease-associated CD21low B cells

Dysregulated STAT3 signalling is correlated with antibody-mediated autoimmunity and B- cell neoplasia, but its effect on B cells is underexplored. Here we address this in children with STAT3 gain-of-function (GOF) syndrome and in mice with STAT3T716M, the most common STAT3 GOF syndrome human mutation, or STAT3K658N, a dimerization interface mutation responsible for STAT3 GOF syndrome in two children. The main B cell consequence of overactive STAT3 was accumulation of CD19high CD21low atypical memory B cells in humans and of CD21low CD23low B cells in mice resembling age-associated B cells expressing T-bet, CD11c and plasma cell differentiation genes. Overactive STAT3 within B cells increased expression of many genes in the B cell receptor and T cell help pathways, increased the tolerogenic receptor CD22, but opposed B cell tolerance checkpoints and increased formation of T-bet+ B cells upon BCR and CD40 stimulation. These results reveal overactive STAT3 as a central driver of a key class of disease-associated B-lymphocytes in humans and mice.

Synergistic immunotherapy of glioblastoma by dual targeting of IL-6 and CD40

Immunologically-cold tumors including glioblastoma (GBM) are refractory to checkpoint blockade therapy, largely due to extensive infiltration of immunosuppressive macrophages (Mϕs). Consistent with a pro-tumor role of IL-6 in alternative Mϕs polarization, we here show that targeting IL-6 by genetic ablation or pharmacological inhibition moderately improves T-cell infiltration into GBM and enhances mouse survival; however, IL-6 inhibition does not synergize PD-1 and CTLA-4 checkpoint blockade. Interestingly, anti-IL-6 therapy reduces CD40 expression in GBM-associated Mϕs. We identify a Stat3/HIF-1α-mediated axis, through which IL-6 executes an anti-tumor role to induce CD40 expression in Mϕs. Combination of IL-6 inhibition with CD40 stimulation reverses Mϕ-mediated tumor immunosuppression, sensitizes tumors to checkpoint blockade, and extends animal survival in two syngeneic GBM models, particularly inducing complete regression of GL261 tumors after checkpoint blockade. Thus, antibody cocktail-based immunotherapy that combines checkpoint blockade with dual-targeting of IL-6 and CD40 may offer exciting opportunities for GBM and other solid tumors.

Regulation of Bcl-XL by non-canonical NF-κB in the context of CD40-induced drug resistance in CLL

In chronic lymphocytic leukemia (CLL), the lymph node (LN) microenvironment delivers critical survival signals by inducing the expression of anti-apoptotic Bcl-2 members Bcl-XL, Bfl-1, and Mcl-1, resulting in apoptosis blockade. We determined previously that resistance against various drugs, among which is the clinically applied BH3 mimetic venetoclax, is dominated by upregulation of the anti-apoptotic regulator Bcl-XL. Direct clinical targeting of Bcl-XL by, e.g., Navitoclax is however not desirable due to induction of thrombocytopenia. Since the actual regulation of Bcl-XL in CLL in the context of the LN microenvironment is not well elucidated, we investigated various candidate LN signals to drive Bcl-XL expression. We found a dominance for NF-κB signaling upon CD40 stimulation, which results in activation of both the canonical and non-canonical NF-κB signaling pathways. We demonstrate that expression of Bcl-XL is first induced by the canonical NF-κB pathway, and subsequently boosted and continued via non-canonical NF-κB signaling through stabilization of NIK. NF-κB subunits p65 and p52 can both bind to the Bcl-XL promoter and activate transcription upon CD40 stimulation. Moreover, canonical NF-κB signaling was correlated with Bfl-1 expression, whereas Mcl-1 in contrast, was not transcriptionally regulated by NF-κB. Finally, we applied a novel compound targeting NIK to selectively inhibit the non-canonical NF-κB pathway and showed that venetoclax-resistant CLL cells were sensitized to venetoclax. In conclusion, protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with non-canonical NF-κB signaling.

Electron Transport Chain Inhibition Suppresses CD40 Expression and Sensitizes Chronic Lymphocytic Leukaemia Cells to Venetoclax

Alterations in expression of specifically BCL-XL and MCL-1 dictate sensitivity of CLL cells to the Bcl-2 specific inhibitor venetoclax (VEN). We and others have shown upregulation of these anti-apoptotic proteins by interaction of CLL cells with CD4+ T helper cells within their lymph node microenvironment (LN-ME) mediated by CD40 signalling. We also reported significant metabolic changes of LN-ME activated CLL cells but whether metabolic alterations can be linked to VEN resistance remains unclear. As VEN is increasingly used in early stages of CLL, better understanding and tools to circumvent VEN resistance are highly needed. We aim to reveal the metabolic adaption of CLL to CD40 signalling in connection with VEN resistance. After in vitro CD40 signalling stimulation of peripheral blood (PB) CLL cells, mitochondrial mass and glucose uptake were measured by flow cytometry, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured on Seahorse XF Analyser. The result demonstrated that CD40 stimulation enhances both oxidative phosphorylation (OXPHOS) and glycolysis. This was also confirmed by microarray and metabolomics analyses, as genes and metabolites involved in these two metabolic pathways are significantly upregulated by CD40 stimulation. To find out whether these pathways are linked to VEN resistance, PB CLL cells were treated with OXPHOS or glycolysis inhibitors during CD40 stimulation. Remarkably, OXPHOS inhibition by electron transport chain (ETC) inhibitors (rotenone, antimycin A and oligomycin) counteracted strongly for VEN resistance, while glycolysis inhibition by 2-Deoxy-D-glucose (2DG) did not. The three ETC inhibitors also attenuated CLL activation, ATP production and NAD levels. Interestingly, complex II inhibition of the ETC (TTFA and DMM) did not affect VEN resistance. Regarding BCL-2 family members induced by CD40 ligation, both MCL-1 and BCL-XL were downregulated by these ETC inhibitors. In addition, OXPHOS inhibition strongly elevates glycolysis, and vice versa, which illustrates a strong metabolic plasticity of CLL cells. To further investigate the cross-talk between CD40 signalling, VEN resistance and mitochondrial metabolism, the three main fuels of the TCA cycle were inhibited: pyruvate (by UK5099), glutamine (by DON) and fatty acids (by etomoxir). Even though the OCR and ECAR were slightly decreased by (combinations of) these fuel inhibitors, neither CD40 signalling nor VEN sensitivity was affected. Next, we inhibited PI3K by idelalisib, BTK by ibrutinib and mTOR by rapamycin, which are three downstream targets of CD40 signalling. The results showed that only rapamycin inhibited CD40 activation and metabolic activities, and none of the three inhibitors counteracts VEN resistance. Lastly, we investigated CD40 splicing and overall expression. Interestingly, CD40 stimulation has a huge impact on CD40 expression itself, and these changes were blocked by ETC inhibition. These data indicate that ETC inhibition affects CD40 signals to counteract VEN resistance, by directly affecting the expression of CD40 protein on the cell membrane. In conclusion, after CD40 stimulation, CLL cells become metabolically activated and highly flexible in the use of mitochondrial fuels. The enhanced OXPHOS but not glycolysis contributes to VEN resistance, while ETC inhibition reverses CLL VEN resistance by directly suppressing CD40 expression on CLL. These findings link CLL metabolism directly to CD40 transcription and signalling, which may contribute to clinical VEN resistance. Disclosures van der Windt: genmab: Current Employment. Kater:Abbvie: Research Funding; Roche: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Genentech: Research Funding. Eldering:Genentech: Research Funding; Celgene: Research Funding; Janssen: Research Funding.

Effects of Ibrutinib on Metabolic Alterations and Micro-Environmental Signalling in Chronic Lymphocytic Leukaemia

Introduction. Altered metabolism is one of the hallmarks of cancer. CLL cells circulate between peripheral blood (PB) and lymph nodes (LN) which necessitates high metabolic plasticity. In LN, CLL cells receive proliferative and pro-survival signals from surrounding cells, and become metabolically activated. However, detailed insight into the altered metabolism of LN CLL and how this may be related to therapeutic responses is lacking. As it is technically difficult to obtain direct insight into CLL LN metabolism, we have applied a two-tiered strategy. By using PB samples taken from patients before/after treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR), which drives CLL cells out of the LN, combined with in vitro re-stimulation of TME signals, we indirectly mapped the metabolism of CLL in their TME, as well as the effects of IBR treatment. We hypothesized that the overlapping/distinct metabolites affected by IBR and in vitro stimulations would reflect the actual CLL metabolism in LN. Methods. PB samples were obtained from 7 CLL patients before or after 3 months of ibrutinib treatment. These paired samples were in vitro stimulated via CD40 and B cell receptor (BCR), which are potential key signals within the tumour microenvironment (TME). Seahorse extracellular flux (ECF) analyses, expression of activation markers (CD95, pS6 by FACS), RNA was isolated for expression of Myc (major driver of metabolic reprogramming) and its target genes, and metabolomics by mass-spec was performed. Results. ECF analyses showed that in comparison to BCR stimulated PB CLL cells, stimulation by CD40 resulted in a high increase of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). A prominent effect on OXPHOS and glycolytic activity was confirmed in direct LN samples, and indirectly by marker analyses in LN emigrants using CXCR4/CD5 staining [1]. Subsequent metabolomics analyses showed that metabolic reprogramming following CD40 or BCR stimulation revealed both shared and distinct responses. The affected metabolic pathways, predicted by significantly changed metabolites, were compared in a pairwise fashion; upregulated by CD40 and BCR but downregulated by IBR, respectively. The results demonstrated 5 upregulated pre-defined pathways (KEGG) by both CD40 and BCR triggering: purine metabolism, Warburg effect, lysine degradation, glucose-alanine cycle and glutamate metabolism. In contrast, the following pathways indicated the two signals had distinct functions on regulating metabolism: CD40 signalling mostly regulates amino acid metabolism, tricarboxylic acid cycle (TCA) and mitochondrial metabolism related to oxidative phosphorylation (OXPHOS) and energy production. BCR signalling mainly involves glucose and glycerol metabolism, which are usually related to biosynthesis. CLL cells from IBR-treated patients showed enhanced BCR responsiveness, in line with the increased in surface IgM expression upon IBR [2]. In contrast, IBR treatment suppressed in vitro CD40 activation, which was accompanied by a lower CD40 expression. Metabolomics analyses also demonstrated that CD40 responses decreased but BCR response increased after IBR. Additionally, analyses of Myc and its target genes showed that they are induced after BCR as well as CD40 stimulation. Effects of IBR on Myc (target) expression were variable for BCR and reduced for CD40 stimulation. Conclusions. In vivo IBR treatment suppresses CD40 expression and activation and enhances BCR responsiveness. Metabolic changes of CLL in LN are recapitulated by these two signals, while IBR treatment shows opposite effects, together providing indirect insight into the LN metabolism. In LN, CD40 may play a prominent role to enhance most of the key metabolic pathways, particularly OXPHOS. This is the first study to describe the metabolic network of CLL cells in LN, and the long-term effects of IBR may yield new clues to therapy response and resistance. References 1. Calissano, Carlo, et al. "Intraclonal complexity in chronic lymphocytic leukemia: fractions enriched in recently born/divided and older/quiescent cells." Molecular Medicine 17.11 (2011): 1374-1382. 2. Drennan, Samantha, et al. "Ibrutinib therapy releases leukemic surface IgM from antigen drive in chronic lymphocytic leukemia patients." Clinical Cancer Research 25.8 (2019): 2503-2512. Disclosures Forconi: AbbVie: Honoraria, Other: Fees for cosulting or advisory role, received travel and expenses, Speakers Bureau; Janssen: Honoraria, Other: Fees for cosulting or advisory role, received travel and expenses, Speakers Bureau; Roche: Honoraria; Novartis: Honoraria; Menarini: Other: Fees for cosulting or advisory role; Astra Zeneca: Other: Fees for cosulting or advisory role; Gilead: Research Funding. Kater:Roche: Research Funding; Abbvie: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Janssen: Research Funding. Eldering:Janssen: Research Funding; Celgene: Research Funding; Genentech: Research Funding.

Overcoming primary and acquired resistance to anti-PD-L1 therapy by induction and activation of tumor-residing cDC1s

The ability of cancer cells to ensure T-cell exclusion from the tumor microenvironment is a significant mechanism of resistance to anti-PD-1/PD-L1 therapy. Evidence indicates crucial roles of Batf3-dependent conventional type-1 dendritic cells (cDC1s) for inducing antitumor T-cell immunity; however, strategies to maximize cDC1 engagement remain elusive. Here, using multiple orthotopic tumor mouse models resistant to anti-PD-L1-therapy, we are testing the hypothesis that in situ induction and activation of tumor-residing cDC1s overcomes poor T-cell infiltration. In situ immunomodulation with Flt3L, radiotherapy, and TLR3/CD40 stimulation induces an influx of stem-like Tcf1+ Slamf6+ CD8+ T cells, triggers regression not only of primary, but also untreated distant tumors, and renders tumors responsive to anti-PD-L1 therapy. Furthermore, serial in situ immunomodulation (ISIM) reshapes repertoires of intratumoral T cells, overcomes acquired resistance to anti-PD-L1 therapy, and establishes tumor-specific immunological memory. These findings provide new insights into cDC1 biology as a critical determinant to overcome mechanisms of intratumoral T-cell exclusion.

Ras isoforms: signaling specificities in CD40 pathway

Background Ras are small cellular GTPases which regulate diverse cellular processes. It has three isoforms: H-Ras, K-Ras, and N-Ras. Owing to the N-terminus (1–165 residues) sequence homology these isoforms were thought to be functionally redundant. However, only K-Ras-deficient mice but not H-Ras- and N-Ras-deficient mice show embryonic lethality. Similarly, mutations in a given Ras isoform are associated with a particular type of cancer. Moreover, we have previously reported that Ras isoforms perform unique functions in Leishmania major infection. Thus, Ras isoforms are implicated to have signaling and functional specificity but the mechanism remains to be elucidated. Result Using CD40 as a model receptor, we showed that depending on the strength of signaling, specific Ras isoforms are activated. Weak CD40 signal activates N-Ras, whereas strong signal activates H-Ras and K-Ras. Additionally, we showed that suppression of N-Ras expression reduced CD40-induced extracellular signal–regulated kinase-1/2 (ERK-1/2) activation and Interleukin (IL)-10 production; whereas suppression of H-Ras or K-Ras reduced CD40-induced p38 mitogen-activated protein kinase (p38MAPK) activation and IL-12 production. Furthermore, we showed that Ras isoforms have activator (GEF) specificity as weak CD40 signal-activated N-Ras requires Sos-1/2 whereas strong CD40 signal-activated H-Ras/K-Ras requires Ras-GRP as the guanine-nucleotide exchange factor (GEF) inducing ERK-1/2- or p38MAPK-mediated IL-10 or IL-12 productions, respectively, in macrophages. Silencing of syk reduced CD40-induced N-Ras activation but silencing of lyn inhibited H-Ras and K-Ras activation. In CD40 signaling, Ras isoforms also showed effector specificity; while H-Ras and K-Ras showed specificity for phosphatidyl inositol-3 kinase activation at high dose of CD40 stimulation, N-Ras primarily associated with Raf-1 at low dose of CD40 stimulation. Moreover, fractal analysis showed that functional site surface roughness for H-Ras (SurfaceFD = 2.39) and K-Ras (SurfaceFD = 2.39) are similar but significantly different from N-Ras (SurfaceFD = 2.25). Conclusion The activator and effector specificities of Ras isoforms in CD40 signaling indicated their differential involvement in CD40 pathway and in maintaining the reciprocity. Our observations reveal Ras-regulated signaling outcome and its potential for developing Ras isoform-targeted immunotherapy and prophylaxis. Graphical abstract

Cross-Talk between Cytokine and NF-ĸb Signaling in the CLL Microenvironment Can Affect Sensitivity for Venetoclax

INTRODUCTION. The Bcl-2 inhibitor Venetoclax provides profound reductions in circulating chronic lymphocytic leukemia (CLL) cells in the majority of patients. However, lymph node (LN) responses are less robust, which may be linked to an acquired resistance imposed by pro-survival signals. Prime among these is CD40 stimulation leading to activation of NF-kB, and induction of Bcl-XL expression1. Bcl-XL is a prime determinant of resistance to Venetoclax2 and regulatory mechanisms of its expression are of clinical significance. Cytokines IL-21 and IL-4 are secreted by T helper cells and abundant in the CLL lymph node microenvironment. Importantly, IL-21 and IL-4 play an important role in CLL survival and proliferation3. In the present study, we investigated how signals from T helper cytokines IL-21 or IL-4 affect Bcl-XL expression as a model for the CLL LN microenvironment, specifically in relation to Venetoclax resistance. RESULTS. Following CD40 stimulation, IL-21 and IL-4 show opposing effects on Bcl-XL expression. Correspondingly, this was associated with CD40-induced resistance to Venetoclax which was augmented by IL-4 and reversed by IL-21. We subsequently investigated the rewiring between CD40 activation, differential cytokine signaling and Bcl-XL expression. IL-21 or IL-4 stimulation correspond with differential STAT3 or -6 phosphorylation and STAT3 and -6 have predicted binding sites near the known p65 and p52 binding sites in the Bcl-XL promoter region. Using reporter assays with Bcl-XL promotor constructs we demonstrate competition (through IL-21-induced STAT3) or synergy (through IL-4 induced-STAT6) with CD40-mediated activation of the NF-kB pathway. By applying in situ proximity ligation (isPLA) in primary CLL cells, we showed direct interaction of both (non-)canonical p65 and p52 with STAT3 and STAT6. Moreover, time-course analyses indicated that STAT3 drives NF-kB out of the nucleus, whereas STAT6 keeps NF-kB inside the nucleus, and this distinction controls Bcl-XL expression. These observations suggest that cross-talk between JAK/STAT signaling and NF-kB signaling happens by direct binding to the Bcl-XL promoter and by limiting NF-kB availability at the Bcl-XL promoter. CONCLUSIONS. These data show that protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with JAK/STAT and NF-kB signaling, providing novel therapeutic clues in case of emerging resistance to targeted drugs such as Venetoclax. 1 J. Tromp, S. Tonino, J. Elias, A. Jaspers, D. Luijks, A. Kater, R. van Lier, M. van Oers, E. Eldering. Dichotomy in NF-kB signaling and chemoresistance in IGHV mutated versus unmutated CLL cells upon CD40/TLR9 triggering. Oncogene 2010. 2 R. Thijssen, E. Slinger, K. Weller, C. Geest, T. Beaumont, M. van Oers, A. Kater, E. Eldering. Resistance to ABT-199 induced by microenvironmental signals in chronic lymphocytic leukemia can be counteracted by CD20 antibodies or kinase inhibitors. Haematologica 2015. 3 C. Schleiss, W. Ilias, O. Tahar, Y. Güler, L. Miguet, C. Mayeur-Rousse, L. Mauvieux, L. Fornecker, E. Toussaint, R. Herbrecht, F. Bertrand. M. Maumy-Bertrand, T. Martin, S. Fournel, P. Georgel, S. Bahram, L. Vallat. BCR-associated factors driving chronic lymphocytic leukemia cells in proliferation ex vivo. Scientific Reports 2019. Disclosures Eldering: Celgene: Research Funding; Roche: Research Funding; Janssen Pharmaceutical Companies: Research Funding.