Aberrant activation of NF-κB transcription factors is a hallmark of human lymphomas. Most lymphoma-intrinsic as well as microenvironment-induced NF-κB activation occurs upstream of the key kinase IKK2, therefore affecting additional pathways. Here, we show that canonical NF-κB signaling in mouse B cells, induced through the expression of one or two copies of a constitutively active IKK2 variant, dose-dependently drives lymphomagenesis. The observed phenotype and stereotypic B cell receptor clonality resemble human small lymphocytic lymphoma (SLL) and chronic lymphocytic leukemia (CLL). Stronger IKK2 signaling drives early B1a cell expansion and uniform SLL/CLL-like lymphomagenesis, while intermediate signals cause more heterogeneous malignancies. Mechanistically, constitutive IKK2 signals provide a profound cell-intrinsic competitive advantage to B1a cells and dose-dependently synergize with TCL1 overexpression in driving aggressive CLL. Further, strong constitutive NF-κB activation overcomes critical microenvironmental dependencies of TCL1-driven lymphomas. Our findings establish canonical NF-κB as an oncogenic driver in lymphoma and reveal reduced microenvironment dependency as a key NF-κB-mediated mechanism, thus highlighting its therapeutic relevance.

The importance of B cell receptor (BCR) signaling for the progression of Chronic Lymphocytic Leukemia (CLL) has impressively been demonstrated through the clinical success of small molecule inhibitors interfering with this pathway, providing effective treatment options for a vast majority of patients. The scientific evidence which supported the development of drugs like BTK and PI3K inhibitors largely originate from studies of antigen-induced BCR-signaling. Notably, the presence of a constitutive, so-called tonic BCR-signal was long postulated to co-exist besides antigen-driven activation and is based on the identification of constitutively activated signaling molecules down-stream of the BCR. We here provide direct evidence for the presence of a tonic BCR-signal, which critically relies on the expression of the tyrosine kinase ZAP-70. We have previously established a method to inhibit ZAP-70 expression in primary CLL cells usingRNA-interference in combination with an extended co-culture system. Our previous work demonstrated that ZAP-70 is required for the constitutive gene expression of T cell chemokines CCL3 and CCL4 and contributes to the dysregulation of global protein synthesis and MYC expression in CLL ( Blood, 2021). Applying this method, we here provide evidence for the presence of a constitutive tonic BCR signal, which is dependent on ZAP-70 expression and exclusively found in IGHV unmutated CLL, but not in IGHV mutated cases discordantly expressing ZAP-70. We demonstrate that ZAP-70 is directly involved in the constitutive activation of AKT and GSK-3beta. Furthermore, ZAP-70 expression increases steady state activation of CD19 and the downstream kinase SRC, providing direct evidence for the existence of a ZAP-70 dependent tonic BCR signal in I GHV unmutated CLL only. To identify the underlying signaling events driving this signal, we performed mass spectrometry in combination with proximity ligation assays on CLL cells in the absence of an induced BCR-signal. Unexpectedly, we identified that the majority of ZAP-70 binding proteins do not related to BCR-signaling components, but to cytoskeleton organizing proteins involved in cell migration. Indeed, migration assays demonstrated that the loss of ZAP-70 directly and significantly impaired cell migration induced by CCL19 and CCL21, in keeping with previous data indication an association between ZAP-70 and cell migration. These results were confirmed using a switchable model of ZAP-70, which allowed us to toggle ZAP-70 expression on and off at any given time. To further define the mechanisms of ZAP-70 mediated cell migration, we studied CCL19 and CCL21 signaling in ZAP-70 proficient and deficient cells. Unexpectedly, we identified that ZAP-70 is required for chemokine-mediated activation of AKT. In summary, we here demonstrate that a tonic BCR-signal is present only in IGHV-unmutated CLL and dependent on the expression of ZAP-70. Furthermore, ZAP-70 converges two seemingly independent signaling events, namely chemokine- and tonic BCR signaling. Our data provide further explanations for the more aggressive clinical course of ZAP-70 positive CLL.

Background: Colorectal cancer (CRC) is one of the major causes of cancer-related death worldwide. Aerobic glycolysis precedes obtainment of oncogenic mutations and loss of tumor suppressors, promoting the progress of CRC. Although numerous biomarkers have been identified to be associated with prognosis and survival, a glycolysis-related gene signature in CRC has not been explored. Methods: mRNA expression profiling data in a group of CRC patients (n = 540) was extracted from the Cancer Genome Atlas (TCGA). Gene set enrichment analysis (GESA) was performed to identify gene sets that were significantly different between CRC tissues and normal tissues. Cox proportional hazards regression models were used to identify genes significantly associated with overall survival. Multivariate Cox regression analysis was used to establish a prognostic risk parameter formula. Kaplan–Meier survival estimates and the log-rank test were used to validate the significance of risk parameters for prognosis prediction. Results: Five glycolysis-related genes (ENO3, GPC1, P4HA1, IDUA, ANKZF1) were identified to be significantly associated with overall survival (AUC=0.754). Based on the five‑gene signature, patients with CRC were divided into high and low‑risk subgroups. CRC patients with a low-risk score had better survival benefits than those with a high one (P < 0.001). Conclusions: A five-gene signature associated with glycolysis for predicting the outcome of CRC patients was generated, serving as a valuable prognosis model with high efficiency and potential targeted therapy of CRC patients.

Expression of ZAP-70 in a subset of patients with chronic lymphocytic leukemia (CLL) positively correlates with the absence of immunoglobulin heavy-chain gene ( IGHV ) mutations and is indicative of a more active disease and shorter treatment-free survival. We recently demonstrated that ZAP-70 regulates the constitutive expression of CCL3 and CCL4, activation of AKT, and expression of MYC in the absence of an overt B-cell receptor (BCR) signal, bona fide functions of BCR activation. We, here, provide evidence that these features relate to the presence of a constitutive tonic BCR signal, exclusively found in IGHV - unmutated CLL and dependent on the ZAP-70 -mediated activation of AKT and its downstream target GSK-3 beta. These findings are associated with increased steady-state activation of CD19 and SRC. Notably this tonic BCR signal is not present in IGHV -mutated CLL cells, discordantly expressing ZAP-70. Results of quantitative mass spectrometry and phosphoprotein analyses indicate that this ZAP-70 -dependent, tonic BCR signal regulates CLL cell migration through phosphorylation of LCP1 on serine-5. Indeed, we show that CCL19- and CCL21-induced chemotaxis is regulated by and dependent on the expression of ZAP-70 through its function to enhance CCR7 signaling to LCP1. Thus, our data demonstrate that ZAP-70 converges a tonic BCR signal, exclusively present in IGHV -unmutated CLL and CCR7-mediated chemotaxis.

The MYC oncogene is a potent driver of growth and proliferation but also sensitises cells to apoptosis, which limits its oncogenic potential. MYC induces several biosynthetic programmes and primary cells overexpressing MYC are highly sensitive to glutamine withdrawal suggesting that MYC-induced sensitisation to apoptosis may be due to imbalance of metabolic/energetic supply and demand. Here we show that MYC elevates global transcription and translation, even in the absence of glutamine, revealing metabolic demand without corresponding supply. Glutamine withdrawal from MRC-5 fibroblasts depletes key tricarboxylic acid (TCA) cycle metabolites and, in combination with MYC activation, leads to AMP accumulation and nucleotide catabolism indicative of energetic stress. Further analyses reveal that glutamine supports viability through TCA cycle energetics rather than asparagine biosynthesis and that TCA cycle inhibition confers tumour suppression on MYC-driven lymphoma in vivo. In summary, glutamine supports the viability of MYC-overexpressing cells through an energetic rather than a biosynthetic mechanism.

The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double-cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.

Zeta-chain-associated protein kinase-70 (ZAP-70) is a tyrosine kinase mainly expressed in T cells, NK cells and a subset of B cells. Primarily it functions in T cell receptor (TCR) activation through its tyrosine kinase activity. Aberrant expression of ZAP-70 has been evidenced in different B cell malignancies, with high expression of ZAP-70 in a subset of patients with Chronic Lymphocytic Leukemia (CLL), associating with unfavorable disease outcomes. Previous studies to understand the mechanisms underlying this correlation have been focused on tumor intrinsic mechanisms, including the activation of B cell receptor (BCR) signaling. Recent evidence also suggests that ZAP-70, intrinsically expressed in tumor cells, can modulate the cross-talk between malignant B cells and the immune environment, implying a more complex role of ZAP-70 in the pathogenesis of B cell malignancies. Meanwhile, the indispensible roles of ZAP-70 in T cell and NK cell activation also demonstrate that the autologous expression of ZAP-70 in the immune environment can be a central target in modulation of tumor immunity. Here we review the evidences of the link between ZAP-70 and tumor immunology in the microenvironment in B cell malignancies. Considering an emerging role of immunotherapies in treating these conditions, understanding the distinct molecular functions of ZAP-70 in a broader cellular context could ultimately benefit patient care.

Novel targeted therapies have substantially improved the prognosis of patients with B-cell malignancies. However, a substantial fraction of patients relapse, even after initially achieving deep remissions. Many studies have characterized the interactions between tumor cells and their microenvironment as integral to leukemia/lymphoma homeostasis and for the provision of survival signals, also contributing to drug resistance (referred to as environment-mediated drug resistance [EMDR]). Therapeutic efforts to antagonize microenvironment-emanating survival cues have predominantly focused on perturbation of tumor cell adhesion enabling the physical displacement from protective niches. In an effort to address whether direct stromal targeting could more precisely mitigate EMDR, we antagonized stromal expressed PKC-beta, which we have previously shown to be a stroma-autonomous signaling pathway critical for the survival of malignant B cells (Lutzny et al., Cancer Cell 2013). The dependency on stroma PKC-b was uniformly found for acute (ALL) and chronic (CLL, MCL) B-cell malignancies. In particular, our data demonstrate that stroma PKC-b is of key importance for multidrug resistance of malignant B cells (Park et al., Science Trans Med 2020). Here we demonstrate novel mechanistic insights into stroma-mediated drug resistance in B-cell malignancies. We identified that stroma PKC-b drives a transcriptional program, activating TGF-b and BMP-signaling in tumor cells. Our data show that antagonizing stroma signals with TGF-b inhibitors abrogated upregulation of BCL-XL and overcomes stroma-dependent resistance to venetoclax. This activation operates in parallel to the activation of the transcription factor EB (TFEB) as a downstream target of PKC-b. Interference with these signaling pathways impairs plasma membrane integrity of MSCs by downregulation of numerous adhesion and signaling molecules (e.g., ADAM17), required for the reciprocal stabilization of BCL-XL in tumor cells. The significance of microenvironment PKC-b for drug resistance was demonstrated in vivo, using C57B/6 mice, diseased with EuTCL-1 driven B-cell tumors and treated with venetoclax in combination with or without enzastaurin (PKC-b inhibitor). Combined treatment significantly prolonged survival, based on PKC-b mediated impairment of lysosome biogenesis in vivo. Similarly, concurrent treatment of PKC-b inhibitors with chemotherapy also improved survival in an ALL-PDx model. Our data demonstrate that mitigating EMDR with small-molecule inhibitors of PKC-b or TGF-b signaling enhances the effectiveness of both targeted and nontargeted chemotherapies and, moreover, has the ability to overcome venetoclax resistance in B-cell malignancies in vivo. A clinical trial to test the dual inhibition of stroma and tumor cells in lymphoma patients is in preparation. Citation Format: Eugene Park, Jingyu Chen, Andrew Moore, Maurizio Mangolini, Joseph R. Byod, Hilde Schjerven, James C. Williamson, Paul J. Lehner, Michael Leitges, Alexander Egle, Marc Schmidt-Supprian, Seth Frietze, Ingo Ringshausen. Overcoming venetoclax resistance in B-cell malignancies by antagonism of stromal TGF-beta-mediated drug resistance [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-62.

Novel targeted therapies have substantially improved the prognosis of patients with B cell malignancies. However, a substantial fraction of patients still relapse, even after initially achieving deep remissions. Many studies have characterized the interactions between tumor cells and their microenvironment as integral to leukemia/ lymphoma homeostasis and for the provision of survival signals, also contributing to drug resistance (referred to as environment-mediated drug resistance (EMDR)). Therapeutic efforts to antagonize microenvironment-emanating survival cues have predominantly focused on perturbation of tumour cell adhesion enabling the physical displacement from protective niches (e.g. BCR-inhibitors). In an effort to address whether direct stromal targeting could more precisely mitigate EMDR, we recently characterised the molecular mechanisms underlying tumor-stroma interactions in B cell malignancies and identified a protein kinase C-β (PKC-β) as an essential kinase, required for activation of NF-κB in mesenchymal stromal cells (Lutzny et al Cancer Cell 2013). The dependency on stroma PKC-β was uniformly found for acute (ALL) and chronic (CLL, MCL) B cell malignancies. Importantly, our data further demonstrate that targeting stroma PKC-β is of key importance for multi-drug resistance of malignant B cells and can be used for therapeutic interventions (Park et al Science Trans Med 2020). Here we demonstrate novel mechanistic insights into stroma-mediated drug resistance in B cell malignancies. We identified that stroma PKC-β drives a transcriptional program in tumor cells, dependent on the activation of TGF-β and BMP-signaling, which ultimately leads to the stabilisation of BCL-XL. Our data show that BCL-XL expression in tumor cells is associated with SMAD1-induction by cytotoxic therapies, which simultaneously suppress SMAD4 expression. Importantly, SMAD1 expression was strictly dependent on stromal PKC-β activity. Antagonizing stroma signals with TGF-β inhibitors inhibits SMAD1 induction, abrogates the up-regulation of BCL-XL and overcomes stroma-dependent resistance to Venetoclax and conventional chemotherapy. The TGF-β pathway operates in parallel to the activation of the transcription factor EB (TFEB) as a down-stream target of PKC-β. Interference with these signaling pathways impairs plasma membrane integrity of stromal cells by down-regulation of numerous adhesion and signaling molecules, such as ADAM17, required for the reciprocal stabilization of BCL-XL in tumor cells. The significance of microenvironment PKC-β for drug resistance was demonstrated in vivo, using C57B/6 mice, diseased with EμTCL-1 driven B cell tumors and treated with Venetoclax in combination with or without PKC-β inhibitors. Combined treatment significantly prolonged survival, based on PKC-β mediated impairment of EMDR. Similarly, concurrent treatment of PKC-β inhibitors with chemotherapy also improved survival in an ALL-PDx model Our data demonstrate that mitigating EMDR with small molecule inhibitors of PKC-β or TGF-β signalling enhance the effectiveness of both targeted and non-targeted chemotherapies and moreover, has the ability to overcome Venetoclax resistance in B cell malignancies. Clinical trials with repurposed drugs inhibiting the here described pathways mediating EMDR are in planning. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Midostaurin as inhibitor of stroma PKC-β

Inhibition of stromal cell PKC-β mitigates environment-mediated drug resistance in B cell malignancies.

The nuclear organelle the nucleolus and the transcription factor nuclear factor of kappa-light-chain-enhancer of activated B cells (NF-kappa B) are both central to the control of cellular homeostasis, dysregulated in common diseases and implicated in the ageing process. Until recently, it was believed that they acted independently to regulate homeostasis in health and disease. However, there is an emerging body of evidence suggesting that nucleoli and NF-kappa B signalling converge at multiple levels. Here we will review current understanding of this crosstalk. We will discuss activation of the NF-kappa B pathway by nucleolar stress and induction of apoptosis by nucleolar sequestration of NF-kappa B/RelA. We will also discuss the role of TIF-IA, COMMD1, and nucleophosmin, which are key players in this crosstalk, and the therapeutic relevance, particularly with respect to the antitumour effects of aspirin.

Nucleoli are emerging as key sensors of cellular stress and regulators of the downstream consequences on proliferation, metabolism, senescence, and apoptosis. NF-κB signalling is activated in response to a similar plethora of stresses, which leads to modulation of cell growth and death programs. While nucleolar and NF-κB pathways are distinct, it is increasingly apparent that they converge at multiple levels. Exposure of cells to certain insults causes a specific type of nucleolar stress that is characterised by degradation of the PolI complex component, TIF-IA, and increased nucleolar size. Recent studies have shown that this atypical nucleolar stress lies upstream of cytosolic IκB degradation and NF-κB nuclear translocation. Under these stress conditions, the RelA component of NF-κB accumulates within functionally altered nucleoli to trigger a nucleophosmin dependent, apoptotic pathway. In this review, we will discuss these points of crosstalk and their relevance to anti-tumour mechanism of aspirin and small molecule CDK4 inhibitors. We will also briefly the discuss how crosstalk between nucleoli and NF-κB signalling may be more broadly relevant to the regulation of cellular homeostasis and how it may be exploited for therapeutic purpose.

Nucleoli are emerging as key sensors of cellular stress and regulators of the downstream consequences on proliferation, metabolism, senescence and apoptosis. NF-kB signalling is activated in response to a similar plethora of stresses, which leads to modulation of cell growth and death programs. Although these pathways are distinct, it is increasingly apparent that they converge at multiple levels. Exposure of cells to certain insults causes a specific type of nucleolar stress that is characterised by degradation of the PolI complex component, TIF-IA, and increased nucleolar size. Recent studies have shown that this atypical nucleolar stress lies upstream of cytosolic IkB degradation and NF-kB nuclear translocation. Under these stress conditions, the RelA component of NF-kB accumulates within functionally altered nucleoli to trigger a nucleophosmin dependent, apoptotic pathway. In this review, we will discuss these points of crosstalk and their relevance to the anti-tumour mechanism of aspirin and small molecule CDK4 inhibitors. We will also briefly discuss how NF-kB-nucleoli crosstalk may be more broadly relevant to the regulation of cellular homeostasis and how it may be exploited for therapeutic purpose.

p53 as an effector of nucleolar stress is well defined, but p53 independent mechanisms are largely unknown. Like p53, the NF-κB transcription factor plays a critical role in maintaining cellular homeostasis under stress. Many stresses that stimulate NF-κB also disrupt nucleoli. However, the link between nucleolar function and activation of the NF-κB pathway is as yet unknown. Here we demonstrate that siRNA silencing of PolI complex components stimulates NF-κB signalling. Unlike p53 nucleolar stress response, this effect does not appear to be linked to inhibition of rDNA transcription. We show that specific stress stimuli of NF-κB induce degradation of a critical component of the PolI complex, TIF-IA. This degradation precedes activation of the NF-κB pathway and is associated with an atypical nucleolar architecture. It is mimicked by CDK4 inhibition and is dependent upon upstream binding factor (UBF) and p14ARF. We show that blocking stress effects on TIF-IA blocks their ability to activate the NF-κB pathway. Finally, using ex vivo culture, we show a strong correlation between degradation of TIF-IA and activation of NF-κB in freshly resected, human colorectal tumours exposed to the chemopreventative agent, aspirin. Together, our study provides compelling evidence for a new, NF-κB nucleolar stress response pathway that has in vivo relevance and therapeutic implications.

The nucleolus is a multifunctional organelle that plays a critical role in maintaining cellular homeostasis under stress. However, how nucleoli sense stress and coordinate specific phenotypic outcomes, remains poorly understood. Here, we identify a novel nucleolar stress response pathway that culminates in activation of NF-κB. Using multiple approaches, we show that specific disruption of the PolI complex stimulates NF-κB signalling. Unlike the paradigm of nucleolar stress, this stimulation is not caused by inhibition of rRNA transcription. We identify a novel mechanism by which specific stresses disrupt nucleoli involving CDK4 inhibition and consequently, UBF-p14ARF-dependent degradation of the PolI complex component, TIF-IA. We show this atypical nucleolar stress response is associated with a distinctive nucleolar architecture. Furthermore, we show it lies upstream of NF-κB signalling. Finally, we explore the relevance of this pathway in response to aspirin in human clinical samples and demonstrate a correlation between TIF-IA degradation and NF-κB pathway activation. Together, these data provide a conceptual advance in understanding of nucleolar stress response with therapeutic implications.

Overwhelming evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumour activity and the potential to prevent cancer, particularly colorectal cancer. However, the mechanisms underlying this effect remain hypothetical. Dysregulation of the nuclear factor-kappaB (NF-κB) transcription factor is a common event in many cancer types which contributes to tumour initiation and progression by driving expression of pro-proliferative/anti-apoptotic genes. In this review, we will focus on the current knowledge regarding NSAID effects on the NF-κB signalling pathway in pre-cancerous and cancerous lesions, and the evidence that these effects contribute to the anti-tumour activity of the agents. The nuclear organelle, the nucleolus, is emerging as a central regulator of transcription factor activity and cell growth and death. Nucleolar function is dysregulated in the majority of cancers which promotes cancer growth through direct and indirect mechanisms. Hence, this organelle is emerging as a promising target for novel therapeutic agents. Here, we will also discuss evidence for crosstalk between the NF-κB pathway and nucleoli, the role that this cross-talk has in the anti-tumour effects of NSAIDs and ways forward to exploit this crosstalk for therapeutic purpose.

Paired box protein 5 (PAX5) plays a lineage determination role in B-cell development. However, high expression of PAX5 has been also found in various malignant diseases, including B-lymphoproliferative disorders (B-LPDs), but its functions and mechanisms in these diseases are still unclear. Here, we show that PAX5 induces drug resistance through association and activation of receptor-interacting serine/threonine-protein kinase 2 (RIP2; also known as RIPK2), and subsequent activation of NF-kappa B signaling and anti-apoptosis gene expression in B-lymphoproliferative cells. Furthermore, PAX5 is able to interact with RIP1 and RIP3, modulating both RIP1-mediated TNFR and RIP2-mediated NOD1 and NOD2 pathways. Our findings describe a newfunction of PAX5 in regulating RIP1 and RIP2 activation, which is at least involved in chemotherapeutic drug resistance in B-LPDs.