Constitutive NF-κB Activation Research Articles

MiR-17∼92 is involved in NF-κB activation via targeting the ubiquitin-editing proteins to mediate RIP1 complex polyubiquitinations in ABC-DLBCL

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoma characterized by constitutive NF-κB activation, but whether miR-17∼92 contributes to this activation remains unclear. Herein, we sought to evaluate the role of miR-17∼92 in the process of NF-κB activation in ABC-DLBCL. We found that the expression of miR-17∼92 primary transcript was positively correlated with NF-κB activity, miR-17∼92 activated the NF-κB signaling in ABC-DLBCL, and its over-expression promoted ABC-DLBCL cell growth, accelerated cell G1 to S phase transition and enhanced cell resistance to NF-κB inhibitor. Importantly, miR-17∼92 promoted NF-κB activation through directly targeting multiple ubiquitin-editing regulators to lead to increase the K63-linked polyubiquitination and decrease the K48-linked polyubiquitination of RIP1 complex in ABC-DLBCL. We further found that miR-17∼92 selectively activated IκB-α and NF-κB p65 but not NF-κB p52/p100, and high miR-17∼92 expression was also associated with poorer outcome in ABC-DLBCL patients. Overall, our results showed that miR-17∼92 selectively activated the canonical NF-κB signaling via targeting ubiquitin-editing regulators to lead to constitutively NF-κB activation and poorer outcome in ABC-DLBCL. These findings uncovered an innovative function of miR-17∼92 and previously unappreciated regulatory mechanism of NF-κB activation in ABC-DLBCL. Targeting miR-17∼92 may thus provide a novel bio-therapeutic strategy for ABC-DLBCL patients.

Targeted Therapies in the Treatment of Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a rare, heterogeneous B-cell non-Hodgkin's lymphoma. The standard front-line treatment utilizes chemotherapy, often followed by consolidation with an autologous hematopoietic cell transplant; however, in most patients, the lymphoma will recur and require subsequent treatments. Additionally, mantle cell lymphoma primarily affects older patients and is frequently chemotherapy-resistant, which has further fostered the necessity for new, chemotherapy-free treatment options. In the past decade, targeted therapies in mantle cell lymphoma have been practice-changing as the treatment paradigm shifts further away from relying primarily on cytotoxic agents. Here, we will review the pathophysiology of mantle cell lymphoma and discuss the emergence of targeted, chemotherapy-free treatments aimed at disrupting the abnormal biology driving its lymphomagenesis. Treatments targeting the constitutive activation of NF-kB, Bruton's Tyrosine Kinase signaling, and anti-apoptosis will be the primary focus as we discuss their clinical data and toxicities. Our review will also focus primarily on the emergence and use of targeted therapies in the relapsed/refractory setting but will also discuss the emergence of their use in front-line therapy and in combination with other agents.

Aberrant non-canonical NF-κB signalling reprograms the epigenome landscape to drive oncogenic transcriptomes in multiple myeloma.

In multiple myeloma, abnormal plasma cells establish oncogenic niches within the bone marrow by engaging the NF-κB pathway to nurture their survival while they accumulate pro-proliferative mutations. Under these conditions, many cases eventually develop genetic abnormalities endowing them with constitutive NF-κB activation. Here, we find that sustained NF-κB/p52 levels resulting from such mutations favours the recruitment of enhancers beyond the normal B-cell repertoire. Furthermore, through targeted disruption of p52, we characterise how such enhancers are complicit in the formation of super-enhancers and the establishment of cis-regulatory interactions with myeloma dependencies during constitutive activation of p52. Finally, we functionally validate the pathological impact of these cis-regulatory modules on cell and tumour phenotypes using in vitro and in vivo models, confirming RGS1 as a p52-dependent myeloma driver. We conclude that the divergent epigenomic reprogramming enforced by aberrant non-canonical NF-κB signalling potentiates transcriptional programs beneficial for multiple myeloma progression.

The Zymogen Form of Caspase-1 Is Required to Fine Tune Excessive Cell-Intrinsic Inflammation in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is characterized by the expansion of clonally-derived mutant hematopoietic stem and progenitor cells. Many patients do not respond to standard chemotherapy because AML cells acquire mutations that exploit inflammatory and cell death pathways, allowing them to evade cell death. Targeted therapies are urgently needed to block AML cell pro-survival and inflammatory signaling for more effective outcomes. One promising area of interest is Caspase-1 (CASP1), a cysteine-aspartic acid protease responsible for cleaving the precursors of interleukin-1β (IL-1β) and Gasdermin D, which induces pyroptosis, into their active mature peptides. In myeloid malignancies, IL-1β levels are significantly elevated, and pyroptosis has been linked to ineffective hematopoiesis as observed in MDS, thereby implicating CASP1. CASP1 is typically expressed as a zymogen (Pro-CASP1) that can be cleaved into an active enzyme. In this report, we present a novel and unexpected role for Pro-CASP1, which appears to be independent of its proteolytic function, in MDS/AML leukemic cells. Pyroptosis, an inflammatory cell death mechanism, is driven by CASP1-mediated osmotic lysis and IL-1b secretion. Notably, CASP1 expression is significantly higher in AML patients compared to healthy hematopoietic cells, and elevated CASP1 expression in AML patients correlates with shorter overall survival. In leukemic cells, CASP1 primarily exists in its zymogen form, lacking the ability to induce pyroptosis. It is thus unexpected that elevated CASP1 expression, which typically leads to heightened susceptibility to pyroptotic cell death, does not result in pyroptosis in leukemic cells, and the underlying mechanisms for this evasion remain unknown. To investigate Pro-CASP1's significance in AML cells, we used CRISPR-Cas9 to create Pro-CASP1 knockout (Pro-CASP1 KO) human isogenic MDS and AML cell lines (THP1 and MDSL). Pro-CASP1 KO MDS/AML cells showed reduced cell growth and leukemic colony forming potential in methylcellulose compared to isogenic WT control cells. When Pro-CASP1 KO THP1 AML cells were transplanted into immunocompromised mice, the loss of Pro-CASP1 significantly reduced leukemic cell burden and extended overall survival. We next preformed experiments to probe the mechanism of action of Pro-CASP1 in AML cells. Previous studies suggest that Pro-CASP1 can function as a scaffold to elicit downstream signaling without cleaving into its activated form. The functional defect in Pro-CASP1 KO THP1 AML cells was rescued with full-length Pro-CASP1, but not with a scaffolding mutant of CASP1, indicating a specific dependency of leukemic cells on the zymogen form of CASP1. To precisely delineate which domains of Pro-CASP1 are essential for AML, we performed a tiled CRISPR dropout screen in MDSL and THP1 cells. The results showed similar CRISPR-Knockout High Sensitivity (CKHS) domain footprints in both cell lines mapping to the N-terminal CARD and the Inter-Domain Linker (IDL) domains. These results indicate that Pro-CASP1's proteolytic function was found to be dispensable, whereas the CARD domain, a scaffolding region, was crucial for leukemogenesis. We next investigated how loss of Pro-CASP1 affects inflammatory signaling in AML cells. Deletion of Pro-CASP1 in THP1 AML cells led to excessive and constitutive activation of NF-kB as indicated by nuclear translocation and phosphorylation of p65/RelA. Restoring NF-kB regulation using the inhibitor of kappa B (IkB) super-repressor (IkB-SR) was sufficient to reverse the leukemic progenitor defect and cell viability in Pro-CASP1 KO AML cells. These findings suggest that Pro-CASP1 is required for AML cells to finetune canonical NF-kB activation. Conversely, loss of Pro-CASP1 results in excessive NF-kB activation that is incompatible with AML, consistent with previous observations (Ellegast et al., Cancer Discovery 2022). In summary, Pro-CASP1 serves as a critical signaling hub, fine-tuning NF-kB pathways independently of its proteolytic function in leukemic cells. Its loss exposes leukemic cells to functional and therapeutic vulnerabilities.

Discovery of Novel, First-in-Class Allosteric Modulators of MALT1 Scaffolding Function with Differentiated Pharmacology for NFκB-Driven Malignancies

Constitutive activation of NFκB is an oncogenic driver for many lymphoid and selected solid tumor malignancies. BTK inhibitors, which affect the upstream NFκB pathway, have been successfully used in some diseases, but their utility is limited. The majority of DLBCL patients, for instance, are insensitive to BTK inhibition, supporting the need for alternative mechanisms to block NFκB-driven tumor progression. Knockdown of MALT1, a key component of the CBMT complex (CARD-BCL10-MALT1-TRAF6) involved in direct signal transduction of NFκB, is able to block NFκB-dependent DLBCL tumor growth in BTKi sensitive or resistant tumors, but pharmacologic modulation has been challenged by the dual-functionality of MALT1 in a complexed signalosome. MALT1 functions as the scaffolding component of the CBMT complex. Activated MALT1 recruits multiple proteins and directly activates NFκB canonical signaling. MALT1 also has protease activity that fine-tunes various signaling pathways including NFκB via the cleavage of many substrates. The scaffolding activity of MALT1 is critical for cancer cell survival. Most MALT1 inhibitors discovered so far only block protease activity, which results in incomplete NFκB inhibition, suboptimal anti-tumor activity, and the induction of severe autoimmunity due to protease inhibition. Using our Smart Allostery platform, we elucidated and then drugged a natural hotspot on MALT1 that selectively impacts scaffolding but not protease function. The series of scaffolding modulators demonstrate potent inhibition of MALT1 scaffolding activity with cellular IC 50 values ranging from 15 to 250 nM, while exerting minimal influence on MALT1 protease function. Unlike protease inhibitors, which only partially affect NFκB activity, scaffolding modulators completely inhibit the NFκB downstream signaling manifested by the suppression of NFκB activity with cellular IC 50 values ranging from 2 to 90 nM. Consequently, scaffolding modulators exhibit a superior and broader anti-proliferation profile compared to MALT1 protease inhibitors and BTK inhibitors in a panel of lymphoma cell lines. Pathway and genetic analyses indicate that B-cell lymphoma lines driven by NFκB are especially sensitive to scaffolding modulators. In vivo studies demonstrate a robust, dose-dependent tumor growth inhibition (TGI) by one representative scaffolding modulator in several B-cell lymphoma xenograft models. In a protease inhibitor sensitive ABC-DLBCL model, OCI-LY10, our scaffolding modulator achieved a maximal TGI of 94%, while a clinical MALT1 protease inhibitor showed 62% maximal TGI (p<0.001). In protease resistant NFκB driven DLBCL models, MALT1 scaffolding modulation resulted in tumor regression or stasis. These findings highlight the differentiated pharmacology of our scaffolding modulators. Distinct from protease inhibitors that suppress T cell activation and Treg population in vivo, scaffolding modulators also did not deplete Treg cells nor suppress T cell activation in a 14-day study. This potentially mitigates the risk of development of autoimmune disease caused by chronic MALT1 protease inhibition. In summary, we have discovered novel, first-in-class allosteric modulators of MALT1 scaffolding activities. These scaffolding modulators drive enhanced and broader anti-tumor effect in models resistant to MALT1 protease or BTK inhibitors and do so without immune dysregulation. Scaffolding modulation represents a potential best-in-class therapeutic opportunity to modulate MALT1 in patients with NFκB-driven malignancies.

A Phase 1, Open-Label, Multicenter, Dose-Escalation Study of Sgr-1505 As Monotherapy in Subjects with Mature B-Cell Malignancies

Background and Significance: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Furthermore, a MALT1 inhibitor (JNJ-67856633) showed efficacy in mature B cell malignancies from phase 1 studies (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3) ABC-DLBCL cell lines. SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition may expand therapeutic options for patients with selected B-cell lymphomas, supporting further evaluation of SGR-1505 in clinical trials. Study Design and Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies (figure 1). At present, the study is open to accrual at multiple investigative sites in the US with plans to expand to Europe. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤ 2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1-6 subjects, and at higher dose levels using a conventional 3+3 design.

MALT1 Protease Inhibition Overcomes BTK Inhibitor Resistance and Shows Synergistic Activity with Venetoclax in Models of B Cell Lymphoma and Leukemia

Background Specific B cell malignancies, including the aggressive non-germinal center B cell-like (GCB) subtype of diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), are driven by constitutive activation of the transcription factor NF-kB. Chronic signaling resulting from mutations or antigen-induced activation of the B cell receptor (BCR) pathway drives NF-kB activity in these tumors resulting in sustained proliferation and survival pathways. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) represents a key mediator of the BCR/NF-kB signal transduction pathway responsible for maintaining survival and driving proliferation of NF-kB-addicted B cell tumors by proteolytically cleaving NF-kB antagonists. Aims Pharmacological inhibition of MALT1 protease activity may provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Methods A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results ABBV-MALT1 potently inhibits MALT1 activity in vitro while showing high selectivity against a panel of proteases, kinases and other types of receptors and enzymes. Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-kB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton's tyrosine kinase (BTK) inhibitors. NF-kB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-kB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Summary/Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with DLBCL, CLL and other B cell malignancies. Disclosures All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Dynamic Recruitment of Inhibitory Complexes Controls Oncogenic Signaling in B-Cell Malignancies

Background and Significance: Initiation of B-cell receptor (BCR) signaling, and subsequent antigen-encounter in germinal centers represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) and lymphoma also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B-cells was unclear. High expression levels on the surface of B-ALL cells are associated with poor clinical outcomes in pediatric (COG P9906) and adult (ECOG E2993) B-ALL cohorts. In DLBCL, aggressive disease is associated with CD25-inactivation by shedding and high serum levels of soluble CD25. Results: Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ, immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors and inhibitory phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ and phosphatases, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early stages of B-cell development (e.g. B-ALL) CD25-loss induced hyperactivation of oncogenic signaling followed by cell death. In contrast, genetic deletion of CD25 in models for mature B-cell lymphoma accelerated proliferation of malignant cells. Biochemical and interactome studies revealed a critical role of CD25 in feedback regulation of oncogenic signaling in B-cell malignancies: Signaling from BCR-ABL1 and RAS-oncogenes in B-ALL as well as oncogenic BCR-signaling in DLBCL induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268). Importantly, CD25-phosphorylation stabilized its expression at the cell membrane and opposed both inactivation by CD25-shedding and internalization. Moreover CD25-S 268 tail-phosphorylation enabled CD25 to recruit inhibitory receptors, including CD22, that bear ITIM-motifs for the activation of inhibitory phosphatases (e.g. SHP1, Figure 1). Thereby, the positively charged cytoplasmic tail of CD25 engages negatively charged residues in the cytoplasmic tails of the BCR signaling chain CD79B and the inhibitory CD22-ITIM. Molecular dynamics simulations revealed that the ternary complex between CD25, CD79B and CD22 enables recruitment and activation of the phosphatase SHP1 within reach of its substrate, the BCR signaling chain CD79B. Thereby, the SH2-domain of SHP1 engages the CD22-ITIM motif, while its phosphatase domain makes contact with the CD79B-ITAM to dephosphorylate and terminate BCR-signaling. This complex, while engaging and activating SHP1, is held together by CD25 ( Figure 1). To test functional consequences of destabilization of this complex, we generated a knockin mouse model carrying a single point mutation (S268A) in the cytoplasmic tail of CD25. The CD25 S268A knockin mutation abolished recruitment and activation of SHP1. In addition, the S268A-mutant failed to assemble CD22 and CD79B in complex with SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function and failure to terminate oncogenic signaling resulted in constitutive activation of ERK and NF-κB, autonomous Ca 2+-oscillations. Hyperactivation of oncogenic signaling resulted in exhaustion and cell death in B-ALL cells, as opposed to excessive proliferation and acceleration of disease in mature B-cell lymphoma. Conclusions: While CD25 has an established function in IL2 signal transduction in T- and NK-cells, these findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and provides an explanation for the common feature of CD25 shedding and high serum levels of functionally inactive soluble CD25 in aggressive B-cell lymphomas.

A First-in-Human Phase 1 Study of ABBV-525, a Small-Molecule MALT1 Inhibitor, in Patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Background and Significance Many B-cell malignancies are characterized by chronic activation of the B-cell receptor (BCR) and nuclear factor kappa B (NF-κB) signaling pathways. In many non-Hodgkin lymphoma (NHL) subtypes, constitutive NF-κB activation results from genetic mutations and/or auto-antigen stimulation of the BCR pathway. While agents targeting the BCR pathway are effective treatments for many B-cell malignancies, patients (pts) often have progressive disease due to acquired resistance to BCR-targeted agents. Thus, there is an unmet medical need for novel therapeutic targets and treatment approaches. The mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is an enzymatic and scaffolding node in the BCR pathway and a key mediator of the BCR/NF-κB signal transduction pathway. Its action is downstream, or in some instances parallel to, resistance mechanisms to existing NHL therapies, including BTK C481S and PLCG2 mutations, which are known acquired mechanisms of resistance to covalent Bruton's tyrosine kinase (BTK) inhibitors in pts with chronic lymphocytic leukemia (CLL). MALT1 inhibition represents a novel therapeutic approach for malignancies with chronic activation of NF-κB, including those pts with acquired resistance to upstream targets. ABBV-MALT1 is a small-molecule, tool compound, MALT1 inhibitor. Preclinical studies of ABBV-MALT1 have demonstrated potent activity against MALT1-relevant biomarkers, including inhibition of BCR pathway signal transduction and reduced NF-κB gene activation in non-germinal center B-cell diffuse large B-cell lymphoma (non-GCB DLBCL) cell lines. ABBV-MALT1 has also shown activity in a range of preclinical lymphoma models as both monotherapy and in combination with venetoclax, including robust antitumor activity in malignant B-cell models that are resistant to BTK inhibitors. This phase 1 study evaluates the clinical candidate MALT1 inhibitor ABBV-525 as monotherapy in pts with relapsed or refractory (R/R) B-cell NHL. Study Design and Methods This is a first-in-human, phase 1, open-label, multicenter study of single-agent ABBV-525 in pts with R/R B-cell NHL (NCT05618028). Eligibility criteria are listed in Table 1. The primary objectives are to evaluate safety, tolerability, and pharmacokinetics (PK) of ABBV-525 and to determine the recommended phase 2 dose (RP2D). The secondary objective is to assess the preliminary efficacy of ABBV-525 in subsets of pts with R/R B-cell NHL. Exploratory objectives include evaluating biomarkers predictive of response and pharmacodynamic biomarkers, including the effects of ABBV-525 on BCR pathway signal transduction and NF-κB gene activation. This is a 3-part study (Figure 1): dose escalation (part 1), dose optimization (part 2), and dose expansion (part 3). Part 1 aims to establish the maximum administered dose/maximum tolerated dose of ABBV-525 and is primarily guided by a Bayesian optimal interval design. Part 2 aims to identify the RP2D of ABBV-525. Part 3 will further characterize the safety profile of ABBV-525 at the RP2D. Approximately 150 pts are planned for enrollment, including those with various R/R mature B-cell lymphomas in part 1, pts with documented diagnosis of R/R CLL in part 2, and pts with documented diagnosis of non-GCB DLBCL in part 3. ABBV-525 is orally administered until disease progression, intolerable toxicity, or other study discontinuation criteria are met. Safety assessments include dose-limiting toxicities, adverse events (per National Cancer Institute Common Terminology Criteria for Adverse Events v5.0), clinical laboratory parameters, vital signs, and electrocardiogram variables. PK parameters, including maximum observed plasma concentration (C max), time to C max, and area under the concentration-time curve will be analyzed using noncompartmental methods. Efficacy will be assessed through response per disease-specific criteria (including International Workshop on Chronic Lymphocytic Leukemia, International Workshop on Waldenstrom's Macroglobulinemia, and Lugano classification). Duration of response will be evaluated by Kaplan-Meier estimates. Biomarker data will be analyzed as change from baseline and summarized for each scheduled postbaseline visit. Pts are being enrolled in 25 sites across the USA, Australia, Belgium, France, Germany, Israel, Spain, and UK. As of August 1, 2023, 2 pts had been treated.

Dimethyl fumarate treatment in relapsed and refractory cutaneous T-cell lymphoma: a multicenter phase 2 study

AbstractTargeted therapies for cutaneous T-cell lymphoma (CTCL) are limited and curative approaches are lacking. Furthermore, relapses and drug induced side effects are major challenges in the therapeutic management of patients with CTCL, creating an urgent need for new and effective therapies. Pathologic constitutive NF-κB activity leads to apoptosis resistance in CTCL cells and, thus, represents a promising therapeutic target in CTCL. In a preclinical study we showed the potential of dimethyl fumarate (DMF) to block NF-κB and, specifically, kill CTCL cells. To translate these findings to applications in a clinical setting, we performed a multicentric phase 2 study evaluating oral DMF therapy in 25 patients with CTCL stages Ib to IV over 24 weeks (EudraCT number 2014-000924-11/NCT number NCT02546440). End points were safety and efficacy. We evaluated skin involvement (using a modified severity weighted assessment tool [mSWAT]), pruritus, quality of life, and blood involvement, if applicable, as well as translational data. Upon skin analysis, 7 of 23 (30.4%) patients showed a response with >50% reduction in the mSWAT score. Patients with high tumor burden in the skin and blood responded best to DMF therapy. Although not generally significant, DMF also improved pruritus in several patients. Response in the blood was mixed, but we confirmed the NF-κB–inhibiting mechanism of DMF in the blood. The overall tolerability of the DMF therapy was very favorable, with mostly mild side effects. In conclusion, our study presents DMF as an effective and excellently tolerable therapeutic option in CTCL to be further evaluated in a phase 3 study or real-life patient care as well as in combination therapies. This trial was registered at www.clinicaltrials.gov as #NCT02546440.

Bi-allelic mutation of CARD11 resulting in fatal immune dysregulation with dramatic B cell expansion

Abstract Caspase recruitment domain family member 11 (CARD11) functions as a scaffold to bridge antigen receptors with downstream immune signaling pathways that enable antigen-dependent lymphocyte activation. Given its pleiotropic role in regulating adaptive immunity, different genetic variants in CARD11 can result in diverse phenotypic consequences in humans. Here we describe a patient harboring a novel bi-allelic CARD11 mutation that results in potentially fatal immune dysregulation with massive B cell expansion. He presented with splenomegaly, amenia, leukocytosis and thrombocytopenia within the first year of life. A sibling showed similar symptoms before passing away in infancy. Due to parental consanguinity, an autosomal recessive inborn error of immunity was suspected; whole exome sequencing revealed a homozygous missense mutation in the coiled-coil (CC) domain of CARD11 (p.Arg331Pro, R331P). Functional testing revealed the R331P mutation caused a mild gain-of-function effect relative to wild-type CARD11 in driving constitutive NF-kB activation, suggesting that R331P may disrupt interactions needed to maintain CARD11 autoinhibition in resting lymphocytes. Dramatic B cell lymphocytosis noted in the patient was not present in his heterozygous parents, indicating that 2 mutant alleles are required for this phenotype. In contrast, the patient’s T cells responded poorly to TCR stimulation with severely reduced proliferation. Collectively, these results indicate that the novel R331P CARD11 mutation manifests as the first known recessive form of B cell Expansion with NF-kB and T cell Anergy (BENTA disease), and illustrate how rare monogenic inborn errors of immunity provide a unique discovery platform for basic immunology.

Unraveling astrocyte behavior in the space brain: Radiation response of primary astrocytes.

Exposure to space conditions during crewed long-term exploration missions can cause several health risks for astronauts. Space radiation, isolation and microgravity are major limiting factors. The role of astrocytes in cognitive disturbances by space radiation is unknown. Astrocytes' response toward low linear energy transfer (LET) X-rays and high-LET carbon (12C) and iron (56Fe) ions was compared to reveal possible effects of space-relevant high-LET radiation. Since astronauts are exposed to ionizing radiation and microgravity during space missions, the effect of simulated microgravity on DNA damage induction and repair was investigated. Primary murine cortical astrocytes were irradiated with different doses of X-rays, 12C and 56Fe ions at the heavy ion accelerator GSI. DNA damage and repair (γH2AX, 53BP1), cell proliferation (Ki-67), astrocytes' reactivity (GFAP) and NF-κB pathway activation (p65) were analyzed by immunofluorescence microscopy. Cell cycle progression was investigated by flow cytometry of DNA content. Gene expression changes after exposure to X- rays were investigated by mRNA-sequencing. RT-qPCR for several genes of interest was performed with RNA from X-rays- and heavy-ion-irradiated astrocytes: Cdkn1a, Cdkn2a, Gfap, Tnf, Il1β, Il6, and Tgfβ1. Levels of the pro inflammatory cytokine IL-6 were determined using ELISA. DNA damage response was investigated after exposure to X-rays followed by incubation on a 2D clinostat to simulate the conditions of microgravity. Astrocytes showed distinct responses toward the three different radiation qualities. Induction of radiation-induced DNA double strand breaks (DSBs) and the respective repair was dose-, LET- and time-dependent. Simulated microgravity had no significant influence on DNA DSB repair. Proliferation and cell cycle progression was not affected by radiation qualities examined in this study. Astrocytes expressed IL-6 and GFAP with constitutive NF-κB activity independent of radiation exposure. mRNA sequencing of X-irradiated astrocytes revealed downregulation of 66 genes involved in DNA damage response and repair, mitosis, proliferation and cell cycle regulation. In conclusion, primary murine astrocytes are DNA repair proficient irrespective of radiation quality. Only minor gene expression changes were observed after X-ray exposure and reactivity was not induced. Co-culture of astrocytes with microglial cells, brain organoids or organotypic brain slice culture experiments might reveal whether astrocytes show a more pronounced radiation response in more complex network architectures in the presence of other neuronal cell types.

Abstract 4330: A CD5 signature identifies diffuse large B-cell lymphomas (DLBCLs) sensitive to Bruton’s tyrosine kinase (BTK) inhibition

Abstract Introduction: Gene expression profiling has identified DLBCLs that bear transcriptional similarity to either non-malignant germinal center B-cells (GCB-DLBCL) or activated B-cells (ABC-DLBCL, i.e. non-GCB-DLBCL). This cell-of-origin (COO) classification has guided precision medicine strategies, including the use of ibrutinib, a BTK inhibitor (BTKi), with ABC-DLBCLs thought to be sensitive to BTKi due to their constitutive activation of NF-kB downstream of the B-cell receptor (BCR). However, the phase III Phoenix trial testing the addition of ibrutinib to R-CHOP chemoimmunotherapy surprisingly failed to demonstrate a survival benefit in non-GCB-DLBCLs. By employing a newly defined genetic classification algorithm (LymphGen), distinct genetic DLBCL subtypes that benefit from this regimen were identified. However, LymphGen is not utilized in the clinic and fails to classify >40% of DLBCLs. To overcome these challenges, we sought to identify a straightforward biomarker of BTKi sensitivity. CD5 was selected as a potential candidate, given its role as a negative regulator of BCR signaling and expression in other BTKi-responsive B-cell lymphomas. Methods: CD5 IHC was performed on a cohort of 405 DLBCLs, most with paired RNA and targeted mutational sequencing. Comparison of the transcriptomes of CD5+ and CD5− DLBCLs was used to construct a 60-gene CD5 signature (CD5sig), which was applied to large genomic DLBCL datasets, including pre-treatment biopsies from the Phoenix trial (n = 584) to evaluate its utility in predicting improved response to R-CHOP + ibrutinib. Results: Twenty-seven DLBCLs were identified as CD5+ by IHC (6% of cases; 12% of non-GCB). Consistent with previous reports, CD5+ DLBCLs were enriched for a non-GCB COO and exhibited inferior progression-free survival to R-CHOP. CD5+ DLBCLs upregulated genes related to BCR signaling and had a significantly higher incidence of CD79B BCR-activating mutations. A CD5 signature (CD5sig) was created from differentially expressed genes between CD5 IHC+ and CD5 IHC− non-GCB-DLBCLs. When applied to an external dataset of 349 non-GCB-DLBCLs, CD5sig+ DLBCLs (15% of cases) were enriched for BCR-activating mutations and were majority MCD (MYD88 and CD79B) or unclassified by LymphGen. CD5sig+ DLBCLs were largely devoid of BCL10 activating mutations known to drive BTKi resistance. When applied to the Phoenix study, CD5sig+ DLBCLs selectively benefitted from R-CHOP + ibrutinib by event-free and overall survival. This survival difference was preserved even among LymphGen-unclassified DLBCLs. Conclusions: Here, we identify CD5 expression as a simple and useful biomarker to identify high-risk, BCR-driven DLBCLs beyond those identified through genetic classification. Based upon unique transcriptional features of CD5 IHC+ DLBCLs, we developed a novel CD5 signature that identifies DLBCLs vulnerable to BTKi therapy. Citation Format: Alan Cooper, Sravya Tumuluru, Kyle Kissick, Girish Venkataraman, Nikita Kotlov, Aleksander Bagaev, Andrew Lytle, Gerben Duns, David W. Scott, Christian Steidl, Brendan Hodkinson, Srimathi Srinivasan, Justin Kline, James Godfrey. A CD5 signature identifies diffuse large B-cell lymphomas (DLBCLs) sensitive to Bruton’s tyrosine kinase (BTK) inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4330.

The NF-κB Transcriptional Network Is a High-Dose Vitamin C-Targetable Vulnerability in Breast Cancer

Breast cancer (BC) is the most common cancer type among women with a distinct clinical presentation, but the survival rate remains moderate despite advances in multimodal therapy. Consequently, a deeper understanding of the molecular etiology is required for the development of more effective treatments for BC. The relationship between inflammation and tumorigenesis is well established, and the activation of the pro-inflammatory transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is frequently identified in BC. Constitutive NF-κB activation is linked to cell survival, metastasis, proliferation, and hormonal, chemo-, and radiotherapy resistance. Moreover, the crosstalk between NF-κB and other transcription factors is well documented. It is reported that vitamin C plays a key role in preventing and treating a number of pathological conditions, including cancer, when administered at remarkably high doses. Indeed, vitamin C can regulate the activation of NF-κB by inhibiting specific NF-κB-dependent genes and multiple stimuli. In this review, we examine the various NF-κB impacts on BC development. We also provide some insight into how the NF-κB network may be targeted as a potential vulnerability by using natural pro-oxidant therapies such as vitamin C.

TPL2, a New Prognostic Factor and a Potential Therapeutic Target in ABC-DLBCL

Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is a subtype of DLBCL, characterized by the constitutive activation of NF-κB and JAK/STAT3 pathways on the background of the chronic active BCR signaling. Being featured by mutations in MYD88 and/or CD79B, it potentially comprises the MCD/C5 subtype in the recent genetics-based categorization. In light of the inferior prognosis of this subtype to germinal center B-cell (GCB) like DLBCL, identification of subtype-specific strategies has been sought. Tumor progression locus 2 (TPL2) is an intracellular serine/threonine kinase that is reported to be activated downstream of TLRs/MYD88 signaling and to play roles in oncogenesis. However, the involvement of TPL2 in B-cell malignancies is less defined despite relatively higher transcript expression than in solid tumors. Results: To address the impact of TPL2 protein levels on DLBCL phenotypes, we performed an IHC analysis on 79 FFPE samples of DLBCL diagnosed and treated at our institution (48 non-GCB and 31 GCB according to Hans algorithm). When divided into two groups, one with strong TPL2 expression (TPL-high) and the other with no/weak expression (TPL2-low), the ratio of TPL2-high did not differ between non-GCB and GCB (67% vs 64%). However, TPL2-high had a significantly higher percentage of positive B symptom (42% vs 11%, p=0.005) and extra-nodal involvement (75% vs 52%, p=0.047) compared to TPL2-low, and these trends were observed more specifically in non-GCB. Moreover, it is noteworthy that TPL2-high showed a significantly lower overall survival in non-GCB (p=0.037), but did not in GCB (p=0.999) (Figure 1). Intriguingly, the analysis combined with pSTAT3 (Y705) staining revealed that TPL2-high accompanied by positive pSTAT3 tended to display particularly poor prognosis in non-GCB. Then, we analyzed the mechanism by which TPL2 is involved in the tumorigenesis of ABC-DLBCL using TMD8, a well-described ABC-DLBCL cell line with MYD88L265P and CD79BY196 mutations. A specific TPL2 inhibitor significantly affected the cell proliferation of TMD8 cells, whereas did not on the cells with GCB phenotype. Of note, TPL2 inhibitor induced G1 cell-cycle arrest via downregulation of CyclinE and CDK2 and upregulation of p27 in western blot analysis, which was confirmed by quantitative PCR. Next, we investigated the downstream signaling of TPL2 in ABC-DLBCL. Western blot analysis revealed that TPL2 inhibitor as well as genetic TPL2 knock down suppressed the activation of NF-κB components and STAT3, and the expression of c-MYC. These results implied the involvement of TPL2 in IL-6/IL-10 autocrine loop and resultant JAK/STAT3 activation. As expected, treatment with TPL2 inhibitor downregulated IL-6/IL-10 mRNA by quantitative PCR and accordingly decreased the levels of IL-6/IL-10 in the cell culture supernatant of treated TMD8 cells. Therefore, we speculated that TPL2 promoted the oncogenic activation of NF-κB and STAT3 pathways via IL-6/IL-10 upregulation in ABC-subtype. Lastly, we evaluated the physiological function of TPL2 by western blot analysis using 293T. Although an overexpression TPL2 alone did not elicit NF-κB and STAT3 activation, when co-expressed with MYD88L265P TPL2 obviously activated these pathways, whereas dominant negative TPL2 failed to demonstrate those effects even with MYD88L265P co-expression. These findings indicated that TPL2 could possibly exert its tumorigenicity in MYD88L265P-dependent manner in ABC-DLBCL. Conclusion: In association with the importance as an unfavorable prognostic factor, our results suggested that TPL2 might mediate the crosstalk between the oncogenic MYD88L265P and the activation of NF-κB and STAT3 pathways, coordinately contributing to the tumorigenesis of ABC-DLBCL. From this standpoint, TPL2 could emerge as a new therapeutic target in ABC-DLBCL. Figure legend Overall survival of non-GCB (A) and GCB (B) DLBCL patients, with regard to the TPL2 expression: TPL2-low (red line) or TPL2-high (black line). Overall survival was determined by the Kaplan-Meier method and compared by using the log-rank test. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Central Nervous System Relapse in Patients with Primary Mediastinal Large B-Cell Lymphoma: Incidence and Risk Factors in the Rituximab Era

Background: Central nervous system relapses (CNSR) are uncommon in primary mediastinal large B-cell lymphoma (PMLBCL) with a reported incidence of 2.3-3.8% in the Rituximab era. Clinical risk factors for CNSR have been recognized in diffuse large B-cell lymphoma (DLBCL), but may not be applicable in PMLBCL, a pathobiologically distinct entity with constitutive activation of NFκB and JAK-STAT pathways and Programmed Death (PD) Ligand 1 and 2 overexpression. Aim: To evaluate the incidence of first CNSR events without prior systemic progression and explore prognostic associations with baseline characteristics, CNS-IPI, and induction immunochemotherapy. Methods: This is a retrospective study of a multinational cohort of 596 adult PMLBCL patients treated with R-CHOP or DA-EPOCH-R immunochemotherapy with or without radiotherapy between 2000 and early 2022. The cumulative incidence of first CNSR (CI-CNSR) was estimated considering the competing risks of death from any cause or prior systemic disease relapse/progression. Results: Baseline characteristics are shown in Table 1. Only 17 patients (2.9%) received CNS prophylaxis [high-dose methotrexate (HD-MTX) 9, intrathecal MTX±other 7, both 1]. Kidney and/ or adrenal involvement at diagnosis was highly correlated with advanced stage, ≥2extranodal sites and high CNS-IPI (4-6) (p<0.001), with the vast majority of the latter also having kidney and/or adrenal involvement. With a 55-month median follow-up [interquartile range (IQR) 32-97 months], all 10 first CNSR events(9 isolated and 1 associated with systemic relapse) were recorded within 2 years of diagnosis [median time7.5 months, IQR 6-8, range 5-13 months], for a 2-year CI-CNSR of 1.78% (95% CI 0.9-3.2%). Two of 10 CNSR cases had received CNS prophylaxis with HD-MTX. All CNSR were parenchymal and only 2/8 were successfully salvaged (2 still under treatment). Four patients with CNSR had kidney involvement (plus adrenal in 2/4). Two of seven patients with both kidney and adrenal involvement and 2/ 17 with kidney infiltration only relapsed in the CNS. Kidney [subhazard ratio (SHR) 15.4, p<0.001], adrenal (SHR 13.6, p=0.001), any kidney or adrenal involvement (SHR 12.5, p<0.001), and high CNS-IPI (4-6, SHR 13.0, p<0.001) were associated with CNSR in univariate analysis. All 4 patients with high CNS-IPI who experienced CNSR (out of 35), had also kidney and/or adrenal involvement. Looser but significant associations were observed with high IPI (2-5; SHR 6.3, p=0.009), advanced stage (III/IV; SHR 5.5, p=0.007), impaired performance status (≥2; SHR 6.6, p=0.005), and ≥2 extranodal sites (SHR 5.4, p=0.009) but not chemotherapy backbone (R-CHOP or DA-EPOCH-R), CNS prophylactic therapy, age, gender, B-symptoms, LDH elevations or other baseline labs, serositis or PMLBCL-specific prognostic indices (stage IV/ extranodal plus bulky or high LDH≥2x). Conclusion: In PMLBCL, CNSR is rare and appears to be primarily associated with kidney and/or adrenal involvement. CNS-IPI is also strongly prognostic but highly correlated with kidney and/or adrenal involvement. No late (>2 years) occurrences were seen and 9/10 relapses were isolated in the CNS. No inferences regarding the value of primary CNS prophylaxis can be made. Even in this very large series multivariate is not reliable due to the small number of events. A larger multinational effort is warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DDDR-04. CA-4948 IN COMBINATION WITH BRAF/MEK INHIBITION IN MELANOMA BRAIN METASTASES

Abstract Melanoma brain metastases (MBM) remain a primary driver of melanoma morbidity and mortality. It is estimated that as many as 60% of all metastatic melanoma patients will develop MBM during the course of their disease. Once found, dual checkpoint inhibitor therapy or BRAF/MEK inhibition in BRAF mutated cancers, are the best options currently available. Even with these treatments, the response to therapy is less than what is seen with disease outside the. In BRAF mutated cancers, which constitute 50% of all patients, initial response to targeted therapy is rapid but resistance to therapy develops 3 months sooner than resistance outside of the brain. Novel strategies to prevent onset of resistance or to work in concert with BRAF/MEK inhibition are needed for this treatment population. The exact mechanism of resistance to targeted therapy in MBM is not known, but mutations in EphA2, PI3K, pAKT, and re-activation of MAPK signaling through secondary mechanisms have all been identified as drivers of resistance. One pathway of MAPK reactivation is through activation of phospho-ERK (eIF2alpha kinase) which also activates the JNK pathway. Our group has previously shown that MBM express high levels of the inflammatory transcription factor IRAK-4, the terminal chaperone of the inflammatory myddosome downstream of TLR and IL1R signally. This increase in IRAK-4 results in constitutive activation of NF-KB, JNK, and MAPK. We have additionally shown the novel oral IRAK-4 inhibitor CA-4948 can reach therapeutically relevant concentrations past the blood brain barrier in both tumor-bearing and naïve mice. We additionally show CA-4948 inhibits MAPK expression in melanoma and suppresses the escape pathways pERK and JNK signaling which can contribute to the prevention of resistance to targeted therapy. This study supports further investigation into the combination potential of CA-4948 with BRAF/MEK inhibitors as triplet therapy to delay intracranial resistance.

Hepatitis B x (HBx) as a Component of a Functional Cure for Chronic Hepatitis B.

Patients who are carriers of the hepatitis B virus (HBV) are at high risk of chronic liver disease (CLD) which proceeds from hepatitis, to fibrosis, cirrhosis and to hepatocellular carcinoma (HCC). The hepatitis B-encoded X antigen, HBx, promotes virus gene expression and replication, protects infected hepatocytes from immunological destruction, and promotes the development of CLD and HCC. For virus replication, HBx regulates covalently closed circular (ccc) HBV DNA transcription, while for CLD, HBx triggers cellular oxidative stress, in part, by triggering mitochondrial damage that stimulates innate immunity. Constitutive activation of NF-κB by HBx transcriptionally activates pro-inflammatory genes, resulting in hepatocellular destruction, regeneration, and increased integration of the HBx gene into the host genome. NF-κB is also hepatoprotective, which sustains the survival of infected cells. Multiple therapeutic approaches include direct-acting anti-viral compounds and immune-stimulating drugs, but functional cures were not achieved, in part, because none were yet devised to target HBx. In addition, many patients with cirrhosis or HCC have little or no virus replication, but continue to express HBx from integrated templates, suggesting that HBx contributes to the pathogenesis of CLD. Blocking HBx activity will, therefore, impact multiple aspects of the host–virus relationship that are relevant to achieving a functional cure.

Isoimperatorin down-regulates epithelial mesenchymal transition through modulating NF-κB signaling and CXCR4 expression in colorectal and hepatocellular carcinoma cells

Worldwide, metastatic spread of tumor is the major cause of cancer-related deaths. There are many anti-metastatic agents that can target metastasis-related pathways, but there are relatively few studies on agents targeting C-X-C chemokine receptor type 4 (CXCR4) signaling axis. Here, we have investigated whether Isoimperatorin (IIPT), derived from Angelica dahurica can modulate CXCR4 signaling axis-related epithelial-to-mesenchymal transition (EMT) and tumor metastasis process. We evaluated the influence of IIPT on CXCR4, HER2, MMPs, EMT markers, and NF-κB signaling pathway by using Western blot analysis. The cellular invasion and migration were observed by Boyden chamber and wound healing assays. IIPT has a down-regulatory effect on CXCR4, HER2, and MMP-9/2. On the contrary, imperatorin (IPT) as compared to IIPT did not alter the expression of CXCR4. IIPT down-regulated the protein levels and RNA levels of mesenchymal markers, twist, snail, and enhanced the levels of different epithelial markers. IIPT also inhibited cell migration, invasion, and proliferation. Furthermore, IIPT negatively regulated constitutive NF-κB activation and inhibited the translocation of phospho-p65 and p65 into the nuclei. IIPT can potentially function as a novel anti-metastatic agent by inhibiting EMT and metastasis process via inhibition of NF-κB activation and CXCR4 expression in colorectal and hepatocellular carcinoma cells.

ADAR3 activates NF-κB signaling and promotes glioblastoma cell resistance to temozolomide

The RNA binding protein ADAR3 is expressed exclusively in the brain and reported to have elevated expression in tumors of patients suffering from glioblastoma compared to adjacent brain tissue. Yet, other studies have indicated that glioblastoma tumors exhibit hemizygous deletions of the genomic region encompassing ADAR3 (10p15.3). As the molecular and cellular consequences of altered ADAR3 expression are largely unknown, here we directly examined the impacts of elevated ADAR3 in a glioblastoma cell line model. Transcriptome-wide sequencing revealed 641 differentially expressed genes between control and ADAR3-expressing U87-MG glioblastoma cells. A vast majority of these genes belong to pathways involved in glioblastoma progression and are regulated by NF-κB signaling. Biochemical and molecular analysis indicated that ADAR3-expressing U87-MG cells exhibit increased NF-κB activation, and treatment with an NF-κB inhibitor abrogated the impacts of ADAR3 on gene expression. Similarly, we found that increased cell survival of ADAR3-expressing cells to temozolomide, the preferred chemotherapeutic for glioblastoma, was due to increased NF-κB activity. Aberrant constitutive NF-κB activation is a common event in glioblastoma and can impact both tumor progression and resistance to treatment. Our results suggest that elevated ADAR3 promotes NF-κB activation and a gene expression program that provides a growth advantage to glioblastoma cells.