B-cell Receptor Activation Research Articles

Immunophenotyping of Peripheral Blood Cells in Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib.

Chronic lymphocytic leukemia (CLL) is a B-cell-derived hematologic malignancy whose progression depends on active B-cell receptor (BCR) signaling. Despite the spectacular efficacy of Ibrutinib, an irreversible inhibitor of Bruton tyrosine kinase (BTK), resistance can develop in CLL patients, and alternative therapeutic strategies are therefore required. Cancer immunotherapy has revolutionized cancer care and may be an attractive approach in this context. We speculated that characterizing the immune responses of CLL patients may highlight putative immunotherapeutic targets. Here, we used high-dimensional spectral flow cytometry to compare the distribution and phenotype of non-B-cell immune populations in the circulating blood of CLL patients treated with Ibrutinib displaying a complete response or secondary progression. Although no drastic changes were observed in the composition of their immune subsets, the Ibrutinib-resistant group showed increased cycling of CD8+ T cells, leading to their overabundance at the expense of dendritic cells. In addition, the expression of 11 different surface checkpoints was similar regardless of response status. Together, this suggests that CLL relapse upon Ibrutinib treatment may not lead to major alterations in the peripheral immune response.

First-in-class inhibitor of HSP110 blocks BCR activation through SYK phosphorylation in diffuse large B-cell lymphoma.

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is driven by aberrant activation of the B-cell receptor (BCR) and the TLR/MyD88 signaling pathways. The heat-shock protein HSP110 is a candidate for their regulation as it stabilizes MyD88. However, its role in overall BCR signaling remains unknown. Here, we used first-in-class HSP110 inhibitors to address this question. HSP110 inhibitors decreased the survival of several ABC-DLBCL cell lines in vitro and in vivo, and reduced the phosphorylation of BCR signaling kinases, including BTK and SYK. We identified an interaction between HSP110 and SYK and demonstrated that HSP110 promotes SYK phosphorylation. Finally, the combination of the HSP110 inhibitor with the PI3K inhibitor copanlisib decreases SYK/BTK and AKT phosphorylation synergistically, leading to suppression of tumor growth in cell line xenografts and strong reduction in patient-derived xenografts. In conclusion, by regulating the BCR/TLR signaling pathway, HSP110 inhibitors are potential drug candidates for ABC-DLBCL patients.

E41K mutation activates Bruton’s tyrosine kinase by stabilizing an inositol hexakisphosphate-dependent invisible dimer

Bruton’s tyrosine kinase (BTK) regulates diverse cellular signaling of the innate and adaptive immune system in response to microbial pathogens. Downregulation or constitutive activation of BTK is reported in patients with autoimmune diseases or various B-cell leukemias. BTK is a multidomain protein tyrosine kinase that adopts an Src-like autoinhibited conformation maintained by the interaction between the kinase and PH-TH domains. The PH-TH domain plays a central role in regulating BTK function. BTK is activated by binding to PIP3 at the plasma membrane upon stimulation by the B-cell receptor (BCR). The PIP3 binding allows dimerization of the PH-TH domain and subsequent transphosphorylation of the activation loop. Alternatively, a recent study shows that the multivalent T-cell-independent (TI) antigen induces BCR response by activating BTK independently of PIP3 binding. It was proposed that a transiently stable IP6-dependent PH-TH dimer may activate BTK during BCR activation by the TI antigens. However, no IP6-dependent PH-TH dimer has been identified yet. Here, we investigated a constitutively active PH-TH mutant (E41K) to determine if the elusive IP6-dependent PH-TH dimer exists. We showed that the constitutively active E41K mutation activates BTK by stabilizing the IP6-dependent PH-TH dimer. We observed that a downregulating mutation in the PH-TH domain (R28H) linked to X-linked agammaglobulinemia impairs BTK activation at the membrane and in the cytosol by preventing PH-TH dimerization. We conclude that the IP6 dynamically remodels the BTK active fraction between the membrane and cytoplasm. Stimulating with IP6 increases the cytosolic fraction of the activated BTK.

Abstract 7586: TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL)

Abstract DLBCL originate from germinal center B cells (GCB). The ABC-DLBCL subtype, characterized by activation of B-cell receptor (BCR) signaling and downstream NF-kB pathway, carries a poor outcome. Up to 30% of ABC-DLBCL present TOXLOF mutations, suggesting that TOX function as a tumor suppressor. Our analysis of TOX expression along the germinal center B-cell differentiation program (naïve → GCB → plasma cells) showed that TOX expression paralleled NF-kB activity with highest levels in plasma cells (P<0.001, vs. either naïve and GCB). We thus propose that TOX acts as a transcriptional gatekeeper in GCB cells to prevent early activation of NF-kB thus enabling differentiation into plasma cells while restraining lymphomagenesis. We generated a conditional GCB TOX KO mouse model, induced the GC reaction with a T-cell antigen and found that TOX KO (vs. TOX WT, 10 mice per condition) had impaired differentiation of plasma cells (p<0.001) and hyperproliferative Ki67+ GCB cells (p<0.05). We then generated (by CRISPR) an isogenic TOXLOF ABC-DLBCL cell line (TMD8) and found upregulation of genes in the NF-kB pathway (q-value <0.05, vs. TMD8). Furthermore, TMD8-TOXLOF cells showed increased secretion of autocrine cytokines IL6 and IL10, and higher STAT3 phosphorylation (p<0.05 for all comparisons, vs. TMD8-TOXWT cells) all compatible with NF-kB activation. Tumor microenvironmental cues can hyperactivate BCR and downstream NF-kB in ABC-DLBCL. We thus implanted isogenic TMD8 cells in mice (n = 10) and determined pathway activation by phospho-flow. We found massive activation of SYK, BTK, PLCG2 (from the BCR) and p65 (from NF-kB) in TMD8-TOXLOF vs. TMD8-TOXWT tumors (all p<0.01), and vs. these cell lines cultured in vitro (all p<0.01). To determine whether these features can be therapeutically exploited, we conducted a viability screening using a panel of 20 clinical-phase targeted therapies relevant to ABC-DLBCL. We found that BTK inhibitor acalabrutinib (BCR pathway) and MALT1 inhibitor JNJ67690246 (NF-kB pathway) were 2-3 fold more active in TMD8-TOXLOF vs. TMD8-TOXWT cells (all p<0.05). We then xenografted isogenic TMD8 cells (n = 10 mice per condition) and, when tumors reached 150 mm3, we randomized mice to vehicle vs. acalabrutinib 30 mg/kg/day by oral gavage (human dose equivalent). Acalabrutinib was significantly more active in TMD8-TOXLOF tumors (vs. TMD8-TOXWT, p=0.0008), which translated into significantly prolonged survival (p<0.05). This acalabrutinib effect was accompanied by significant reduction of BTK, SYK and PLCG2 phosphorylation in TMD8-TOXLOF tumors (p<0.0001, all comparisons). Thus, TOXLOF leads to hyperactivation of the BCR and NF-kB signaling pathways in ABC-DLBCL cells, rendering them hypersensitive to BCR-targeted therapies, delineating a potential mechanism-based therapeutic approach for ABC-DLBCL TOXLOF patients. Citation Format: Eloisi Caldas Lopes, Maria Revuelta, Rossella Marullo, Leandro Cerchietti. TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7586.

Abstract 649: SOX11 regulates BCR signaling via the PAX5/CD19 axis in MCL

Abstract Background: Mantle cell lymphoma (MCL) is an incurable lymphoid neoplasm, comprising about 6% of all non-Hodgkin lymphoma (NHL). The transcription factor SOX11 is overexpressed in the majority of MCL patients and linked to a more aggressive disease course and poorer clinical outcomes. In MCL, naïve B cells experience continuous proliferation because of the dysregulation of the B-cell receptor (BCR) pathway. Our previously published data (Kuo et. al., Blood 2018) identified that SOX11 aberrantly upregulated BCR signaling in transgenic mouse models. The current study describes the exact mechanism by which SOX11 regulates the BCR pathway using a combination of functional and pharmacological approaches. Methods: To investigate the alterations in signaling pathways regulated by SOX11, we utilized a small molecule SOX11 inhibitor (Jatiani et. al., CCR 2021), performed CRISPR/CAS9 knockout of SOX11, 3X FLAG TAG overexpression, as well as conducted western blot and flow-cytometry experiments. Results: Our prior published results (Kuo et. al., Oncogene 2015), as well as others (Vegliante et. al., Blood 2013), have shown that PAX5 is a direct target of SOX11. We confirmed PAX5 downregulation after SOX11 depletion in three human MCL cell lines (JEKO, MINO, Z138) using CRISPR-CAS9. Decrease of PAX5 expression was associated with reduction in CD19 expression and a concurrent reduction in phospho-BTK (Bruton tyrosine kinase). Conversely, overexpression of SOX11 could rescue PAX5/CD19 expression and restore p-BTK levels. Furthermore, the treatment of SOX11 expressing cells with small molecule SOX11-specific inhibitors resulted in a reduction in PAX5, CD19 and BCR activation (p-BTK). Conclusion: Taken together, these findings delineate PAX5 as the direct target and PAX5/CD19 axis as the downstream pathway by which SOX11 regulates B-cell receptor (BCR) signaling in MCL. Notably, our treatment using a SOX11 inhibitor successfully replicated the effects observed with genetic knockdown. In conclusion, our findings provide valuable insights into the molecular mechanisms by which SOX11 contributes to MCL pathogenesis. The further development of pharmacological inhibitors targeting SOX11 holds promise as a beneficial therapeutic strategy for MCL patients, as these will block signaling upstream of current resistance mechanisms. Citation Format: Rudra Prasad Dutta, Heng-Huan Lee, Violetta V Leshchenko, Ravi Prakash Shukla, Yang Liu, Jian Jin, Michael Wang, Samir Parekh. SOX11 regulates BCR signaling via the PAX5/CD19 axis in MCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 649.

Abstract 5322: Discovery of CRD1601, a potent and selective HPK1 inhibitor with robust in vivo anti-cancer activity

Abstract Background: Haematopoietic Progenitor Kinase (HPK-1, MAP4K1) is a member of the STE20 family of serine/threonine kinases, expressed predominantly in hematopoietic cells. HPK-1 functions as a negative regulator of T-cell and B-cell receptor activation by triggering proteasomal degradation and disrupting signalosome complexes downstream of TCR and BCR. HPK1 inhibition is a novel IO strategy that could combine with existing chemotherapies and immunotherapies. Methods: Curadev used a pharmacophore based approach to discover novel small molecule HPK-1 inhibitors using in vitro enzymatic and phenotypic cellular screens using primary cells. CRD1601, the clinical candidate was selected based on its ability to enhance human T-cell proliferation through the induction of IL2 and IFNγ. Syngeneic tumor models were used to assess anti-tumor activity of selected compounds as a monotherapy or combination therapy. Results: CRD1601 is a potent HPK1 inhibitor with a single digit nanomolar IC50 value in enzymatic assay and potent cellular activity in the SLP76 assay. A single dose of CRD1601 in mice treated with an anti-CD3 antibody caused systemic enhancement of pro-inflammatory cytokines such as IL2, IFN gamma and reduced pSLP76 levels in the spleen. Further, CRD1601 reversed the immunosuppressive effect of PGE2 and NECA. CRD1601 demonstrated significant single agent activity in murine tumor models while also synergizing with chemotherapy and immunotherapy. Conclusions: Inhibition of HPK-1 is a promising therapeutic modality that could augment the effects of existing anti-cancer treatments including immunotherapy. CRD1601 is a potent and selective HPK-1 inhibitor with favorable drug like properties that has been selected from a rich portfolio of active compounds and shows promising in vivo activity in tumor models. Citation Format: Monali Banerjee, Ritesh Shrivastava, David Pryde, Sandip Middya, Sabyasachi Debnath, Anindita Middya, Dharmendra Yadav, Nidhi Rawat, Rajib Ghosh, Manisha Padaliya, Sourav Basu, Arjun Surya. Discovery of CRD1601, a potent and selective HPK1 inhibitor with robust in vivo anti-cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5322.

Antigen-independent, autonomous B cell receptor signaling drives activated B cell DLBCL.

Diffuse large B cell lymphoma of activated B cell type (ABC-DLBCL), a major cell-of-origin DLBCL subtype, is characterized by chronic active B cell receptor (BCR) signaling and NF-κB activation, which can be explained by activating mutations of the BCR signaling cascade in a minority of cases. We demonstrate that autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia (CLL), can explain chronic active BCR signaling in ABC-DLBCL. 13 of 18 tested DLBCL-derived BCR, including 12 cases selected for expression of IgM, induced spontaneous calcium flux and increased phosphorylation of the BCR signaling cascade in murine triple knockout pre-B cells without antigenic stimulation or external BCR crosslinking. Autonomous BCR signaling was associated with IgM isotype, dependent on somatic BCR mutations and individual HCDR3 sequences, and largely restricted to non-GCB DLBCL. Autonomous BCR signaling represents a novel immunological oncogenic driver mechanism in DLBCL originating from individual BCR sequences and adds a new dimension to currently proposed genetics- and transcriptomics-based DLBCL classifications.

B-cell receptor signaling activity identifies patients with mantle cell lymphoma at higher risk of progression

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.

Membrane-bound CD95 ligand modulates CD19-mediated B cell receptor signaling and EBV activation.

Post-transplant lymphoproliferative disorders (PTLDs) are associated with Epstein-Barr virus (EBV) infection in transplant recipients. Most of lymphoblastoid cell lines (LCLs) derived from EBV-immortalized B cells or PTLDs are sensitive to CD95-mediated apoptosis and cytotoxic T cell (CTL) killing. CD95 ligand (CD95L) exists as a transmembrane ligand (mCD95L) or a soluble form (sCD95L). Using recombinant mCD95L and sCD95L, we observed that sCD95L does not affect LCLs. While high expression of mCD95L in CTLs promotes apoptosis of LCLs, low expression induces clathrin-dependent CD19 internalization, caspase-dependent CD19 cleavage, and proteasomal/lysosomal-dependent CD19 degradation. The CD95L/CD95-mediated CD19 degradation impairs B cell receptor (BCR) signaling and inhibits BCR-mediated EBV activation. Interestingly, although inhibition of the caspase activity restores CD19 expression and CD19-mediated BCR activation, it fails to rescue BCR-mediated EBV lytic gene expression. EBV-specific CTLs engineered to overexpress mCD95L exhibit a stronger killing activity against LCLs. This study highlights that engineering EBV-specific CTLs to express a higher level of mCD95L could represent an attractive therapeutic approach to improve T cell immunotherapy for PTLDs.

ZAP-70 augments tonic B-cell receptor and CCR7 signaling in IGHV–unmutated chronic lymphocytic leukemia

AbstractExpression 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-3β. 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.

CD5 Gene Signature Identifies Diffuse Large B-Cell Lymphomas Sensitive to Bruton's Tyrosine Kinase Inhibition.

A genetic classifier termed LymphGen accurately identifies diffuse large B-cell lymphoma (DLBCL) subtypes vulnerable to Bruton's tyrosine kinase inhibitors (BTKis), but is challenging to implement in the clinic and fails to capture all DLBCLs that benefit from BTKi-based therapy. Here, we developed a novel CD5 gene expression signature as a biomarker of response to BTKi-based therapy in DLBCL. CD5 immunohistochemistry (IHC) was performed on 404 DLBCLs to identify CD5 IHC+ and CD5 IHC- cases, which were subsequently characterized at the molecular level through mutational and transcriptional analyses. A 60-gene CD5 gene expression signature (CD5sig) was constructed using genes differentially expressed between CD5 IHC+ and CD5 IHC- non-germinal center B-cell-like (non-GCB DLBCL) DLBCLs. This CD5sig was applied to external DLBCL data sets, including pretreatment biopsies from patients enrolled in the PHOENIX study (n = 584) to define the extent to which the CD5sig could identify non-GCB DLBCLs that benefited from the addition of ibrutinib to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). CD5 expression was observed in 12% of non-GCB DLBCLs. CD5+ DLBCLs displayed transcriptional features of B-cell receptor (BCR) activation and were enriched for BCR-activating mutations known to correlate with BTKi sensitivity. However, most CD5+ DLBCLs lacked canonical BCR-activating mutations or were LymphGen-unclassifiable (LymphGen-Other). The CD5sig recapitulated these findings in multiple independent data sets, indicating its utility in identifying DLBCLs with genetic and nongenetic bases for BCR dependence. Supporting this notion, CD5sig+ DLBCLs derived a selective survival advantage from the addition of ibrutinib to R-CHOP in the PHOENIX study, independent of LymphGen classification. CD5sig is a useful biomarker to identify DLBCLs vulnerable to BTKi-based therapies and complements current biomarker approaches by identifying DLBCLs with genetic and nongenetic bases for BTKi sensitivity.

Sending positive signals and good (calcium) vibes.

In this issue of JEM, Yada et al. (https://doi.org/10.1084/jem.20222178) demonstrate that effective antibody affinity selection in germinal centers relies on the store-operated calcium entry (SOCE) component of the B cell receptor (BCR) signaling network. Therefore, active BCR signaling is as relevant to positive selection as the function of BCRs as endocytic receptors, answering a question that had puzzled experts for a while. These findings transform our understanding of the mechanisms supporting adaptive immune responses (to vaccines, for example) and have important implications for interpreting the genomics and pathogenesis of germinal center-derived B cell lymphomas.

A CD5 Gene Signature Identifies Diffuse Large B-Cell Lymphomas Sensitive to Brutonʼs Tyrosine Kinase Inhibition

Introduction: Diffuse large B-cell lymphomas (DLBCLs) with a non-germinal center B cell-like (non-GCB) cell-of-origin are frequently driven by genetic alterations that culminate in constitutive B-cell receptor (BCR) signaling, which has inspired the exploration of Bruton's tyrosine kinase inhibitors (BTKi) in these lymphomas. However, the phase III PHOENIX study that randomized untreated, non-GCB DLBCL patients to R-CHOP plus placebo or ibrutinib failed to meet its primary endpoint of event-free survival (Younes et al. 2019), which suggests that cell-of-origin alone is an insufficient biomarker to predict BTKi sensitivity in DLBCL. More recently, a DLBCL genetic classifier termed LymphGen has identified distinct subtypes (MCD and N1) of non-GCB DLBCL that benefit from the addition of BTKi to R-CHOP (Wilson et al. 2021). However, genetic classifiers are complex and difficult to implement in routine clinical settings and may fail to capture all DLBCLs that benefit from BTKi. Therefore, we sought to identify a straightforward biomarker of BTKi responsiveness in DLBCL with greater precision than cell-of-origin but with broader inclusivity than current genomic platforms, such as LymphGen. We hypothesized that CD5 - a surrogate marker of BCR activation - may effectively identify BCR-driven, non-GCB DLBCLs that are sensitive to BTKi therapy, and evaluated the extent to which CD5 protein expression and a transcriptionally defined CD5 gene signature could accurately identify BCR-activated DLBCLs with potential susceptibility to BTKi-based therapies. Methods: CD5 immunohistochemistry (IHC) was performed on a cohort of 406 diagnostic DLBCL samples, which were considered CD5+ if >=30% of lymphoma cells exhibited unequivocal membranous staining. A majority of DLBCL samples had available RNA-sequencing and targeted mutational sequencing data. A comparison of differentially expressed genes between CD5+ and CD5- DLBCLs was performed in order to construct a 60-gene CD5 signature (CD5sig), which was applied to large genomic DLBCL datasets, including pre-treatment biopsies from patients enrolled on PHOENIX (n = 584) to evaluate the utility of the CD5sig in identifying DLBCLs that benefitted from the addition of ibrutinib to R-CHOP. Results: Twenty-six of 406 DLBCLs were identified as CD5+ by IHC (6% of all DLBCLs; 12% of non-GCB DLBCLs). CD5 IHC+ DLBCLs were majority non-GCB cell-of-origin and were associated with inferior progression-free survival (PFS) to R-CHOP (50% 3-year PFS), compared with CD5 IHC- DLBCLs, consistent with previous reports. Gene set enrichment analysis revealed that CD5 IHC+ DLBCLs exhibited transcriptional features of BCR activation, and mutational analysis demonstrated that CD5 IHC+ DLBCLs were enriched for CD79B BCR-activating mutations known to correlate with BTKi sensitivity. Many CD5 IHC+ DLBCLs, however, lacked canonical BCR-activating mutations or were classified as “Other” by LymphGen. A CD5 gene signature (CD5sig; Figure 1A) was developed that recapitulated these findings in independent DLBCL datasets (NCI, Duke), where ~13% of non-GCB DLBCLs were classified as CD5sig+. Together, these results suggest that CD5 signature expression captures DLBCLs with both a genetic and non-genetic basis for BCR dependence. Supporting this notion, CD5sig+ DLBCL patients (< 60 years) derived a selective and striking event-free and overall survival advantage from the addition of ibrutinib to R-CHOP in the PHOENIX study ( Figure 1B), independent of LymphGen classification. Conclusions: We demonstrate that CD5 IHC and a novel CD5 gene signature identify high-risk, BCR-driven DLBCLs. Importantly, the CD5 signature also identifies DLBCL patients with a selective survival advantage to BTK inhibitor-based therapy, independent of LymphGen classification. In conclusion, the CD5 signature expands upon LymphGen classification as a biomarker of BTKi response by accurately identifying DLBCLs with both genetic and non-genetic bases for BTKi response. The CD5 signature and/or CD5 IHC should be prospectively evaluated in BTKi-based clinical trials for non-GCB DLBCLs.

The DNA Sensor AIM2 Determines B Cell Fate after BCR Activation in Chronic Graft-Vs-Host Disease

Prevention and treatment of chronic graft-vs-host disease (cGVHD), a debilitating late immune toxicity in patients after allogeneic hematopoietic stem cell transplantation (allo-HCT), who are otherwise cured of hematological malignancies, remains a major challenge. T cells incite and promote cGVHD through coordinated T-B cell responses. Alloantibody production ( Srinivasan et al, Blood 2012, Jin et al, Blood 2016) and aberrant B Cell Receptor (BCR) signaling ( Allen et al, Blood 2014, Flynn et al, Blood 2015) play fundamental roles in the pathology of cGVHD. B cell-intrinsic pathways in cGVHD remain incompletely understood, limiting our therapeutic capacity for patients. In a single cell RNA sequencing study, we found that transcript levels of the DNA sensor Absent in Melanoma 2 (AIM2) were significantly increased in CD27+ BCR-activated B cell clusters in circulating as well as in tissue-resident cGVHD B cells ( Poe et al, JCI Insight 2023). After confirming AIM2 expression is restricted to the nucleus, we also previously showed that AIM2-expressing B cells were augmented after BCR and interferon-gamma (IFNγ) and/or Toll-like receptor (TLR)-7 or 9 stimulation ( Visentin J, Blood 2022;140 Supp 1), suggesting a context-dependent role for AIM2 after allo-HCT. Our new findings now afford the hypothesis that an AIM2-BCR signaling axis determines B cell fate after allo-HCT. To address this hypothesis, we first examined whether AIM2 modulates the promotion of antibody-secreting cells (ASCs) after BCR activation using a hapten-carrier immunization model. We compared numbers of hapten-specific ASCs from immunized WT and AIM2-KO mouse splenocytes using enzyme-linked immunosorbent spot (ELISPOT). We compared numbers of ASCs on day 3 post-immunization, when early germinal center (GC) formation is known to occur. We found that hapten-specific IgM ASCs were significantly increased in WT splenocytes compared to AIM2-KO (p=0.002). By contrast, when we looked at day 8, when mature GC formation and rapid ASC proliferation is known to occur, we found that AIM2-KO immunized mice had significantly lower numbers of hapten-specific IgM (p=0.005) and IgG (p=0.004) ASCs in AIM2-expressing mice (Figure 1a). Our results suggest a role for AIM2 in regulating the survival of short-lived plasmablast-like ASCs after high affinity antigen encounter and maturation of germinal centers. To further investigate whether AIM2 affects expansion of GC-derived (GL7+CD95+) ASCs, we stimulated B cells purified from wildtype (WT) or global AIM2-deficient (AIM2-KO) mice in vitro with lipopolysaccharide (LPS) and IL-4 +/- BCR stimulation. We found that AIM2-deficient B cells, especially GC-like cell populations, expanded significantly in an anti-IgM concentration-dependent manner up to 12-fold (p=0.03) after 72 hr (Figure 1b). By contrast, B cell expansion was constrained in AIM2-sufficient B cells. This expansion was unlikely to be due to differences in proliferation alone, as we did not detect differential Ki67 expression in stimulated B cells with or without AIM2. Together, these data suggest a role for AIM2 in modulating BCR signaling. Finally, we examined the role of AIM2 in BCR-mediated cell death after chronic antigen exposure using our previously described bone marrow transplant (BMT) model, where recipients of bone marrow and splenocytes (BM+Spl) exhibit cGVHD manifestations ( Poe et al, JCI Insight 2018, Jia et al, Blood 2021). We have previously generated C57BL/6J mice with conditional deficiency of AIM2 only in B cells (AIM2 fl/flMb1 Cre/+, CKO), and used these in our BMT model as donors. At day 58 post BMT, we performed ex vivo stimulation of splenic B cells from BMT recipients. While B cells from CKO BM+Spl recipients expanded ex vivo in an anti-IgM concentration-dependent manner, B cells from AIM2-sufficient BM+Spl recipients demonstrated limited responsiveness ex vivo to BCR stimulation. This extends our in vitro observations by suggesting a role for AIM2 in B cell tolerance after chronic antigen exposure. Ongoing experiments will determine if B cell-intrinsic AIM2 contributes to cGVHD pathogenesis. Thus far, our data reveal a novel role for AIM2 as a B cell survival checkpoint after BCR activation. Further delineation of context-dependent and synergistic AIM2-BCR signaling after allo-HCT will lead to strategies that block the development of cGVHD without adversely affecting anti-tumor activity.

The Unique BCR Inhibiting Properties of BMS-986205 in Chronic Lymphocytic Cells

B cell receptor (BCR) signaling is recognised as a central pathway in the pathogenesis of chronic lymphocytic leukaemia (CLL), the most common leukemia in the western world. In CLL, the BCR pathway is activated by antigens in the tissue microenvironment to promote the maintenance and expansion of leukemic cells. The impressive inhibition of clonal expansion of CLL cells and the disease control achieved with tyrosine kinase inhibitors that block BCR signaling provided further evidence of the role of BCR in CLL. Indoleamine 2,3-dioxygenase 1 (IDO1) is the inducible and rate-limiting enzyme involved in the catabolism of tryptophan to kynurenine. It was discovered as a modulator of the innate immune response during infection. Subsequent discoveries have implicated IDO1 as a player in acquired immune tolerance. IDO1 activity has been identified in several types of cancer cells as well as in their tumor-surrounding microenvironment. We have previously investigated and characterized the impact of IDO1 expression in CLL. Our results show that CLL microenvironmental stimuli positively modulate IDO1 expression in leukemic cells and that increased IDO1 activity leads to sustained survival of malignant lymphocytes and impaired drug sensitivity through the autocrine/paracrine activation of the aryl hydrocarbon receptor by kynurenine (Atene CG et al. 2022). To assess the functional and clinical significance of IDO1 enzymatic activity in CLL, we used several inhibitors that are currently in late-stage clinical trials. Using BMS-986205 (BMS), an irreversible inhibitor of IDO1 with the best cell-based potency, in primary patient cells, we observed not only a reduction in kynurenine production due to enzymatic inhibition of IDO1, but also lower levels of IDO1 RNA and protein following BCR stimulation with α-IgM. As we have previously shown that BCR activation can induce IDO1 expression, we treated CLL cells with increasing doses of BMS for 3 hours and then added α-IgM to the culture. BMS is able to counteract the pro-survival effect of BCR triggering, leading to significant apoptosis of CLL cells, while the viability of CLL cells in the absence of external stimuli was not significantly modulated. Focusing on the complex signaling cascade activated by BCR engagement, we observed that BMS impaired the phosphorylation of upstream kinases such as SYK and BTK, but also CD79a, CD19 and BLNK, in addition to downstream effectors such as AKT. We therefore investigated which factors that negatively regulate the BCR pathway are modulated by BMS treatment. By analyzing both mRNA and protein levels, we measured a significant induction of the phosphatases PTPN6, PTPN22 and SHIP1. Since BMS also showed a downregulation of total SYK expression, we are analyzing the expression of the proto-oncogene CBL, an E3 ubiquitin ligase known to be involved in the negative regulation of SYK. Further studies should be undertaken to determine whether, in addition to its role as an IDO1 inhibitor, BMS may also have the ability to indirectly modulate and disrupt BCR signaling. Efforts are underway to identify the proteome-wide direct target of BMS in CLL cells. In conclusion, our limited and preliminary data suggest that BMS could be considered as a potential therapeutic strategy to target CLL cells with a double hit: by blocking the pro-survival effect of BCR signaling, one of the driving forces for CLL cell survival and proliferation, and by inhibiting IDO1/kynurenine action to restore sensitivity to key leukemia treatments.

Mechanistic Elucidation of the Tumor-Promoting Role of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 in B-Cell Receptor Signaling in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a highly aggressive and incurable type of B-cell non-Hodgkin's lymphoma. B cell receptor (BCR) signaling plays an important role in the pathogenesis of MCL; however, the underlying mechanisms are unknown. Our genome-wide screens identified that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1/CC1) is a central component of BCR signaling and is essential for MCL survival in vitro and in vivo. Among the twelve alternatively spliced human CC1 isoforms, we found that CC1-4L, the longest CC1 isoform is overexpressed in MCL. Structurally, it has the N-terminal domain and three type 2 constant ectodomains, a transmembrane sequence and a signaling cytoplasmic domain with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs or Y493/520). These ITIMs of CC1 can inhibit signals in T cells, NK cells, neutrophils and granulocytes. However, its role in B cells and BCR signaling is not clear. In this study, we have elucidated a novel mechanistic function of CC1 in promoting oncogenic BCR signaling in MCL. First, we studied the role of CC1 in lipid raft formation, which is one of the early events during BCR signaling activation. To study this, we used three panels of MCL lines, Jeko-1-NTC (non-template control), Jeko-1-gCC1 (CC1-knockout) and Jeko-1-gCC1-4L (CC1-knockout restored with CC1-4L isoform). Using high-resolution confocal microscopy, we observed that CC1-expressing Jeko-1 MCL cell line showed significantly increased lipid raft formation when compared to CC1-knockout cells. However, CC1-deficient cells restored with the CC1-4L isoform exhibited increased lipid raft assembly. Further, we also observed that CC1-expressing MCL lines showed increased cytoskeletal assembly, which was observed via increased F-actin and filamin A staining in CC1-expressing MCL lines when compared to CC1-knockout cells. Second, we analyzed the phosphorylation levels of proteins involved in the BCR signaling cascade in a panel of genetically engineered MCL lines, i.e., Jeko-1-NTC, Jeko-1-gCC1, Jeko-1-gCC1-4L, Jeko-1-gCC1-4S (CC1-knockout restored with CC1-4S or short cytoplasmic tail isoform) and Jeko-1-gCC1-mutants (CC1-knockout cells restored with N-terminal domain deletion mutant and ITIM-mutant or YY/FF 493/520). We observed that Jeko-1-NTC and Jeko-1-gCC1-4L cell lines showed significantly increased phosphorylation levels of BCR signaling components such as phospho-SRC (Y416), phospho-SYK (Y319), phospho-AKT (S473) and phospho-BTK (Y223) when compared to Jeko-1-gCC1, Jeko-1-gCC1-4S and Jeko-1-gCC1 mutant MCL lines. This shows that CC1 positively regulates or activates oncogenic BCR signaling in MCL and the N-terminal domain and cytoplasmic tail are required for this function. Next, we analyzed whether CC1 could interact with the BCR signaling components such as CD79B, LYN and SYK using immunoprecipitation (IP) and proximity ligation assay (PLA) in MCL lines. Further, we analyzed the kinetics of BCR activation in order to infer the role of SHP-1 binding to the ITIMs of CC1. Our IP and PLA experiments demonstrated that in Jeko-1 and Mino MCL lines, at the peak (i.e., at 5 and 15 min) of BCR activation, there was significantly increased interaction between CC1 and the BCR-pathway components (CD79B, LYN and SYK), and this interaction subsided by 30 min of IgM activation. However, in the case of CC1 and SHP-1, the interaction was stable throughout the time points and increased by 30 min of IgM activation. These interactions were disrupted in the CC1-N-terminal domain deletion and ITIM mutant MCL lines when compared to the CC-4L expressing MCL line. Finally, we analyzed the interaction between CD79B and SYK in CC1-4L, CC1-ITIM mutant and N-domain deletion mutant expressing MCL lines. Using PLA, we observed increased interaction between CD79B and SYK in CC1-4L expressing cell line compared to other cell lines indicating that functional CC1 is required for the recruitment of SYK to CD79B. In summary, CC1 stabilizes lipid rafts following BCR engagement by anchoring to F-actin and Filamin A. CC1 recruits and activates LYN and SYK at the BCR complex at the peak of BCR activation and subsequently recruits SHP-1 for signal downregulation. These activities of CC1 require intact ITIMs and the N-terminal ligand-binding domain. Hence, CC1 drives oncogenic BCR signaling in MCL and forms an important candidate for MCL immunotherapy.

Evidence of Defective Cell-Autonomous Signalling By B Cell Receptors in CLL Stereotyped Subset #1

B cell receptor (BCR)-mediated signalling is central in the pathogenesis of chronic lymphocytic leukaemia (CLL), and represents a target for pharmacological intervention. Notably, so far characterized CLL-derived BCRs are capable of triggering cell-autonomous intracellular signalling through homotypic interactions without requirement for exogenous antigens. Approximately 41% of all CLL cases can be classified in subsets based on homologous, otherwise known as stereotyped, BCRs expressed by the malignant clones, sharing remarkably similar sequences in the VH CDR3 region and highly restricted immunoglobulin heavy and/or light variable genes. Since cases within a subset display clinicobiological consistency in contrast with the pronounced heterogeneity of CLL at large, analysis of the mechanism of BCR-mediated signalling can unveil correlations between molecular details of BCR activation and the course of the disease. Our present study focused on BCRs of stereotyped subset #1, the second largest stereotyped subset, known for a particularly aggressive clinical course. Cases belonging to subset #1 express unmutated IGHV genes of phylogenetic clan I (namely, IGHV1, IGHV5 and IGHV7 subgroup genes) partnered by light chains encoded by the IGKV1-39/1D-39 gene. First, we used X-ray crystallography to determine the high-resolution crystal structures of the BCR Fab fragments from three subset #1 cases utilizing the IGHV1-2, IGHV1-3, and IGHV5-10 genes partnered with IGKV1-39/1D-39 to obtain information on the combining sites' structures and possible modes of intermolecular interactions that may mediate cell-autonomous signalling. Analysis of the experimental structures revealed that, despite the high sequence homology of the three proteins, the heavy and light chain CDR loops in the three BCR Fabs are arranged differently to shape binding cavities with distinct dimensions and polarity, thus suggesting that distinct antigens may be the physiological target of each subgroup. Unlike BCRs from other subsets previously characterised, the intermolecular contacts between the subset #1 Fab molecules in the three crystals involve diverse residues that are not indicative of a shared homotypic interaction. In addition, the surfaces involved in the contacts are small and the computed energies for the protein-protein contacts observed are suggestive at best of very weak interactions. In accordance to this, when we used analytical ultracentrifugation along with chromatographic and calorimetric methods to detect BCR Fab oligomerization in solution, no evidence of self-association was obtained, thus indicating that the receptors in solution either do not interact, or are characterized by self-dissociation constants that exceed the sensitivity limit of the techniques employed. To assess whether the cellular environment could promote self-association and signalling activity of the full-length BCR, we expressed subset #1 BCRs in the model triple knockout (TKO) pre-B-cell line, designed to measure Ca 2+ ion influx using cytofluorimetry. All subset #1 BCRs tested were unable to mobilize autonomously the Ca 2+ ions, despite being fully expressed on the cell surface and capable of signalling upon stimulation with anti-BCR antibody as a proxy for exogenous ligand. This result is in sharp contrast with the behaviour of all other CLL-derived BCRs so far characterised in this system that could activate a cell-autonomous signal as evidenced from a persistent Ca 2+ ion influx in the transduced cells without requiring external stimuli. Hence, subset #1 BCRs differ from the so far characterized BCRs from CLL cases belonging to other stereotyped subsets, including clinically aggressive subset #2 and its satellite subset #169 as well as the clinically indolent stereotyped subset #4. In conclusion, our combined biochemical and cellular analysis demonstrates that a constitutively active cell-autonomous signalling may not be a unifying feature of all CLL clones as previously proposed, and that other mechanisms may be at the roots of the BCR-mediated signal required for leukemic cell proliferation and development of CLL.

Combined Inhibition of Protein Kinase CSNK2 and BET Proteins As a Novel Therapeutic Strategy for Mantle Cell Lymphoma

Currently available therapeutic approaches for Mantle Cell Lymphoma (MCL), including the Bruton Tyrosine Kinase (BTK) inhibitors ibrutinib, acalabrutinib and zanubrutinib, are not curative. Therefore, the discovery of novel molecules amenable of drug targeting is a research priority in the MCL field. Bromodomain-containing protein 4 (BRD4) is a member of the Bromodomain (B) and extra terminal (ET) (BET) protein family that regulate chromatin modifications and assembly of transcriptional complexes. Inhibition of BET by available compounds have shown promising anticancer effect. In MCL, BET inhibitors, such as INCB054329 or JQ-1, have been shown to increase apoptosis through downregulation of the AKT-mTOR, ERK, and other B Cell Receptor (BCR)-triggered cascades. We and others have previously demonstrated that the S/T kinase CSNK2 supports chronic BCR activation in MCL by upregulating the AKT/PI3K and NF-κB pathways. Inactivation of CSNK2 results in efficacious induction of MCL cell death. Notably, BRD4 activity is regulated by CSNK2 and double inhibition of BRD4 and CSNK2 has shown significant activity in other tumor models. The most effective and tested CSNK2 chemical inhibitor is CX4945 (silmitasertib), but very recently a novel compound, SGC-CK2, has been developed. No data are available on the effects of SGC-CK2 on MCL cells. In this work, we have evaluated the effects of the combined inhibition of CSNK2 and BET proteins on MCL cell growth and survival signaling pathways. CSNK2 inhibitors employed were CX4945 and SGC-CK2. CSNK2 gene silencing was achieved with the generation of anti- CSNK2 short hairpin-RNA IPTG-inducible MCL cell clones. BET inhibitors used were JQ-1 and INCB054329. Cell survival, apoptosis and proliferation were investigated by AnnexinV/PI staining and FACS analysis, by Western blot (WB) detection of PARP cleavage and Mcl1 expression and by other cell proliferation assays. The synergic action of BET and CK2 inhibitors was assessed by the Chou-Talalay combination index method. CK2 expression and survival-related signaling components were analyzed by WB, qRT-PCR and protein expression assays. CX4945 and the novel inhibitor SGC-CK2 displayed a potent anti-tumor activity on MCL cells as judged by the effective induction of apoptosis and proliferation arrest. A synergistic effect of CSNK2 and BET inhibitors was observed in all the MCL cell lines tested, (Jeko-1, Rec-1, including Granta-519 that are known to be much less sensitive to ibrutinib) and with all the inhibitors tested. The same results were obtained in the CSNK2 gene silencing models. Preliminary data showed that the cooperative cytotoxic effect of CK2 inhibition and BET inhibition on MCL apoptosis was observed also in MCL patient derived B cells, but not in healthy B lymphocytes form buffy coat. CSNK2 inactivation was associated with strong down-regulation of c-Myc and Mcl-1 proteins and with hampered activation of NF-κB and PI3K/AKT-dependent signaling. Surprisingly, BET protein inhibition caused increased Mcl-1 protein expression and augmented Ser 529 p65-NF-kB phosphorylation in Granta-519 and Rec-1 cells. Remarkably, CK2 inactivation led to a robust reduction of the BET inhibitor-induced increase of Mcl1, and NF-kB Ser 529 phosphorylation, thus counteracting BET-inhibitors-evoked compensatory pathways that could favor apoptosis resistance. Therefore, combined CSNK2 and BET proteins inhibition could represent an innovative strategy for chemotherapy and BTKi-resistant MCL.

MS-553 Therapy Inhibits BCR Signaling and Increases Survival Dependence on BCL-2 in BTK-Inhibitor Resistant CLL

B-cell receptor (BCR) signaling pathway inhibitors and B-cell lymphoma-2 (BCL-2) antagonist venetoclax (Ven) have transformed the treatment of chronic lymphocytic leukemia (CLL), including in high-risk patients with 17p deletion and/or TP53 mutations and complex karyotype. Despite the durable remissions observed with inhibitors of Bruton's Tyrosine Kinase (BTKi), a significant proportion of patients develop resistance with acquired mutations in BTK and its downstream target phospholipase Cγ2 ( PLCG2). Patients who progress on BTKi including ibrutinib (ibr) often receive Ven but eventually relapse, underscoring an urgent need for new treatment strategies. Protein kinase C-β (PKC-ß) is a protein downstream of BCR signaling (BTK-PLCG2) and is essential for CLL cell survival and proliferation in vivo. Here we investigate MS-553, an inhibitor of PKC-ß, with a focus of combination strategies in CLL patient samples including relapsed/refractory CLL. We show that CLL cells in contact with stroma upregulate PKC-ßII, its immediate downstream effectors, pGSK and pERK as well as ß-catenin in CLL patients including those with mutant TP53 and 17p (n=8). Correspondingly, MS-553 (5µM, 48h) exposure reduced levels of p-PKC-ßII and its downstream effectors (p-ERK, p-GSK) in a dose dependent manner regardless of TP53 mutation in both CLL cells and those supported by NK.Tert (stromal cell line derived from bone marrow, BM, and mimics BM microenvironment). Pre-treatment (48h, 5µM MS-553) of stromal NK.Tert cells with MS-553 prevented the upregulation of PKC-ßII within adjacent CLL cells implicating the stroma as a major source of PKC-ß activation in CLL cells. We also tested whether MS-553 could inhibit PKC-ß signaling in CLL cells exposed to phorbol myristate acetate (PMA-400nM, 90 min), which simulates BCR activation. MS-553 (5µM, 48h) exposure caused downregulation of BTK effectors (pPKC-ßII, pERK, pGSK), BCL-2 family member proteins (MCL-1 and BCLxl), and ß-catenin (n=10). In parallel experiments, we used BH3 profiling to show that CLL cells' (n=8) exposure to MS-553 in vitro developed increased dependence on BCL-2 and BCLxl for survival (Bim, Bad, XXA1_y4ek, 5-fold± 2-fold increase) in both wild type (WT) and mutant (Mut) CLL cells, suggesting a mechanistic rationale for synergy with Ven. To confirm this, CLL cells were exposed to MS-553, Ven or a combination of MS-553 and Ven. ~3-fold and 9-fold synergistic increase in the mitochondrial dysfunction (measured by cytochrome C release, 12h, 5µM MS-553, 1nM Ven) was observed in stromal supported WT and Mut CLL respectively in MS-553 versus Ven+MS-553 treated group. Similarly, ~3-fold increase was observed in WT and Mut CLL alone group and more than 10-fold increase was found in stromal supported WT and Mut group in cell death determined as percent apoptotic index (measured by annexin V-PI staining, 18h, 5µM MS-553, 1nM Ven) in MS-553 versus Ven+MS-553 group respectively. Finally, we validated our preclinical findings in CLL samples obtained from four patients on MS-553 therapy (NCT03492125, Range = cycles 0-19, MS-553 28 days/cycle). All patients showed an increase in survival dependence towards BCL-2 and BCLxl by Cycle 3-6 of MS-553 therapy. This was accompanied by downregulation of ß-catenin, pPKC-ßII, pGSK, pERK along with BCLxl and MCL-1 after Cycle 10 or later of MS-553 therapy (data summary table1). Overall, our data provides clear evidence that MS-553 inhibits BCR signaling in vitro and in patients on MS-553 therapy. It also provides a rationale for combinations with Ven as a treatment partner based on its ability to increase survival dependence on BCL-2.

Phase 2 Response-Adapted Study of Ibrutinib with Temozolomide, Etoposide, Liposomal Doxorubicin, Dexamethasone, and Rituximab (TEDDI-R) for Secondary CNS Lymphoma

Background: Secondary CNS lymphomas (SCNSL) are aggressive and there are few treatment options. CNSLs often have underlying chronic active B-cell receptor signaling that responds to BTK inhibitors, but the response duration is short. TEDDI-R achieves durable remissions in primary DLBCL of the CNS (PCNSL), but the molecular profile of SCNSL tumors that are BTK-responsive is unknown. We present results from an ongoing response-adapted study of ibrutinib with TEDD-R in SCNSL [NCT03964090]. Methods: Pts with aggressive B-cell lymphomas with CNS +/- systemic involvement are eligible if age ≥18, PS<3, and adequate organ function. Pts are untreated with CNS involved or relapsed in the CNS after therapy. Prior BTKi is allowed. HIVis excluded. Baseline tests: brain MRI, PET/CT of body, bone marrow (BM), CSF flow cytometry, and fundoscopic exam. Pts receive isavuconazole as fungal prophylaxis throughout therapy. Pts first receive ibrutinib 560mg x 14d in a treatment window to determine ibrutinib-responsiveness. Pts with ≥20% reduction after ibrutinib receive TEDDI-R, and if <20% reduction receive TEDD-R (no ibrutinib). Chemotherapy is 4 cycles x21d as an outpatient and includes intrathecal chemotherapy. There is no maintenance therapy or planned consolidation. Response is assessed after cycles 2 and 4. Complete responses (CR) are confirmed with FDG-PET brain/body, CSF analysis, and BM. Surveillance scans are q3m for 1y, q4m x 1y, q6m x 1y, then annually. The primary endpoint is PFS. Results:50 pts enrolled between June 2019 and March 2023 with median age 63 (range 26-89), and 15 (30%) aged ≥70. 17 pts (34%) were female. 28 (56%) pts had DLBCL: 21 (42%) non-GCB, 5 (10%) GCB, 1 (2%) PMBL, and 1 (2%) unknown. 16 (32%) pts had HGBL: 10 (20%) with MYC/ BCL2-R and 6 (12%) with MYC/ BCL6-R. Two (4%) pts each had MCL and EBV-LPD, and 1 pt each had plasmablastic (2%) and Burkitt (2%). Five (10%) pts were treatment naïve and 45 (90%) were relapsed or refractory. 25 (50%) had synchronous CNS and peripheral disease, and 25 (50%) were isolated to the CNS. All relapsed or refractory pts had prior anthracycline, 29 (58%) prior HD-MTX, and 8 (18%) prior CAR-T. Forty-three pts completed the ibrutinib window and were evaluable for responsiveness; 25 (58%) were ibrutinib-responsive and 18 (42%) were ibrutinib-resistant (Figure 1). The overall response rate (ORR) to TEDDI-R (n=28) was 96%, including a CR rate of 71%. The overall response rate to TEDD-R (n=20) was 35%, with a CR rate of 25%. G3 or G4 neutropenia occurred in 43% and 31% of cycles, respectively, and febrile neutropenia occurred in 11%. G3 or G4 thrombocytopenia occurred in 31% and 12% of cycles, respectively, and G3 anemia occurred in 37%. G3 non-hematologic AEs (% pts) included UTI (20%), hypokalemia (22%), sepsis (14%), diarrhea (16%), hypotension (12%), fatigue (10%), anorexia (10%), adrenal insufficiency (10%), and atrial fibrillation (8%). Few G4 AEs were noted. No opportunistic infections (including Aspergillus) were observed. 14 (28%) pts had hand-foot syndrome. After a median follow-up of 24m, the 1-year PFS and OS of all patients were 37.8% and 59.9%, respectively. Pt deaths were due to disease progression (20 pts, 83%), pneumonia (3 pts, 13%), and a cardiac event (1 pt, 4%). The 1-year PFS and OS for pts with ibrutinib-responsive compared to ibrutinib-resistant tumors were 57.0% vs. 16.7% (p=0.0006) and 73.8% vs. 47.9% (p=0.06) (Figure 2). Most (86%) ibrutinib-responsive DLBCL/HGBL tumors were CD10-negative. CD10-negative DLBCL/HGBL pts had an ORR of 77% and a CR rate of 70%. The 1-year PFS and OS for pts with CD10-negative DLBCL/HGBL were 50.5% and 65.0%, respectively. There was no difference in PFS or OS in pts aged <70 versus ≥70 (p=0.87 and p=0.74, respectively). We performed comprehensive genomic profiling on 27 DLBCL/HGBL tumors according to the LymphGen algorithm. 80% (n=4) MCD, 75% (n=3) BN2, 50% (n=2) EZB, and 50% (n=7) unclassified tumors were responsive to ibrutinib. Conclusions: Pts with ibrutinib-responsive SCNSL achieve a high rate of CR to TEDDI-R, and preliminary data suggest these remissions are durable. Most ibrutinib-responsive tumors are CD10-negative and enriched for MCD and BN2. TEDDI-R can be delivered as an outpatient to pts of all ages.