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Diffuse large B-cell lymphoma (DLBCL) can be subdivided into activated B-cell like (ABC) and germinal center B-cell-like (GCB) DLCBL. Self-antigen engagement of B-cell receptors (BCRs) in ABC tumors induces their clustering, thereby initiating chronic active signaling and activation of NF-kB and PI3 kinase. Constitutive BCR signaling is essential in some GCB tumors but primarily activates PI3 kinase. We devised genome-wide CRISPR-Cas9 screens to identify regulators of IRF4, a direct transcriptional target of NF-kB and an indicator of proximal BCR signaling in ABC DLBCL. Unexpectedly, inactivation of N-linked protein glycosylation by the oligosaccharyltransferase-B (OST-B) complex reduced IRF4 expression. OST-B inhibition of BCR glycosylation reduced BCR clustering and internalization while promoting its association with CD22, which attenuated PI3 kinase and NF-kB activation. By directly interfering with proximal BCR signaling, OST-B inactivation killed models of ABC and GCB DLBCL, supporting the development of selective OST-B inhibitors for the treatment of these aggressive cancers.

Supplemental Table 7. Targeting epigenetic resistance to BTK inhibitor treatment in ABC DLBCL, relates to Fig7

Supplemental Table 1. Epigenetic Ibrutinib Resistance in ABC DLBCL lines, , relates to Fig. 1

Supplemental Information- Figures & Legends (Supplemental Tables loaded separately)

Supplemental Table 6. RAC2 protein interactions are a marker of epigenetic ibrutinib resistance

Supplemental Table 4. Altered dependencies in ibrutinib resistant ABC DLBCL, relates to Fig. 4

Supplementary Methods

Supplemental Table2. Tracking the evolution of ibrutinib resistance phenotypes, relates to Fig. 2

Oligo and Primer Tables

Supplemental Table 5. RAC2 as a mediator of epigenetic ibrutinib resistance, , relates to Fig5

The use of Bruton tyrosine kinase (BTK) inhibitors to block B-cell receptor (BCR)–dependent NF-κB activation in lymphoid malignancies has been a major clinical advance, yet acquired therapeutic resistance is a recurring problem. We modeled the development of resistance to the BTK inhibitor ibrutinib in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma, which relies on chronic active BCR signaling for survival. The primary mode of resistance was epigenetic, driven in part by the transcription factor TCF4. The resultant phenotypic shift altered BCR signaling such that the GTPase RAC2 substituted for BTK in the activation of phospholipase Cγ2, thereby sustaining NF-κB activity. The interaction of RAC2 with phospholipase Cγ2 was also increased in chronic lymphocytic leukemia cells from patients with persistent or progressive disease on BTK inhibitor treatment. We identified clinically available drugs that can treat epigenetic ibrutinib resistance, suggesting combination therapeutic strategies.Significance:In diffuse large B-cell lymphoma, we show that primary resistance to BTK inhibitors is due to epigenetic rather than genetic changes that circumvent the BTK blockade. We also observed this resistance mechanism in chronic lymphocytic leukemia, suggesting that epigenetic alterations may contribute more to BTK inhibitor resistance than currently thought.See related commentary by Pasqualucci, p. 555.This article is highlighted in the In This Issue feature, p. 549

Supplemental Table 3. Epigenetic retuning of oncogenic signaling in ibrutinib resistance, relates to Fig. 3

Summary: BCL10, a key activator of NF-κB downstream of oncogenic B-cell receptor signaling, is mutated in nearly 40% of the BN2/C1 genetic subtype of diffuse large B-cell lymphoma, but how these mutations function to augment signaling and their relevance to targeted precision medicine agents remains unclear. In this issue of Cancer Discovery, Xia and colleagues demonstrate distinct mechanisms of oncogenic signaling regulation and therapeutic vulnerabilities among different recurrent BCL10 mutations. See related article by Xia et al., p. 1922 (1).

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

Burkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.

The integrin lymphocyte function–associated antigen 1 (LFA-1) helps to coordinate the migration, adhesion, and activation of T cells through interactions with intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. LFA-1 is activated during the engagement of chemokine receptors and the T cell receptor (TCR) through inside-out signaling, a process that is partially mediated by phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). To evaluate potential roles of PI3K in LFA-1 activation, we designed a library of CRISPR/single guide RNAs targeting known and potential PIP 3 -binding proteins and screened for effects on the ability of primary mouse T cells to bind to ICAM-1. We identified multiple proteins that regulated the binding of LFA-1 to ICAM-1, including the Rap1 and Ras GTPase-activating protein RASA3. We found that RASA3 suppressed LFA-1 activation in T cells, that its expression was rapidly reduced upon T cell activation, and that its activity was inhibited by PI3K. Loss of RASA3 in T cells led to increased Rap1 activation, defective lymph node entry and egress, and impaired responses to T-dependent immunization in mice. Our results reveal a critical role for RASA3 in T cell migration, homeostasis, and function.

SUMMARYReference atlases, molecular and spatial maps of mammalian tissues, are critical resources for discovery efforts and translational research. Their utility is dependent on operationalizing the resulting data by identifying cell types, histological patterns, and predictive biomarkers underlying health and disease. The human lymph node (LN) offers a compelling use case because of its importance in immunity, structural and cellular diversity, and neoplastic involvement. One hematological malignancy, follicular lymphoma (FL), evolves from developmentally blocked germinal center B cells residing in and trafficking through these tissues. To promote survival and immune escape, tumor B cells undergo significant genetic changes and extensively remodel the lymphoid microenvironment. Here, we present an integrated portrait of healthy and FL LNs using multiple genomic and advanced imaging technologies. By leveraging the strengths of each platform, we identified several tumor-specific features and microenvironmental patterns enriched in individuals who experience early relapse, the most high-risk of FL patients.

Oncogenic mutations within the RAS pathway are common in multiple myeloma (MM), an incurable malignancy of plasma cells. However, the mechanisms of pathogenic RAS signaling in this disease remain enigmatic and difficult to inhibit therapeutically. We employed an unbiased proteogenomic approach to dissect RAS signaling in MM by combining genome-wide CRISPR-Cas9 screening with quantitative mass spectrometry focused on RAS biology. We discovered that mutant isoforms of RAS organized a signaling complex with the amino acid transporter, SLC3A2, and MTOR on endolysosomes, which directly activated mTORC1 by co-opting amino acid sensing pathways. MM tumors with high expression of mTORC1-dependent genes were more aggressive and enriched in RAS mutations, and we detected interactions between RAS and MTOR in MM patient tumors harboring mutant RAS isoforms. Inhibition of RAS-dependent mTORC1 activity synergized with MEK and ERK inhibitors to quench pathogenic RAS signaling in MM cells. This study redefines the RAS pathway in MM and provides a mechanistic and rational basis to target this novel mode of RAS signaling.

Diffuse large B-cell lymphoma (DLBCL) encompasses a group of aggressive B-cell non-Hodgkin lymphomas with striking genetic heterogeneity and variable clinical presentations. Among these is primary mediastinal B-cell lymphoma (PMBL), which has unique clinical and molecular features resembling Hodgkin lymphoma. Treatment of DLBCL is usually curative, but identifiable subsets at highest risk for treatment failure may benefit from intensified chemotherapy regimens and/or targeted agents added to frontline therapy. Recent comprehensive genomic analyses have identified distinct genetic subtypes of DLBCL with characteristic genetic drivers and signaling pathways that are targetable. Immune therapy with chimeric antigen receptor T cells and checkpoint inhibitors has revolutionized the treatment of relapsed or refractory disease, and antibody drug conjugates have weaponized otherwise intolerable cytotoxic agents. Ongoing clinical trials are further refining the specificity of these approaches in different genetic subtypes and moving them from the setting of recurrent disease to frontline treatment in high-risk patient populations.

The use of Bruton tyrosine kinase (BTK) inhibitors to block B-cell receptor (BCR)–dependent NF-κB activation in lymphoid malignancies has been a major clinical advance, yet acquired therapeutic resistance is a recurring problem. We modeled the development of resistance to the BTK inhibitor ibrutinib in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma, which relies on chronic active BCR signaling for survival. The primary mode of resistance was epigenetic, driven in part by the transcription factor TCF4. The resultant phenotypic shift altered BCR signaling such that the GTPase RAC2 substituted for BTK in the activation of phospholipase Cγ2, thereby sustaining NF-κB activity. The interaction of RAC2 with phospholipase Cγ2 was also increased in chronic lymphocytic leukemia cells from patients with persistent or progressive disease on BTK inhibitor treatment. We identified clinically available drugs that can treat epigenetic ibrutinib resistance, suggesting combination therapeutic strategies. Significance: In diffuse large B-cell lymphoma, we show that primary resistance to BTK inhibitors is due to epigenetic rather than genetic changes that circumvent the BTK blockade. We also observed this resistance mechanism in chronic lymphocytic leukemia, suggesting that epigenetic alterations may contribute more to BTK inhibitor resistance than currently thought. See related commentary by Pasqualucci, p. 555. This article is highlighted in the In This Issue feature, p. 549

T follicular helper (Tfh) cells provide signals to initiate and maintain the germinal center (GC) reaction and are crucial for the generation of robust, long-lived antibody responses, but how the GC microenvironment affects Tfh cells is not well understood. Here we develop an in vivo T cell-intrinsic CRISPR-knockout screen to evaluate Tfh and Th1 cells in an acute viral infection model to identify regulators of Tfh cells in their physiological setting. Using a screen of druggable-targets, alongside genetic, transcriptomic and cellular analyses, we identify a function of HIF-1α in suppressing mTORC1-mediated and Myc-related pathways, and provide evidence that VHL-mediated degradation of HIF-1α is required for Tfh development; an expanded in vivo CRISPR screen reveals multiple components of these pathways that regulate Tfh versus Th1 cells, including signaling molecules, cell-cycle regulators, nutrient transporters, metabolic enzymes and autophagy mediators. Collectively, our data serve as a resource for studying Tfh versus Th1 decisions, and implicate the VHL-HIF-1α axis in fine-tuning Tfh generation.

Signals emanating from the B cell receptor (BCR) promote proliferation and survival in diverse forms of B cell lymphoma. Precision medicine strategies targeting the BCR pathway have been generally effective in treating lymphoma, but often fail to produce durable responses in diffuse large B cell lymphoma (DLBCL), a common and aggressive cancer. New insights into DLBCL biology garnered from genomic analyses and functional proteogenomic studies have identified novel modes of BCR signaling in this disease. Herein, we describe the distinct roles of antigen-dependent and antigen-independent BCR signaling in different subtypes of DLBCL. We highlight mechanisms by which the BCR cooperates with TLR9 and mutant isoforms of MYD88 to drive sustained NF-κB activity in the activated B cell-like (ABC) subtype of DLBCL. Finally, we discuss progress in detecting and targeting oncogenic BCR signaling to improve the survival of patients with lymphoma.

Genomic analyses of diffuse large B cell lymphoma (DLBCL) are revealing the genetic and phenotypic heterogeneity of these aggressive lymphomas. In part, this heterogeneity reflects the existence of distinct genetic subtypes that acquire characteristic constellations of somatic genetic alterations to converge on the DLBCL phenotype. In parallel, functional genomic screens and proteomic analyses have identified multiprotein assemblies that coordinate oncogenic survival signaling in DLBCL. In this review, we merge these recent insights into a unified conceptual framework with implications for the design of precision medicine trials in DLBCL.

Primary central nervous system lymphoma (PCNSL) is confined to the brain, eyes, and cerebrospinal fluid without evidence of systemic spread. Rarely, PCNSL occurs in the context of immunosuppression (eg, posttransplant lymphoproliferative disorders or HIV [AIDS-related PCNSL]). These cases are poorly characterized, have dismal outcome, and are typically Epstein-Barr virus (EBV)-associated (ie, tissue-positive). We used targeted sequencing and digital multiplex gene expression to compare the genetic landscape and tumor microenvironment (TME) of 91 PCNSL tissues all with diffuse large B-cell lymphoma histology. Forty-seven were EBV tissue-negative: 45 EBV- HIV- PCNSL and 2 EBV- HIV+ PCNSL; and 44 were EBV tissue-positive: 23 EBV+ HIV+ PCNSL and 21 EBV+ HIV- PCNSL. As with prior studies, EBV- HIV- PCNSL had frequent MYD88, CD79B, and PIM1 mutations, and enrichment for the activated B-cell (ABC) cell-of-origin subtype. In contrast, these mutations were absent in all EBV tissue-positive cases and ABC frequency was low. Furthermore, copy number loss in HLA class I/II and antigen-presenting/processing genes were rarely observed, indicating retained antigen presentation. To counter this, EBV+ HIV- PCNSL had a tolerogenic TME with elevated macrophage and immune-checkpoint gene expression, whereas AIDS-related PCNSL had low CD4 gene counts. EBV-associated PCNSL in the immunosuppressed is immunobiologically distinct from EBV- HIV- PCNSL, and, despite expressing an immunogenic virus, retains the ability to present EBV antigens. Results provide a framework for targeted treatment.

The role of Fas in germinal center (GC) homeostasis is controversial. Razzaghi et al. show that Fas is a strong cell-intrinsic regulator of the GC and that its loss defines an aggressive subtype of GC-derived lymphoma., Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B cell lymphoma (DLBCL). Although GC-derived DLBCL has better overall outcomes than other DLBCL types, some cases are refractory, and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes cell death in the light zone, likely via T follicular helper (Tfh) cell–derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. FAS alterations occurred most commonly in GC-derived DLBCL, were associated with inferior outcomes and an enrichment of Tfh cells, and co-occurred with deficiency in HVEM and PD-L1 that regulate the Tfh–B cell interaction. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in GC-derived lymphoma., Graphical Abstract

Significance MYC is a transcriptional regulator that controls much of the coding and noncoding transcriptome. It is also an oncoprotein that functions as a driver in numerous human cancers. The mechanism of this oncogenic activity is not known, but it probably involves specific MYC-regulated target genes. Here we systematically identify and characterize MYC-regulated, long noncoding RNAs (lncRNAs) that are required for MYC-driven cellular proliferation in human lymphoid cells. We use targeted transcriptional repression, adding new tools to this technology that will facilitate the functional analysis of any transcriptional regulator., MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.

SummaryTumors frequently subvert MHC class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance. To better define the regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of novel genes that positively and negatively modulate MHC-I cell surface levels. Identified genes cluster in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Many genes exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors display genetic alterations in multiple regulators. We establish that the HSP90 co-chaperone SUGT1 is a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhances DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.

The KLHL14 gene acquires frequent inactivating mutations in mature B cell malignancies, especially in the MYD88 L265P , CD79B mutant (MCD) genetic subtype of diffuse large B cell lymphoma (DLBCL), which relies on B cell receptor (BCR) signaling for survival. However, the pathogenic role of KLHL14 in DLBCL and its molecular function are largely unknown. Here, we report that KLHL14 is in close proximity to the BCR in the endoplasmic reticulum of MCD cell line models and promotes the turnover of immature glycoforms of BCR subunits, reducing total cellular BCR levels. Loss of KLHL14 confers relative resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib and promotes assembly of the MYD88-TLR9-BCR (My-T-BCR) supercomplex, which initiates prosurvival NF-κB activation. Consequently, KLHL14 inactivation allows MCD cells to maintain NF-κB signaling in the presence of ibrutinib. These findings reinforce the central role of My-T-BCR–dependent NF-κB signaling in MCD DLBCL and suggest that the genetic status of KLHL14 should be considered in clinical trials testing inhibitors of BTK and BCR signaling mediators in DLBCL.

Background: Follicular lymphoma (FL) shows marked variation in clinical course including spontaneous regression and histologic transformation (HT). Watchful waiting (W&W) is routinely applied to pts with newly diagnosed FL, but monitoring strategies are not standardized. Pts with progression within 1-2y of diagnosis have worse outcomes, but the biologic basis is unclear and biologic-based classifiers are not routinely applied at diagnosis. Circulating tumor DNA (ctDNA) is a highly tumor-specific biomarker that is prognostic in aggressive B-cell lymphomas, but its ability to serially monitor FL remains undefined. We applied a next-generation sequencing assay to identify tumor clonotypes for serial monitoring of peripheral blood in pts with untreated FL as part of an ongoing prospective clinical trial [NCT03190928]. Methods: Pts with grade I-II or 3A FL are eligible if evaluable disease on CT or FDG-PET, age ≥18, ECOG ≤2, no evidence of HT, and no prior systemic therapy. Pts undergo W&W until they meet uniform protocol-defined treatment criteria and remain on study until second-line therapy. Baseline testing includes labs, peripheral blood flow cytometry, BM biopsy/aspirate, CT and FDG-PET scans, and research biopsy. Pt have clinic visits every 4m for 2y, every 6m in years 3-5, then annually. CT scans are every 8m for 2y, then annually. FDG-PET scans are at baseline, at 2y, and any time of suspected progression. Peripheral blood samples including Streck tubes (plasma) and PBMCs are drawn at each clinic visit and stored. For ctDNA analysis, tumor DNA was amplified from FFPE using locus-specific primer sets for the Ig heavy-chain and light-chain loci along with BCL1/BCL2 translocations (Adaptive Biotechnologies). Amplified products were sequenced and tumor clonotypes were identified in plasma and PBMCs. Serial tracking of ctDNA was done in plasma and blinded to clinical outcomes. Results: 77 pts enrolled between July 2017 and July 2021. Median age was 57 (range 24-83) including 14 (18%) low-risk, 29 (38%) intermediate-risk, and 34 (44%) high-risk by FLIPI. Fourteen (18%) pts had stage I-II disease. Forty-three (56%) pts had monoclonal B-cells on peripheral blood flow cytometry. Twenty-nine (38%) pts progressed requiring frontline therapy including 7 (9%) pts with HT. Twenty-five (32%) pts were monitored ≥2y with no progression including 10 (13%) pts with evidence of at least some spontaneous regression by CT. Twenty (26%) pts were on study for Conclusions: ctDNA quantified from plasma in FL mirrors TMTV. Serial monitoring of ctDNA in patients without therapy demonstrated various patterns of fluctuation, including some patients in which ctDNA became undetectable coincident with spontaneous clinical regressions. ctDNA thus provides a non-invasive platform to monitor the natural history of FL, enabling future studies of tumor immune surveillance in this disease. Figure 1 Figure 1. Disclosures Jacob: Adaptive Biotechnologies: Current Employment, Current equity holder in publicly-traded company. Bagaev: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Meerson: BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Postovalova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Kudryashova: BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Kotlov: BostonGene Corp: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Fowler: BostonGene: Current Employment, Current holder of stock options in a privately-held company.

Introduction: Immune tolerance and evasion plays a significant role in the pathogenesis of EBV+ lymphoproliferative disorders (LPD) and non-Hodgkin lymphomas (NHL). Programmed cell death protein-1 (PD-1) is a signaling molecule on the surface of T-cells that suppresses the cytotoxic effects of T-cells on tumor cells. PD-L1 expression is a marker of poor prognosis in aggressive lymphomas and most EBV+ LPDs demonstrate high levels of PD-L1 expression. Chronic viral infections, such as EBV, also result in T-cell exhaustion that can be reversed by PD-1 blockade. Nivolumab is a fully human IgG4 monoclonal anti-PD-1 antibody which has demonstrated activity and favorable safety in lymphoid malignancies. We hypothesized that PD-1 blockade may reverse the inactivation of tumor-specific effector T-cells and result in anti-tumor responses in EBV+ LPD and NHL. Methods: Relapsed/refractory (R/R) EBV+ LPD and B-cell NHL pts age ≥ 12y with adequate organ function are eligible. Untreated pts are eligible if EBV+ LPD. Exclusions include prior use of PD-1/PD-L1/PD-L2/CD137/CTLA-4 antibodies, prior solid organ transplant and HIV. Pts with immunodeficiency or autoimmune illness are eligible if not requiring steroids or immunosuppression. CNS involvement is permitted if no seizure activity within 4 weeks of study. Nivolumab 480mg IV is given every four weeks for up to 2y. Pts who achieve CR discontinue nivolumab after 1y of treatment. Baseline evaluation includes CT, PET, MRI brain, flow cytometry of peripheral blood and CSF, BM biopsy along with optional tumor biopsy. CT scans are performed after cycles 3, 6, 13 and 19 and end of treatment (EoT). PET is performed after cycles 1, 3 and EoT. Surveillance CT scans are performed q3m for 1y, q6m for yrs 2-5, and annually thereafter. Results: 9 pts, 7 (78%) R/R and 2 (22%) untreated, enrolled between April 2018 and May 2021; 5 (56%) with EBV+ LPD [4 G1-2 lymphomatoid granulomatosis (LYG) and 1 chronic active EBV disease (CAEBV)] and 4 (44%) with EBV+ NHL (all DLBCL, NOS). Median age was 48y (range 30-63) and all pts (100%) had stage III/IV disease. Four pts (44%) had elevated LDH (all DLBCL). Median baseline CD4 and CD8 count (cells/mcL) was 378 (range 99-984) and 86 (range 22-1237), respectively, for LPD and 190 (range 133-255) and 90 (range 9-630), respectively, for NHL. Median EBV VL at baseline (Log10 IU/mL) was 2.55 (range 0-6.78) and 2.53 (range 0-5.33) for LPD and NHL, respectively. Eight (89%) pts had extranodal disease with pulmonary involvement most common in 6 (67%). Median prior therapies were 1 (range 0-1) and 2 (range 1-4) for LPD and NHL pts, respectively. Three (43%) R/R pts were refractory (i.e., Of 9 pts enrolled, 7 were evaluable for response (1 NHL pt died prior to restaging and 1 NHL pt has not yet been restaged). In 6 measurable pts, tumor reduction was observed in 67% (Fig 1A). ORR and CR rate was 57% (4/7) and 43% (3/7), respectively; 60% (3/5) and 40% (2/5) in LPD and 50% (1/2) and 50% (1/2) in NHL. Median TTR was 3.0m with 3 (75%) of 4 responses ongoing from 6.9m to 35.2m after first response (Fig 1B). Most common adverse events (AEs) (% pts) included maculopapular rash (38%), ALT elevation (25%), AST elevation (25%), CPK elevation (25%) and fatigue (25%). One pt discontinued therapy due to G2 immune-mediated myositis that required prolonged steroid therapy. >G3 AEs included AST elevation in 1 (13%) pt with no G4/G5 or serious adverse events. With a median potential follow up of 12.6m, 12-month PFS and OS was 50.8% (95% CI: 15.7-78.1) and 75.0% (95% CI: 31.5-93.1). In LPD pts, 12-month PFS and OS was 80% (95% CI: 20.4-96.9) and 100%. Three (75%) NHL pts progressed and 2 (50%) died of disease progression. One NHL pt stopped therapy due to apparent disease progression after 2 cycles but later developed CR without further therapy and remains in remission 35.2m after stopping therapy. Conclusion: Nivolumab appears safe in pts with EBV+ LPD and NHL without unexpected toxicities. Preliminary clinical activity, including CRs, is noted in pts with EBV+ LPD and NHL. Additional pts are needed for a better assessment of true activity in these rare entities and correlates of response including PD-1/PD-L1 expression and/or 9p24.1 alterations are ongoing and will be presented at the meeting. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Nivolumab for EBV+ LPD and EBV+ NHL.

Background: Diffuse large B-cell lymphoma (DLBCL) subtypes have differential response to BTK inhibitors (BTKi). Ibrutinib with R-CHOP improves survival in DLBCL subsets, but toxicity is limiting. Precise characterization of BTKi-responsive tumors enhances pt selection. Acalabrutinib (acala) is a BTKi with activity in DLBCL, but the molecular correlates of acala response are unknown. Circulating tumor DNA (ctDNA) is a prognostic biomarker in DLBCL including early changes during chemotherapy. PhasED-Seq is a novel ctDNA method that lowers the error profile of mutation detection by requiring the concordant detection of two separate mutations on an individual cell-free DNA molecule (Kurtz et al. Nat Biotechnol 2021). We employed a response-adapted study of acala for up to 14d prior to frontline therapy for aggressive B-cell lymphoma to determine the molecular profile of BTKi-responsive tumors. We report preliminary results including dynamic changes in ctDNA from this ongoing trial [NCT04002947]. Methods: Pts with untreated aggressive B-cell lymphoma and any HIV status are eligible if age ≥18, ≥stage 2, PS ≤2, and adequate organ function. Pts with PMBL, unmeasurable lesions, or active CNS disease are excluded. Screening includes labs, CT and FDG-PET, BM, and CSF with flow cytometry. Pts first receive acala 100mg twice daily x 14d. Pts with Results: 34 pts enrolled between August 2019 and July 2021 and completed the acala window. Median age was 64 (range 28-85) including 13 (38%) < 60y, 14 (41%) 61-69, and 7 (21%) ≥70y. Three (9%) pts had HIV and 17 (50%) were high-risk by IPI. The median diagnosis to treatment was 22.5d (4-53). IHC subtypes by Hans included 17 (50%) non-GCB, 16 (47%) GCB, and 1 (3%) T-cell/histiocyte-rich large B-cell lymphoma (TRLBCL). Four (12%) pts were high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 (HGBL-DH). Fifteen (44%) pts responded to acala during the window, while 19 (56%) pts had no response (Figure 1A). Acala responses were seen across DLBCL subtypes including 7 (47%) pts with non-GCB, 7 (47%) pts with GCB, and 1 (7%) pt with TRLBCL. Twenty pts had RNA sequencing to confirm cell-of-origin including 10 responders which included 7 (70%) GCB, 2 (20%) ABC, and 1 (10%) Unclassified. Notably, 13 (86%) BTKi-responsive tumors were CD10 negative and only 2 (18%) CD10+ tumors were BTKi-responsive. ctDNA dynamics strongly correlated with CT response as the log-fold change in ctDNA (hGE/mL) at the end of the window correlated with change on CT (r=0.75, p=0.0013). Remarkably, ctDNA dynamics after only 7d also correlated with change on CT (r=0.82, p=0.00006)(Figure 1B-C). Interestingly, one pt had improved symptoms and a 20-fold drop in ctDNA, but no corresponding CT changes suggesting that ctDNA changes may precede CT changes in some cases. Twenty-nine (85%) pts completed all planned cycles of therapy while 5 pts stopped chemotherapy early due to myelosuppression (n=3), CHF (n=1), and MI (n=1). Toxicity across 156 cycles was mostly hematologic. G3/G4 neutropenia occurred in 50% and 38% of cycles and febrile neutropenia in 10% of cycles. G3/G4 thrombocytopenia occurred in 22% and 12% of cycles. No increase in infections, atrial fibrillation, or bleeding were observed in pts treated with acala. All 27 pts who completed therapy achieved a CR. Two pts (1 acala responder) have relapsed from CR and 1 pt died of an MI. After a median follow-up of 9.2m the estimated 1-year PFS was 84.9% (95% CI: 58-95). Conclusions: Acalabrutinib prior to frontline therapy has activity in GCB, non-GCB, and HGBL-DH: confirmed by gene expression profiling. CD10+ GCB tumors are mostly acala-resistant. Toxicity is mainly hematologic and manageable across age groups including pts with HIV. ctDNA correlates with CT change and may predict response to targeted agents as early as 7 days. Updated clinical results within genetic subtypes will be presented at the meeting. Figure 1 Figure 1. Disclosures Chabon: Foresight Diagnostics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Lurain: CTI Biopharma: Research Funding; EMD-Serrono: Research Funding; Merck: Research Funding; BMS-Celgene: Research Funding; Janssen: Research Funding. Bagaev: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Postovalova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Meerson: BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Kudryashova: BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Kotlov: BostonGene Corp: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Fowler: BostonGene: Current Employment, Current holder of stock options in a privately-held company. Kurtz: Roche: Consultancy; Foresight Diagnostics: Consultancy, Current holder of stock options in a privately-held company; Genentech: Consultancy. Alizadeh: CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Bristol Myers Squibb: Research Funding; Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Background: Mantle cell lymphoma (MCL) is a biologically and clinically heterogenous B-cell lymphoma that is generally incurable with standard therapies. Novel targeted agents can disrupt key survival pathways in MCL such as regulation of apoptosis (BCL2: venetoclax), B-cell receptor signaling (BTK: ibrutinib), and NF-κB survival pathways (IRF4/SPIB: lenalidomide). As monotherapy, these agents fail to induce deep responses and doublet/triplet regimens often require continuous or maintenance therapy. ViPOR has been shown to be safe and active in non-MCL NHL pts without significant tumor lysis syndrome (TLS) (Melani et al. Blood. 2020; 136(Supplement 1):44-45). We hypothesized that combining agents that target multiple survival pathways with ViPOR will leverage efficacy and time-limited, cyclic dosing will limit toxicities in MCL. Methods: Relapsed/refractory (R/R) and untreated MCL pts with adequate organ function were eligible. In R/R MCL, a phase I "3+3" design was used to determine the maximum tolerated dose (MTD) of 2 dose-levels of dose-escalated venetoclax (200mg and 400mg) PO D2-14 (starting C2) in combination with fixed-dose ibrutinib 560mg PO D1-14, prednisone 100mg PO D1-7, obinutuzumab 1000mg IV D1-2, and lenalidomide 15mg PO D1-14. A phase II expansion in untreated MCL was included at the MTD. ViPOR q21d x 6C was given without maintenance or consolidation. All pts were admitted C2 for 12d venetoclax escalation and TLS monitoring. All pts received TLS prophylaxis (ppx) with IVFs and allopurinol as well as PCP and G-CSF ppx. VTE ppx was per investigator discretion. Baseline CT, PET, BM, and tumor biopsy was performed with CT scans after cycles 1, 2, 4, and 6 and PET after cycle 6 or at time of suspected complete response (CR). Surveillance CT was performed q3m for 1y, q4m x 1y, q6m x 1y, then annually x 2y. Plasma for ctDNA was collected at baseline, prior to each treatment cycle, at each follow-up visit, and at disease progression. Results: 11 pts have been enrolled and treated; 9 (82%) R/R in dose-escalation and 2 (18%) untreated in dose-expansion. Median age was 71y (range 57-79) with 73% >65y and 64% male. Low, intermediate, and high-risk MIPI occurred in 18%, 64%, and 18% of pts, respectively. Stage IV disease was seen in 91%, with BM involvement in 73%, extranodal disease in 82%, and both in 64%. Disease bulk >5cm occurred in 45% of pts. Blastoid morphology, Ki-67 >30%, and TP53 IHC >50% occurred in 27%, 36%, and 18% of pts, respectively. Median prior therapies in R/R pts was 3 (range 1-4) with 44% receiving prior BTKi, 11% receiving prior CAR-T, and 78% refractory (i.e., No dose-limiting toxicities (DLTs) occurred in 9 evaluable pts in the dose-escalation cohort; thus, venetoclax 400mg was used in expansion. G3-4 heme AEs (% cycles) included neutropenia (13%), anemia (11%), and thrombocytopenia (9%). No cases of febrile neutropenia occurred across 46 total cycles. G3-4 non-heme AEs (% pts) included hypokalemia in 3 (33%) pts as well as fatigue, hypomagnesemia, elevated bilirubin, atrial fibrillation, lung infection, and syncope in 1 (11%) pt each. No laboratory or clinical TLS occurred. Dose reductions and delays occurred in 5% and 15% of cycles, respectively. Of 11 pts enrolled, 10 are evaluable for response (1 pt has not yet been restaged) with an overall response rate (ORR) and CR rate of 100% (10/10) and 80% (8/10), respectively (Fig 1A). Of 8 pts who have completed therapy, all 8 (100%) have achieved CR, including all 4 post-BTKi pts, 1 post-CAR-T pt, and 6 refractory pts. With a median potential f/u of 5.2m, median TTR and DOR was 0.7m and not reached, respectively, with 9 (90%) responses ongoing ranging from 0.3m to 14.5m after first response (Fig 1B). One pt with R/R blastoid MCL relapsed in the CNS 9.9m after initial response. Median PFS and OS were both not reached with 10 (91%) pts alive and progression-free and 1 relapse and death from progression at 11.9m. Conclusion: ViPOR is safe in MCL without significant TLS or DLTs using a 12d venetoclax ramp-up on C2 and venetoclax 400mg was taken forward in expansion. Most common G3-4 AEs were hematologic with no febrile neutropenia observed when given with G-CSF ppx. High preliminary activity is noted in MCL pts with fixed-duration ViPOR x 6C, including CRs in refractory, post-BTKi, and post-CAR-T pts. Molecular and ctDNA analyses are ongoing and will be presented at the meeting. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Off-label use of ViPOR in relapsed/refractory and untreated MCL.

Background: Secondary CNS B-cell lymphomas (SCNSL) are aggressive lymphomas with a very poor prognosis. Genetic subtypes of DLBCL with CNS tropism are enriched for chronic active B-cell receptor signaling and may respond to BTK inhibition (BTKi). CLL, MCL, and transformed lymphomas can also involve the CNS and are BTKi responsive. TEDDI-R achieves durable remissions in relapsed/refractory primary DLBCL of the CNS (PCNSL) but the profile of SCNSL tumors that are ibrutinib-responsive is unknown. We present preliminary results from a response-adapted trial of ibrutinib with TEDD-R in SCNSL. Methods: Pts with aggressive B-cell lymphomas with secondary CNS involvement are eligible if age ≥18 and adequate organ function. Pts must have relapsed after frontline therapy or can be untreated if brain parenchyma involved. Prior BTKi is allowed, but HIVinfection and EBV+ lymphomas are excluded. Baseline tests include brain MRI, FDG-PET brain and body, CSF with flow cytometry, Ommaya, and eye exam. Pts receive isavuconazole starting at 200mg BID x 3d prior to ibrutinib to prevent fungal infections associated with TEDDI-R and then 200mg daily unless ibrutinib stopped. Pts first receive ibrutinib 560mg daily x 14d in a window. If ≥20% reduction after ibrutinib, pts receive TEDDI-R for 4 cycles every 21d with IT cytarabine. Pts with Results: 16 pts with a median age 67 (range 40-79) enrolled between June 2019 and July 2020. 15 (94%) pts had DLBCL comprising 9 (60%) non-GCB, 5 (33%) GCB, and 1 (7%) transformed from MZL. One pt had plasmablastic lymphoma. Eight (50%) pts had a MYC-rearrangement including 4 (25%) with both MYC and BCL2 or BCL6 rearrangements. Eight (50%) pts had isolated CNS disease and 8 (50%) had synchronous CNS and peripheral disease. All pts relapsed after a median of 2 (range 1-4) prior therapies and all (100%) pts received prior anthracycline. Seven pts (44%) had prior CNS prophylaxis and 8 pts (50%) had prior HD-MTX based salvage therapy. Toxicity was evaluated across 35 cycles. G3 and G4 neutropenia occurred in 49% and 29% of cycles, respectively, while febrile neutropenia occurred in 9% of cycles. The median (range) duration of neutropenia was 6 (1-13) days. Five (14%) cycles were complicated by ≥G3 infection, but no opportunistic infections (including Aspergillus) were observed. One pt developed a bacterial infection during cycle 1 and died. G3 and G4 thrombocytopenia occurred in 34% and 23% of cycles, respectively, and 1 pt developed G3 hematuria. G3 mucositis occurred in 9% of cycles and palmar-plantar-erythrodysesthesia led to dose reductions of liposomal doxorubicin in 5 (36%) pts. Of 15 pts who completed the 14d ibrutinib window and were evaluable, 8 (53%) were ibrutinib-responsive and 7 (47%) were ibrutinib-resistant (Figure 1). Clinical responses were concordant across anatomic compartments; of 8 pts with both CNS and peripheral disease, 5 (63%) responded to ibrutinib in both compartments while 3 (37%) did not respond in either compartment. All 8 ibrutinib responders had a non-GCB phenotype and six (75%) achieved CR. One died of treatment-related toxicity after a PR and 1 is still on therapy. Only two (29%) pts with ibrutinib-resistant tumors achieved PR and none have achieved CR. After a median follow-up of 5.1m, a landmark analysis starting after the ibrutinib window demonstrated the PFS for pts with ibrutinib-responsive compared to ibrutinib-resistant tumors was not reached vs. 0.9m (95% CI: 0.1-2m)(p=0.002)(Figure 2). Conclusions: Patients with SCNSL tumors that are ibrutinib-responsive achieve a high rate of complete response to TEDDI-R in both CNS and peripheral disease. Patients with tumors that are ibrutinib-resistant also respond poorly to TEDD-R. Toxicity is mainly hematologic, and no Aspergillus infections have occurred with the use of isavuconazole prophylaxis. Updated clinical results from this ongoing study (NCT03964090) and will be presented at the meeting. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: ibrutinib for use in secondary cns lymphoma as part of a clinical trial

Introduction Follicular lymphoma (FL) has a highly variable clinical course. Chemoimmunotherapy can induce durable remissions, but ~20% relapse early. The PI3K pathway is central to FL biology and multiple PI3K inhibitors (PI3Ki) are approved for FL, but the molecular profile of tumors most sensitive to PI3Ki is unknown. Further, PI3Ki are dosed indefinitely which contributes to toxicity and cost. Copanlisib is an IV inhibitor of PI3Kα and δ isoforms with high activity in relapsed FL. We hypothesized that patients with FL tumors most sensitive to PI3Ki will achieve deep and durable remissions after a fixed duration of copanlisib-based therapy. Here we report preliminary results of a response-adapted study using copanlisib and rituximab as frontline therapy for FL. Methods Pts with untreated grade 1-2, 3A FL, ≥stage 2, and any tumor burden are eligible if they meet criteria for need of systemic therapy that includes symptoms, increasing size of nodes, critical organ involvement, or impending organ compromise. No prior systemic therapy other than radiation is permitted. Frozen or archival tissue is required. Eligibility includes age ≥18 and adequate organ function unless involved by FL. Active HIV, CMV, Hep B or C, and autoimmune conditions requiring therapy are excluded. All pts receive PCP prophylaxis. Pts first receive copanlisib 60mg on days 1, 8, and 15 of a 28-day window to test activity of monotherapy. On-treatment tumor biopsies are optional after the window. Following the window, pts receive 6 cycles of copanlisib 60mg on days 1, 8, and 15 of a 28 day cycle along with rituximab 375mg weekly x 4 then on day 1 of each cycle. Streck tubes for circulating tumor DNA (ctDNA) are collected weekly during the window, after each cycle, and during surveillance. FDG-PET and CT scans are performed at baseline, after the window, and after cycles 3 and 6 to determine response. Patients with complete response (CR) after 6 cycles stop therapy. Patients with partial response (PR) after 6 cycles receive another 6 cycles of combination therapy with the copanlisib reduced to day 1 and 15 of each cycle. Non-responding patients will receive standard chemotherapy. The primary endpoint is the overall rate of CR with secondary endpoints of safety and duration of CR. Exploratory objectives include identification of a signature that predicts PI3Ki response. Results Ten pts have been enrolled and completed the copanlisib window. Median age was 50y (range, 28-77) including 3 (30%) over age 70. Seven (70%) pts had high-risk FLIPI scores ≥3 and two (20%) pts had Grade 3A FL. Comorbid conditions included prediabetes in 4 (40%) and hypertension in 4 (40%). All 10 (100%) pts had tumor reductions during copanlisib monotherapy with a median reduction of 41% (range 16-62%) (Figure 1). Four pts have completed 6 cycles of copanlisib and rituximab and all 4 (100%) have responded including 2 (50%) who achieved CR. In the two pts who achieved a PR after 6 cycles, one had a 90% tumor reduction and one had only persistent minimal residual disease in the bone marrow by flow cytometry. Toxicity was evaluated in 10 pts across 58 cycles and the most common have included rash (50%), diarrhea (50%) and mucositis (40%) which have all been G1 or G2 and successfully managed with supportive care and did not recur with subsequent cycles. One pt developed grade 3 neutropenia that responded to growth factors and did not recur. Four pts had dose delays due to rash (N=3) and lung infection (N=1). One pt had copanlisib reduced to 45mg due to recurrent rash and elevated liver tests. No pt has discontinued therapy. One pt required oral diabetic medications during therapy that were stopped after therapy completed. One pt had asymptomatic PCP pneumonia diagnosed during the copanlisib window prior to starting prophylaxis that was successfully treated while therapy continued. Conclusion Copanlisib is highly active in untreated FL and the first 10 (100%) patients all had tumor reductions after the first cycle of copanlisib monotherapy, including patients with high tumor bulk. Combination of copanlisib and rituximab can induce complete responses after only 6 cycles and without indefinite therapy. The safety profile includes rash and diarrhea that respond to supportive care and lessen on subsequent cycles. Updated clinical results from this ongoing trial (NCT03789240) along with the molecular profile of FL tumors will be presented at the meeting. Disclosures No relevant conflicts of interest to declare.

Background: Primary DLBCL of the CNS (PCNSL) relies on chronic active B-cell receptor (BCR) signaling. Ibrutinib targets BCR signaling through BTK inhibition (BTKi), which may also impair innate immunity. We showed that ibrutinib and temozolomide, etoposide, liposomal doxorubicin, dexamethasone, rituximab (TEDDI-R) induces durable remissions in relapsed/refractory PCNSL but 7 (39%) pts developed Aspergillus infections without fungal prophylaxis. Newer triazoles are effective against Aspergillus but inhibit ibrutinib clearance through CYP3A4. Isavuconazole has less effect on CYP3A4 and less hepatotoxicity than voriconazole. We hypothesized that ibrutinib and isavuconazole could be safely co-administered in TEDDI-R and ameliorate the risk of Aspergillus while maintaining efficacy. We studied escalating doses of ibrutinib in TEDDI-R with isavuconazole to determine the safety profile, ibrutinib PK, and clinical activity in relapsed/refractory PCNSL. Methods: Pts with relapsed/refractory PCNSL, age ≥18, ECOG PS ≤2, and adequate organ function were enrolled. Previous BTKi, HIV, EBV+, and pregnancy were excluded. Pts had baseline MRI brain, FDG-PET brain and body, Ommaya placed, CSF with flow cytometry, and eye exam. Isavuconazole 200mg BID x 3d started prior to ibrutinib then 200mg daily. Three dose levels of ibrutinib (280mg, 420mg, 560mg) were given continuously through each cycle. Pts received up to 6 cycles of TEDDI-R with IT cytarabine. No one received maintenance or consolidation. If a DLT occurred in the first 3 pts at a given ibrutinib dose level, 3 more pts were treated before escalating. Full safety and PK data was reviewed after two dose levels prior to escalating. An expansion of 10 pts was planned at the highest ibrutinib dose level to confirm safety and clinical activity. Surveillance for fungal infections included chest CT mid-cycle 1 and after each cycle along with Beta-D glucan and aspergillus galactomannan in blood and CSF. Brain MRI was performed after cycles 1, 2, 4, and 6 to determine response and screen for CNS Aspergillus. All remissions by MRI were confirmed with FDG-PET and CSF analysis. Surveillance brain MRI were q3m for 1y, q4m x 1y, q6m x 1y, then annually. Primary objective was to identify the highest dose of ibrutinib safely co-administered with isavuconazole in TEDDI-R that achieves adequate PK concentrations. Results: 13 relapsed/refractory PCNSL pts enrolled between 11/2018 and 06/2020. 10 (77%) pts were male and the median age was 65 (range 46-77), including 3 pts ≥age 70. 13 (100%) pts had prior high-dose MTX, and 2 (15%) pts had prior autologous stem cell transplant (ASCT). Three evaluable pts received ibrutinib 280mg, 3 pts received ibrutinib 420mg, and 6 pts received ibrutinib 560mg. One pt in the 280mg cohort was not evaluable. Toxicity was evaluated in 13 pts across 49 cycles and the toxicity was mainly hematologic. G3 and G4 neutropenia occurred in 45% and 37% of cycles, respectively, while febrile neutropenia occurred in 8% of cycles. The median (range) duration of neutropenia was 4.5 (1-12) days. One pt with prior ASCT stopped after 4 cycles due to myelosuppression. Four (8%) cycles were complicated by ≥G3 infection, but no opportunistic infections (including Aspergillus) were observed. G3 and G4 thrombocytopenia occurred in 22% and 8% of cycles, respectively, and 1 pt developed melena with no overt GI bleeding. ≥G3 mucositis occurred in 6% of cycles and 1 patient stopped therapy after 5 cycles due to recurrent mucositis. Palmar-plantar-erythrodysesthesia led to dose reductions of liposomal doxorubicin in 9 (69%) pts, but only 1 G3 event occurred. Twelve pts were evaluable for response, and 11 (92%) pts have responded and all after receiving only 1 cycle (Figure 1). All 8 (100%) pts who have completed at least 4 cycles have achieved CR and the other 4 remain on therapy. Six (75%) pts who achieved CR remain in remission while 2 (25%) pts relapsed within 3 months of stopping therapy. After a median potential f/u of 5.2 months, the 1-year PFS is estimated at 60.0% (95% CI, 12.6-88.2) and the OS is 100%. Conclusions: Ibrutinib 560mg was safely co-administered with isavuconazole in TEDDI-R for relapsed/refractory PCNSL. No DLTs were observed, no cases of Aspergillus occurred, and no new safety signals. The first 8 (100%) patients who have completed therapy achieved complete response. Updated clinical results from this ongoing study (NCT02203526) will be presented at the meeting. Disclosures Lai: Abbvie: Consultancy; Agios: Consultancy; Jazz: Speakers Bureau; Macrogenics: Consultancy; Astellas: Speakers Bureau.

Background: Aggressive B-cell non-Hodgkin lymphoma (NHL) can be cured with chemoimmunotherapy; however, those who fail primary therapy and those with indolent NHL are rarely curable. Targeted agents can disrupt key survival pathways in NHL such as regulation of apoptosis (BCL2: venetoclax), B-cell receptor signaling (BTK: ibrutinib), and NF-κB survival pathways (IRF4/SPIB: lenalidomide). These agents are active as monotherapy but fail to induce deep responses and require continuous therapy. Also, genetically defined subtypes of NHL that best respond to these targeted agents are undefined. Synergistic cytotoxicity has been shown with these targeted therapies and corticosteroids in DLBCL cell lines. We hypothesized that combining agents that target multiple survival pathways will leverage efficacy and time-limited, cyclic dosing will limit toxicities. Methods: Relapsed/refractory (R/R) B-cell NHL pts, excluding MCL and CLL/SLL, with adequate organ function were eligible. A phase I "3+3" design was used to determine the maximum tolerated dose (MTD) of 4 dose-levels (DLs) of dose-escalated venetoclax (200mg, 400mg, 600mg, and 800mg) PO D2-14 (starts cycle 2 for DL1) in combination with fixed-dose ibrutinib 560mg PO D1-14, prednisone 100mg PO D1-7, obinutuzumab 1000mg IV D1-2, and lenalidomide 15mg PO D1-14. A phase II expansion in R/R DLBCL and FL was included at the MTD. Up to 6 cycles of ViPOR every 21-days was given without maintenance. TLS and PCP prophylaxis was given to all pts and VTE prophylaxis and G-CSF use was per investigator discretion. Baseline CT, PET, BM and tumor biopsy was performed with CT scans after cycles 1, 2, 4, and 6 and PET after cycle 6 or at time of suspected CR. Surveillance CT was performed q3m for 1y, q4m x 1y, q6m x 1y, then annually x 2y. Results: 53 pts were enrolled and treated; 17 in dose-escalation and 36 in dose-expansion. NHL subtypes included DLBCL (23), FL (19), HGBCL "double-hit" (9), and MZL (2). Of 32 aggressive pts, 34% transformed from indolent NHL. Median age was 57y (range 29-83) with stage III/IV disease in 89%, elevated LDH in 68%, and >2 EN sites in 57%. Median prior therapies was 3 (range 1-9) with 45% of pts refractory (i.e. A single dose-limiting toxicity (DLT) of G3 intracranial hemorrhage occurred at DL1 with concomitant enoxaparin and ASA. No other DLTs occurred and venetoclax 800mg was used in expansion. Heme AEs (% cycles) were most common and included thrombocytopenia (23%), neutropenia (23%) and anemia (7%). G-CSF was used in 92% of pts and 89% of cycles with only 3 (6%) cases of febrile neutropenia. Non-heme AEs (% pts) were mainly G1-2 and included diarrhea (67%), hypokalemia (56%), nausea (52%), and rash (42%). Most common G3-4 non-heme AEs included hypokalemia (19%), diarrhea (8%), and a.fib/flutter (6%). G4 TLS occurred in 1 pt with HGBCL after the first venetoclax dose and was successfully treated without further TLS upon continued treatment. Dose reductions and delays occurred in 8% and 9% of cycles, respectively. Of 53 total patients, 51 completed 1C of therapy with restaging CT and tumor reduction occurred in 90% of pts overall (Fig 1A). Of 44 pts who are now off therapy, 43 were evaluable for response with an ORR of 70% and 49% CR, with responses across all DLs and NHL subtypes. In 27 pts with aggressive NHL, ORR was 56% with 37% CR. Based on DLBCL subtype by IHC, ORR and CR rate was 62% (8/13) and 54% (7/13) in non-GCB and 50% (7/14) and 21% (3/14) in GCB DLBCL, respectively. In 16 pts with indolent NHL, ORR was 94% with 69% CR. ORR and CR rate was 52% (11/21) and 29% (6/21) in refractory pts and 86% (19/22) and 68% (15/22) in relapsed pts, respectively. ORR was 40% with 30% CR in 10 patients who failed prior CAR-T and completed ViPOR therapy. With a median potential f/u of 13m, median TTR and DOR was 0.8m and NR, respectively, with 25 (69%) of 36 responses ongoing. 5 pts relapsed after CR, including 2 non-GCB at 3m and 6m, 1 HGBCL at 5m, 1 FL at 6m, and 1 MZL at 16m. Median PFS and OS was 9m and NR, respectively; 20m and NR in indolent NHL, 3m and 13m in GCB, and 7m and 13m in non-GCB DLBCL (Fig 1B). Conclusions: ViPOR is safe without unexpected toxicities observed. Most common AEs were hematologic with rare febrile neutropenia and no severe infections observed when given with G-CSF prophylaxis. ViPOR induces durable CRs without maintenance therapy, including refractory and post CAR-T pts. Molecular analyses are ongoing and will be presented at the meeting. Disclosures Portell: Infinity: Research Funding; Roche/Genentech: Consultancy, Research Funding; Xencor: Research Funding; Kite: Consultancy, Research Funding; TG Therapeutics: Research Funding; AbbVie: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Bayer: Consultancy; BeiGene: Consultancy, Research Funding; Acerta/AstraZeneca: Research Funding. OffLabel Disclosure: Off-label use of the combination of venetoclax, ibrutinib, prednisone, obinutuzumab and lenalidomide in relapsed/refractory B-cell non-Hodgkin lymphoma.

Genome-wide screens are a powerful technique to dissect the complex network of genes regulating diverse cellular phenotypes. The recent adaptation of the CRISPR-Cas9 system for genome engineering has revolutionized functional genomic screening. Here, we present protocols used to introduce Cas9 into human lymphoma cell lines, produce high-titer lentivirus of a genome-wide sgRNA library, transduce and culture cells during the screen, isolate genomic DNA, and prepare a custom library for next-generation sequencing. These protocols were tailored for loss-of-function CRISPR screens in human lymphoma cell lines but are highly amenable for other experimental purposes.

Genome-wide screens are a powerful technique to dissect the complex network of genes regulating diverse cellular phenotypes. The recent adaptation of the CRISPR-Cas9 system for genome engineering has revolutionized functional genomic screening. Here, we present protocols used to introduce Cas9 into human lymphoma cell lines, produce high-titer lentivirus of a genome-wide sgRNA library, transduce and culture cells during the screen, isolate genomic DNA, and prepare a custom library for next-generation sequencing. These protocols were tailored for loss-of-function CRISPR screens in human lymphoma cell lines but are highly amenable for other experimental purposes.

Summary Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.

Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B-cell lymphoma (DLBCL); however, the function of Fas in the GC remains controversial. Although GC-derived DLBCL has better overall outcomes to therapy than other DLBCL types, some cases are refractory and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes B-cell death in the light zone likely via T follicular helper (Tfh) cell-derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. We found that FAS alterations occurred most commonly in the GC-derived genetic subtype of DLBCL, EZB. FAS alterations in EZB were associated with inferior outcomes and an enrichment of Tfh cells. Alterations in FAS co-occurred with deficiency in HVEM and PD-1 ligands that regulate the Tfh-B cell interaction and were associated with increased diversity of B-cell receptor variable genes across samples. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in a distinct subtype of GC-derived lymphoma. Citation Format: Raud Razzaghi, Shreya Agarwal, Nikita Kotlov, Olga Plotnikova, Krystle Nomie, Da Wei Huang, George W. Wright, Moyi Li, Katsuyoshi Takata, Chen Yao, John J. O'Shea, James D. Phelan, Stefania Pittaluga, David W. Scott, Jagan R. Muppidi. Compromised counterselection by FAS creates a lethal subtype of germinal center lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-22.

The development of precision medicine approaches for diffuse large B cell lymphoma (DLBCL) is confounded by its pronounced genetic, phenotypic, and clinical heterogeneity. Recent multiplatform genomic studies revealed the existence of genetic subtypes of DLBCL using clustering methodologies. Here, we describe an algorithm that determines the probability that a patient's lymphoma belongs to one of seven genetic subtypes based on its genetic features. This classification reveals genetic similarities between these DLBCL subtypes and various indolent and extranodal lymphoma types, suggesting a shared pathogenesis. These genetic subtypes also have distinct gene expression profiles, immune microenvironments, and outcomes following immunochemotherapy. Functional analysis of genetic subtype models highlights distinct vulnerabilities to targeted therapy, supporting the use of this classification in precision medicine trials.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer of aberrant B-lymphocytes. Although a portion of DLBCL is curable with standard immunochemotherapy, patients who fail this treatment have a poor prognosis. Recently, cancer genomics has paved the way for better understanding of the genetic basis of lymphoma pathogenesis. Characterization of point mutations and structural alterations has uncovered novel molecular targets for lymphoma therapy and provided a comprehensive view of lymphoma development. By performing multiplatform genomic analysis of DLBCL biopsy samples, we have identified KLHL14 as a recurrent target of somatic mutations in activated B-cell-like (ABC) DLBCL biopsies (10.8% of patients). KLHL14 contains a BTB (broad complex, tramtrack, and bric a brac) domain that can potentially mediate dimerization and binding to Cullin3 (CUL3)-a essential scaffold component of the Cullin-RING-based E3 ubiquitin ligase complexes. KLHL14 also contains kelch repeats that can form a B-propeller tertiary structure that can serve as a substrate-binding domain. KLHL14 is highly expressed in B-cells but is found at low levels in non-immune tissues. Deficiency of KLHL14 in mice leads to embryonic lethality while KLHL14 heterozygous mice show reduction of B-1a cells, suggesting a role for KLHL14 in B-cell homeostasis. Importantly, KLHL14 mutations are highly enriched in tumors belonging to the recently defined MCD (MYD88L265P/CD79B mutation) genetic subtype of DLBCL, the subset of ABC DLBCLs. Somatic mutations primarily localize to the N-terminus of the protein in the BTB domain and BACK (BTB and C-terminal Kelch) domain. However, the impact of these mutations as well as the molecular function of KLHL14 is largely unknown. To investigate the biological effect of KLHL14 loss of function, we used an inducible CRISPR/Cas9 system to delete KLHL14 in ABC DLBCL cell lines and monitored cell growth. Ablation of KLHL14 resulted in an increase in cell proliferation and survival, supporting a role for KLHL14 as a tumor suppressor. Next, we performed a multiplatform -omic analysis (proteomics, phosphoproteomics, ubiquitinomics, high-throughput sequencing) to explore the signaling networks and interactome of KLHL14. Whereas ectopic expression of wild-type KLHL14 altered the dynamics of tyrosine phosphorylation and ubiquitylation events in ABC DLBCL lines, KLHL14 lymphoma-associated mutant alleles had little if any effect, suggesting that they are loss-of-function variants. Gene expression profiling by RNA-sequencing revealed that KLHL14-inactivated cells have a higher NF-kB target gene expression than wild-type cells. Thus, tumor-associated inactivating mutations of KLHL14 depend on a subset of essential NF-kB-related oncoproteins for their survival and this might contribute to the proliferative advantage of DLBCL. In summary, we have uncovered a tumor suppressive function of KLHL14 and found that KLHL14 mutants promote ABC DLBCL survival by increasing NF-kB activity. These findings suggest that tumors with KLHL14 inactivating mutations may serve as a marker of resistance to anti-NF-kB treatment and provide the basis for treating MCD subtype patients with downstream NF-kB pathway inhibitors in the clinical settings. Disclosures Staudt: Nanostring: Patents & Royalties.

BACKGROUND: Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Gene-expression profiling has identified subgroups of DLBCL (activated B-cell-like [ABC], germinal-center B-cell-like [GCB], and unclassified) according to cell of origin that are associated with a differential response to chemotherapy and targeted agents. We sought to extend these findings by identifying genetic subtypes of DLBCL based on shared genomic abnormalities and to uncover therapeutic vulnerabilities based on tumor genetics. METHODS: We studied 574 DLBCL biopsy samples using exome and transcriptome sequencing, array-based DNA copy-number analysis, and targeted amplicon resequencing of 372 genes to identify genes with recurrent aberrations. We developed and implemented an algorithm to discover genetic subtypes based on the co-occurrence of genetic alterations. RESULTS: We identified four prominent genetic subtypes in DLBCL, termed MCD (based on the co-occurrence of MYD88L265P and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Genetic aberrations in multiple genes distinguished each genetic subtype from other DLBCLs. These subtypes differed phenotypically, as judged by differences in gene-expression signatures and responses to immunochemotherapy, with favorable survival in the BN2 and EZB subtypes and inferior outcomes in the MCD and N1 subtypes. Analysis of genetic pathways suggested that MCD and BN2 DLBCLs rely on "chronic active" B-cell receptor signaling that is amenable to therapeutic inhibition. CONCLUSIONS: We uncovered genetic subtypes of DLBCL with distinct genotypic, epigenetic, and clinical characteristics, providing a potential nosology for precision-medicine strategies in DLBCL. (Funded by the Intramural Research Program of the National Institutes of Health and others.).