DLBCL Tumors Research Articles

Abstract B079: Molecular determinants of sensitivity to Polatuzumab-Vedotin

Abstract Purpose: Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the B cell surface antigen CD79B. When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL overall; however, there is noted heterogeneity in response to Pola-V, with germinal center b-cell (GCB) DLBCL showing no added benefit to the addition of Pola-V compared to standard immunochemotherapy. We aimed to identify molecular determinants of sensitivity or resistance to Pola-V. We hypothesized that these might lead us to innovative strategies to improve on-target tumor killing by Pola-V, or to find novel biomarkers that predict drug resistance. Methods: We employed combined drug-sensitization and CD79B-sorted CRISPR-Cas9 screening to identify molecular determinants of sensitivity to CD79B-directed, tumor killing by Pola-V in 9 cell lines representing different molecular subtypes of DLBCL. Results: Our results reveal the striking impact of epitope glycosylation, specifically a2,6 sialylation, on the binding of Pola-V to CD79B and thereby its ability to kill tumor cells. Specifically, we identify the exact glycosylated residues on CD79A and CD79B which create a sialylated glycan shield around the Pola-V binding site, precluding binding to its target. We show how genetic, pharmacological and enzymatic approaches that remove terminal sialic acid residues from these N-linked glycans lead to enhanced tumor killing by Pola-V. We hypothesize and test multiple methods of targeting this pathway in order to enhance Pola-V killing both in vitro and in vivo. Finally, we reveal a previously unappreciated role for the ubiquitin ligase KLHL6 in regulating CD79B protein abundance and surface expression of the B cell antigen receptor (BCR), including how this pathway is used by physiological germinal center B cells and how it is corrupted to enhance BCR expression in GCB DLBCL. Conclusions: These findings unravel the molecular basis of response heterogeneity to Pola-V and identify approaches that might be deployed therapeutically to enhance the efficacy of CD79B-specific tumor killing. In addition, we identify how KLHL6 determines expression of the BCR in both physiological and malignant germinal center B cells, and how KLHL6 mutation may modulate sensitivity of GCB DLBCL tumors to Pola-V. Citation Format: Sean R Corcoran, Jaewoo Choi, Rachel E Fenner, Xin Yu, Sebastian Scheich, Galina Schevchenko, Evangelia K Papachristou, Vivian M Morris, Kamal Kishore, Clive S D'Santos, Stefania Pittaluga, George Wright, Jagan Muppidi, Daniel J Hodson, Louis M Staudt. Molecular determinants of sensitivity to Polatuzumab-Vedotin [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B079.

Molecular Classification of Relapsed DLBCL Reveals Novel Biologic Subgroups

Background Diffuse large B cell lymphoma (DLBCL) is the most common aggressive lymphoid malignancy in adults. Though standard immunochemotherapy regimens can result in clinical remission and cure in a majority of patients, approximately 30% of patients are primary treatment resistant or eventually relapse (rrDLBCL), and their prognosis is very poor. Recent molecular classification by large scale genomic and transcriptomic profiling of newly diagnosed DLBCL tumors has resulted in a deeper understanding of disease drivers, however, the molecular heterogeneity of rrDLBCL is yet to be fully explored. Therefore, in this study, we used an unsupervised approach to better define transcriptional and genetic programs in rrDLBCL samples hypothesizing that molecular stratification within this high-risk group may lead to a better understanding of this disease, which remains an unmet need, and a more personalized therapeutic approach. Methods Data used in this study included RNA (n=143) and whole exome (n=126) sequencing data from available FFPE tumor samples at the time of a relapse (any line of treatment, r1-r10 relapse timepoints included in analysis, one per patient), consented to the Molecular Epidemiology Resource (n=61), banked in the Mayo Lymphoma Biobank (n=50), or consented to the CC-122-ST-001 clinical trial (n=32, NCT01421524). Unsupervised clustering was performed on protein coding gene expression values using the non-negative matrix factorization (NMF) approach. NMF was carried out by performing 200 runs on cluster sizes from 2 to 6. Cell of origin (COO) was determined using the method described by Reddy et al. The tumor microenvironment was analyzed using CIBERSORTx and Lymphoma Microenvironment Classification (LME). Genetic classification was done using LymphGen. Results In order to identify unique transcriptional programs in rrDLBCL, we implemented NMF and consensus clustering to define an ideal quantity of stable patient clusters. We identified 4 rrDLBCL patient clusters (rrC1-rrC4) as the most stable solution, based on consensus clustering selection criteria of iterative NMF runs. While relapse timepoint was not associated with individual clusters, we did observe significant differences in tumor characteristics between the clusters. COO classification found that rrC1 and rrC4 were enriched for GCB-DLBCL while rrC3 was enriched for ABC-DLBCL classification (P < .001). To explore the biologic programs in each cluster, we used pathFindR, which leverages protein-protein interaction networks to identify disease related pathways, rrC1 and rrC4 were defined by pathways involved in cell cycle and mismatch repair; oxidative phosphorylation in rrC2; and MAPK and NF-kB in rrC3. Next, LME classification was done to define patterns in the tumor microenvironment and we found a significant difference between groups (P < .001), with rrC2 and rrC4 tumors enriched for a depleted TME, while rrC1 and rrC3 had a more immune rich or inflammatory TME. This was supported by CIBERSORTx analysis identifying significant differences (P > .001) in abundance of CD8 T cells, memory CD4 T cells, and M1 macrophages between clusters, with rrC4 having the lowest abundance. We then explored genetic patterns in each cluster. Gene level mutation enrichment analysis showed BCL7A mutation exclusively in C4 (6/6, P = .03). The clusters were not associated with LymphGen classification. Lastly, our recently published high risk signature classification that is associated with early clinical failure and is characterized by metabolic dysregulation, a depleted TME, T53 alterations, and poor outcome, was significantly different between the groups (P = .03) with enrichment for high-risk cases in rrC4. In summary, we show for the first time that rrDLBCL patients can be classified into four gene expression clusters that are associated with distinct pathway, TME, and genetic programs. These clusters should now be tested to learn if they can help select patients for newer therapies for rrDLBCL such as CAR-T and bispecific antibodies.

Molecular Determinants of Sensitivity to Polatuzumab-Vedotin in Diffuse Large B-Cell Lymphoma

Purpose: Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the B cell surface antigen CD79B. When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL overall; however, there is noted heterogeneity in response to Pola-V, with germinal center b-cell (GCB) DLBCL showing no added benefit to the addition of Pola-V compared to standard immunochemotherapy. We aimed to identify molecular determinants of sensitivity or resistance to Pola-V. We hypothesized that these might lead us to innovative strategies to improve on-target tumor killing by Pola-V, or to find novel biomarkers that predict drug resistance. Methods: We employed combined drug-sensitization and CD79B-sorted CRISPR-Cas9 screening to identify molecular determinants of sensitivity to CD79B-directed, tumor killing by Pola-V in 9 cell lines representing different molecular subtypes of DLBCL. Results: Our results reveal the striking impact of epitope glycosylation, specifically a2,6 sialylation, on the binding of Pola-V to CD79B and thereby its ability to kill tumor cells. Specifically, we identify the exact glycosylated residues on CD79A and CD79B which create a glycan shield around the Pola-V binding site, precluding binding to its target. We show how genetic, pharmacological and enzymatic approaches that remove terminal sialic acid residues from these N-linked glycans lead to enhanced tumor killing by Pola-V. We hypothesize and test multiple methods of targeting this pathway in order to enhance Pola-V killing both in vitro and in vivo. Finally, we reveal a previously unappreciated role for the ubiquitin ligase KLHL6 in regulating CD79B protein abundance and surface expression of the B cell antigen receptor (BCR), including how this pathway is used by physiological germinal center B cells and how it is corrupted to enhance BCR expression in GCB DLBCL. Conclusions: These findings unravel the molecular basis of response heterogeneity to Pola-V and identify approaches that might be deployed therapeutically to enhance the efficacy of CD79B-specific tumor killing. In addition, we identify how KLHL6 determines expression of the BCR in both physiological and malignant germinal center B cells, and how KLHL6 mutation may modulate sensitivity of GCB DLBCL tumors to Pola-V.

DLBCL-associated NOTCH2 mutations escape ubiquitin-dependent degradation and promote chemoresistance

AbstractDiffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullin–really interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemoresistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane–associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective γ-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.

Abstract 6301: Post-translational modifications regulate sensitivity to Polatuzumab Vedotin in DLBCL

Abstract Diffuse Large B-Cell Lymphoma (DLBCL) is an aggressive malignancy of which one-third of diagnosed patients ultimately relapse and die. New treatment paradigms for relapsed-refractory patients are needed. Most DLBCL tumors are reliant on signaling through their cell surface B-cell receptor (BCR) mediated by ITAMs on CD79A and CD79B to drive anti-apoptotic NF-kB and PI3 kinase signaling. In order to understand how surface BCR is regulated, we performed whole genome CRISPR screens in 9 DLBCL cell lines in which we sorted for high and low surface CD79B or added the CD79B targeted antibody-drug conjugate Polatuzumab-Vedotin (POLA-V). Strikingly, independent of genetic or cell of origin subtype, sgRNAs encoding the entire N-linked glycosylation pathway and specific sugar modification by addition of galactose and sialic acid were found to be enriched in high CD79B fractions and depleted in POLA-V treated samples compared to control. Further testing showed that removing sialic acid by treating with a small molecule inhibitor of sialic acid, genetic perturbation of golgi sugar transporters SLC35A1 and SLC35A2, or the a2,6 sialyltransferase ST6GAL1 resulted in more surface CD79B antibody binding with no significant change in CD79B protein abundance. The specificity of the effect of sialic acid loss on POLA antibody binding was confirmed by removing cell surface sialic acid with exogenous sialidase and observing increased binding of POLA, but not other therapeutic antibodies like Rituximab or CD19 FMC-63. Loss of SLC35A1, SLC35A2, and ST6GAL1 synergized with POLA-V but not the drug conjugate MMAE alone, showing a BCR dependent mechanism of sialic acid loss on POLA-V sensitization. In a further cell of origin subtype analysis of the germinal center B-cell (GCB) DLBCL cell lines, loss of the E3 ligase KLHL6 was found to synergize with POLA-V. Importantly, KLHL6 is heavily mutated across all germinal center derived malignancies, is more highly expressed in germinal center B-cells than other members of the B-cell lineage, and plays an unknown role in the development of autoimmune diseases. Further work will give evidence for the target of KLHL6 mediated ubiquitination and will show how this gene can drive lymphoma and autoimmune disease development in the germinal center. Citation Format: Sean R. Corcoran, Louis M. Staudt, Daniel Hodson. Post-translational modifications regulate sensitivity to Polatuzumab Vedotin in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6301.

The Single-Cell Pathology Landscape of Diffuse Large B Cell Lymphoma

Diffuse large B-cell lymphoma is a heterogeneous disease with established patterns of recurrent molecular and genetic features. However, we have limited understanding of the cellular composition of DLBCL tumors and the underlying biology of the tumor microenvironment (TME). Here we used imaging mass cytometry to simultaneously quantify 2 sets of 34 protein markers across serial sections of tumor tissues. Altogether, 57 unique markers identifying major cell lineages (CD3, CD20, PDPN, CD68, etc.), immune function (IDO, PD1, Granzyme B, etc.) and tumor phenotypes (IRF4, BCL6, p53, etc.) were quantified at single-cell resolution across 545 tumor cores from 328 primary DLBCL tumors. Single-cell clustering and cell community analysis identified 27 distinct cell types and 19 cell communities with 5 overarching patterns of tumor composition and structure (Fig. 1A). "Cold" microenvironments, tumors depleted of infiltrating immune and stromal cells, were the most frequent and accounted for 35.1% (n=115) of the imaged tumors. "Cytotoxic" TMEs (20.7%, n=68) were significantly enriched for CD8 T cells expressing granzyme B and LAG3, as well as CD163+ M2-like macrophages expressing IDO and S100A9. "Stromal" tumors (21.3%, n=70) were enriched for PDPN-positive stroma and M1-like macrophages. The "CD206" TMEs (8.2%, n=27) were enriched for CD206+ M2-like macrophages and Tregs. The "T cell" TMEs (14.6%, n=48) were depleted of macrophages and significantly enriched for CD4 T cells and Tregs. Separately, we also found that 98% (213/217) of cases with replicate tumor cores did not have statistically meaningful differences in tumor composition between cores (chi-squared: p-value >0.05). Multivariate Cox proportional hazard analysis (Fig. 1B) found the TME clusters to be significantly associated with both overall survival (OS; log-rank p < 0.001) and progression-free survival (PFS; log-rank p < 0.005). Notably, the cytotoxic TMEs were found to manifest the most unfavorable clinical outcomes (OS; HR = 2.69; 95% CI: 1.26-5.35; p-value < 0.001), whereas the cold tumors featured a modest trend towards inferior survival (OS; HR=1.5; 95% CI: 0.76-3.01; p-value <0.25). The remaining TME clusters were found to have comparably superior outcomes. Importantly, after correcting for cell of origin (COO) and IPI score, cytotoxic TMEs maintained significant association with inferior OS (HR = 1.78; 95% CI: 1.06-2.98; p-value < 0.05) and PFS (HR=1.85; 95% CI: 1.18-2.90, p-value < 0.01). Furthermore, cytotoxic TMEs were found to be significantly enriched in patients with stage III & IV disease (p-adj <0.001; 76% vs 36%-50% in other TMEs). We developed machine learning frameworks to reproduce the TME classifications with a significantly reduced feature set. Random forest classifiers achieved an overall multiclass classification accuracy of 84% (individual classes 74%-90%) with features from 7 biomarkers. We evaluated 23 phenotypic markers of the malignant B-cell populations for relationships with the TME. Malignant B cells in the cytotoxic TMEs had significantly elevated MHC-I, PD-L1, and vimentin expression (p-adj <0.01) and were enriched with ABC-like DLBCLs (50% vs. 27% in other TMEs; p-adj <0.05). Cold TMEs had significantly elevated BCL2 and Ki67 proliferative index, and lower MHC-I expression. Stroma TMEs were enriched with GCB-like DLBCLs (75.0% vs 53.3% in other TMEs; p-adj <0.01). Consistent with this, we found the BCL2-MYC 'double-hit' signature and the 'EZB-MYC+' LymphGen genetic group to be significantly enriched in cold TMEs (p-adj <0.001), with 68.2% of EZB-MYC+ belonging to the cold cluster. The MCD LymphGen class was also significantly associated with TME compositions (Fisher's test: p-value <0.01); however, it was predominately split between cold (50.0%) and cytotoxic (36.4%) TMEs. There were no MCD cases presenting the 'Stroma' or 'T cell' TME. In summary, we present the first comprehensive assessment of the molecular and cellular landscape of DLBCL by highly multiplexed imaging. Collectively, these results suggest the cold and cytotoxic TMEs may represent a dichotomy of 2 divergent paths to aggressive disease, either extreme immune dysfunction (cytotoxic) or evasion (cold). Identifying TME compositions in patients will improve patient stratification and may provide helpful context for the milieu of immune engaging therapies currently being evaluated for the treatment of DLBCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DTX1 Controls Germinal Center B-Cell Development and Lymphomagenesis

DTX1 is highly expressed in germinal center B (GCB)-cells and is targeted by coding mutations in 17% of DLBCL, including 50% of the BN2 genetic subtype that is enriched for the ABC-like cell of origin subtype (Wright et al., Cancer Cell 2020). DTX1 has also been recognized as a prominent target of AID-mediated mutagenesis, with a high rate of non-coding regulatory element (RE) mutations being observed across DLBCL subtypes. The DTX1 gene encodes a ring finger ubiquitin ligase that regulates NOTCH1 recycling. However, the role of DTX1 in GC B-cell development and lymphomagenesis has not been thoroughly investigated. We characterized mutations within the regulatory element of DTX1 using whole genome sequencing data from ICGC and TCGA, together with H3K27Ac ChIP-sequencing data from tonsillar GCB-cells to define active regulatory elements (REs). We observed non-coding mutations of DTX1-proximal REs in 59% of DLBCL tumors. We characterized these putative REs by knocking GFP into the start codon of the endogenous DTX1 gene and performing CRISPR/Cas9 tiling, using reductions of GFP expression as a surrogate for reduced DTX1 RE activity. We observed a significant loss of GFP expression with DTX1 RE-targeted gRNAs compared control gRNAs, confirming these recurrently mutated regions as active REs. We therefore generated a novel floxed Dtx1 genetically engineered mouse model and crossed with Aid-cre for GCB-specific conditional knock-out (cKO). Immunized mice were euthanized at the peak of the GC reaction and analyzed by flow cytometry and multispectral immunofluorescence (mIF) imaging. Mice with monoalleleic (Dtx1+/-) or bialleleic (Dtx1-/-) cKO of Dtx1 had a significant increase in GCB cell frequency compared to wild-type (Dtx1+/+) littermates, with a significant skew of polarization towards the light zone. This was confirmed by mIF analysis, which showed significantly larger and more abundant GCs, and a significantly higher fraction of Ki67+ cells within the GCs, in Dtx1+/- compared to Dtx1+/+ mice. We therefore investigated whether Dtx1 loss may confer a competitive advantage within the GC reaction by generating Dtx1+/+ / Dtx1+/- mixed chimera models in Rag-deficient recipients. Dtx1+/- cells were significantly over-represented within the GCB compartment and showed a significant light-zone skew, and significantly higher frequencies of proliferating (Edu+) GCB cells within the light-zone relative to Dtx1+/+ cells. We therefore investigated changes in GC development using scRNA-sequencing PNA-enriched GCB cells from SRBC-immunized Dtx1+/+ (n=4) and Dtx1+/- littermates (n=4). This confirmed a significant light-zone polarization within GCB cells, and highlighted a dramatic reduction in pre-memory cells within Dtx1+/- mice. Furthermore, differential gene expression analysis identified significant alterations in light-zone gene expression programs within Dtx1+/- GCB cells. To test whether Dtx1 loss may participate in the early stages of lymphomagenesis, we performed serial re-challenge with SRBCs monthly for 3 months and 10 days later euthanized mice. Histological analysis of major organs revealed a significant increase in the frequency of lymphoid aggregates within the lungs, liver and kidneys, indicating extranodal dissemination. In conclusion, these data demonstrate for the first time that highly-recurrent mutations in DLBCL target an active RE for DTX1, resulting in inhibition of DTX1 expression. Dtx1 haploinsufficiency confers a selective advantage to GCB cells that promotes GC hyperplasia with light-zone polarization and alters GCB cell differentiation trajectories, and can result in extranodal dissemination of Dtx1+/- GCB cells following chronic antigen stimulation.

Dose Response of Bulky Tumors in Relapsed/Refractory Diffuse Large B-Cell Lymphoma

<h3>Purpose/Objective(s)</h3> This study sought to assess whether there is a radiotherapy (RT) dose response for bulky tumors in relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). <h3>Materials/Methods</h3> Patients with r/r DLBCL (age ≥18 years) treated with salvage- or palliative-intent RT (2008-2020) at a single institution were included. Course-level data were examined to assess in-field responses of index lesions. Courses used for either post-therapy consolidation, CNS or skin disease, and TBI conditioning were excluded. Index lesion size ≥7.5 cm was considered bulky. EQD2s (α/β = 10) were calculated to accurately compare biologic effective doses between conventional and hypofractionated (≥2.5 Gy/fraction) schemes. Post-treatment responses of index lesions were classified using Lugano Criteria. Objective response rates (ORR), defined as achieving either CR or PR, were compared between non-bulky and bulky tumors using Fisher's exact test. Freedom from local progression (FFLP) and overall survival (OS, patient-level data) from RT start date were recorded. Bulky disease impacts on FFLP and OS were assessed using Kaplan-Meier and multivariable-adjusted Cox proportional hazard regression analyzes. <h3>Results</h3> 151 r/r DLBCL patients underwent 183 RT courses (median follow-up time: 6 months, IQR: 2-17 months). Median age at RT was 67 years (IQR: 56-72 years) with a male/female ratio of 55%/45%. Non-bulky and bulky tumors were treated in 109 (60%) and 74 (40%) cases, respectively. Intent was classified as salvage or palliative in 68 (37%) and 115 (63%) cases, respectively. Median EQD2 was 33 Gy (IQR: 23-39 Gy) with hypofractionation used in 84 (46%) cases. Of those with post-RT imaging (n = 146, 80%), there was a trend towards lower ORR for bulky vs. non-bulky tumors (50% vs. 65%, p = 0.087; CR: 21% vs. 42%, PR: 29% vs. 23%, SD: 28% vs. 14%, PD: 22% vs. 22%). For bulky tumors, RT regimens with EQD2s >30 Gy were associated with better ORR (≤30 Gy vs. >30 Gy: 27% vs. 64%, p = 0.014), whereas a lower EQD2 cut-off was sufficient for non-bulky tumors (<20 Gy vs. ≥20 Gy: 38% vs. 73%, p = 0.0076). In all courses, bulky tumors were significantly associated with shorter FFLP (median: 5.6 months vs. not reached, HR = 2.30, 95% CI: 1.24-4.26, p = 0.0079) and OS (median: 3.7 vs. 10.1 months, HR = 1.66, 95% CI: 1.06-2.58, p = 0.025). Amongst bulky tumors, there was a trend towards improved FFLP with RT regimens with higher EQD2s (20-30 Gy vs. <20 Gy - median: 4.2 vs. 2.3 months, HR = 0.38, 95% CI: 0.09-1.62, p = 0.19; >30 Gy vs. <20 Gy - median: not reached vs. 2.3 months, HR = 0.34, 95% CI: 0.11-1.01, p = 0.053). <h3>Conclusion</h3> In this study, bulky r/r DLBCL tumors were associated with less favorable outcomes in salvage and palliative settings. If durable local control of bulky tumors is needed, RT regimens using higher EQD2s (>30 Gy) should be considered, including cases where shortened, hypofractionated courses are opted for such as during the SARS-CoV-2 pandemic, bridging to CAR-T cell infusion, or prior to allogeneic stem cell transplantation.

Impact of Molecular Features of Diffuse Large B-Cell Lymphoma on Treatment Outcomes with Anti-CD19 Chimeric Antigen Receptor (CAR) T-Cell Therapy

Impact of Molecular Features of Diffuse Large B-Cell Lymphoma on Treatment Outcomes with Anti-CD19 Chimeric Antigen Receptor (CAR) T-Cell Therapy

Next Generation Sequencing Highlights the Role of Cell Cycle and Apoptosis Dysregulation and Hepatitis B Infection in Refractory Diffuse Large B Cell Lymphoma

About 10-15% DLBCL are primary refractory, the underlying biology aggressiveness contributed to refractoriness remain to be defined. To date, whole-exome (WES) or whole-genome sequences (WGS) obtained from primary or refractory/relapse DLBCL tumors have been published, while the biology study conduct on patients of mixed prognosis failed to characterizing the 10-15% primary refractory cases, and WES or WGS are not applicable in clinical routine practice. To address this issue, we performed target next generation sequencing in 11 primary refractory DLBCL cases. Through pathway enrichment analysis, as expected, the cell cycle/apoptosis and P53 signaling (FDR 10-11~10-9), NF-kB (FDR ~10-9), BCR signaling(FDR ~10-9), PI3K/Akt (FDR ~10-8), MAPK(FDR ~10-10), Wnt (FDR ~10-12), Jak/STAT (FDR ~10-14) signaling were significant mutated. Furthermore, some pathogen infection pathways were also significantly affected, which was not identified as significantly mutated in previous DLBCL WGS studies, including hepatitis B virus infection(HBV) signaling pathway involving BCL-2, MYC, MYD88, CREBBP, FAS, STAT3, TP53, and 4 of the 11 (36.4%) patients were HBsAg-positive. Overall, somatic mutations or copy number aberrations of CD79B (5/11, 45.5%), TP53(4/11, 36.3%), PIM1 (4/11, 36.3%) , MYD88 (3/11, 27.2%), KMT2D (3/11, 27.2%), CREBBP(3/11, 27.2%) and IRF4 (3/11, 27.2%) were the most prevalent aberrations. We separately compared the prevalence of target gene variants in our refractory (ref)-DLBCLs to published cohorts of responsive or de novo or relapse(rel) or relapse/refractory(r/r)-DLBCLs, the profile and frequency was highly concordant to those of relapse ones, while CD79B and cell cycle regulation or apoptosis related genes were more prevalent affected in our refractory cohort than those of responsive or de novo ones(table 2). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Using Targeted Enrichment RNA Sequencing in the Diagnosis of Hematologic Neoplasms in Fresh and Paraffin-Embedded Tissue

Introduction: RNA profiling using microarray has been used extensively in cancer research, but its clinical utilization remains limited due to its poor reproducibility and difficulty in standardization.Next generation sequencing (NGS) technologies have advanced significantly in accuracy, reproducibility and simplicity. Targeted enrichment of RNA and NGS sequencing provides a reliable and reproducible approach for RNA profiling that is amenable to implementation in clinical laboratories. We explored using this approach in evaluating the utility of targeted enrichment RNA NGS sequencing in the diagnosis of various hematologic neoplasms.Methods: RNA from fresh bone marrow or peripheral blood samples from patients with ALL (N=29), AML (N=18), and CLL (N=25) and formalin fixed paraffin embedded (FFPE) samples from DLBCL (N=79), sarcoma (N=12) and various epithelial tumors (N=22). We used the TruSight RNA Pan-Cancer Panel (Illumina, San Diego, CA) for detecting the expression of 1385 genes. The results were standardized so the expression values had mean 0 and standard deviation 1. Fusions and mutations were not considered in this analysis.Results: The generated expression profiles provided simple data on various disease markers including CD3, CD19, CD8 and PAX5 that can be used directly for distinguishing between diseases. However, using statistical analysis we were able to distinguish between various diseases with high accuracy. We first focused on classifying ALL vs AML vs CLL since fresh samples were used in generating the expression data on these samples. Accounting for multiple hypothesis testing and setting false discovery rate (FDR) at 0.05, we found 1074 genes as statistically significant. Using the top 5 principal components, we can build a multinomial logit model with 93.1% accuracy rate based on leave-one-out cross-validation. Using a tree model with only FANCI and CIITA genes levels, we were able to diagnose ALL, AML, and CLL with an 88.9% accuracy rate also based on leave-one-out cross-validation.Similarly for the three FFPE groups (DLBCL, solid epithelial tumor, and sarcoma), setting FDR at 0.05 resulted in 1169 genes that were significantly different across the three groups. Using the top 5 principal components, we can build a multinomial logit model with a 92.0% accuracy rate on the leave-one-out testing. Using a simple tree model and the expression levels of CD79A and MALAT1 genes, we were able to distinguish between the three groups with a 94.7% accuracy rate on leave-one-out testing.Mixing FFPE with fresh tissue samples and analyzing all 6 groups, we found 1361 genes significantly by setting FDR at 0.05 to account for multiple hypothesis testing. A multinomial logit model with the top 5 principal components provided 89.7% accuracy in classifying the 6 disease using the leave-one-out testing.Conclusion: This data suggests that using targeted enrichment RNA sequencing and quantifying the expression of genes provide reliable data that can be used for distinguishing between various diseases. This is applicable to fresh and FFPE tissue. This approach also provides information on fusion genes and mutations. While these sources of information were not used in this model, they can provide additional important information not only to improve the model, but also to provide information on prognosis and potential response to therapy. [Display omitted] DisclosuresMa:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Funari:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Deciphering Discordance between MYC mRNA and MYC IHC in DLBCL: The Role of MYC Exon 2 Mutations and N11S Polymorphism

Deciphering Discordance between MYC mRNA and MYC IHC in DLBCL: The Role of MYC Exon 2 Mutations and N11S Polymorphism

Genomic insights into the pathogenesis of Epstein\u2013Barr virus-associated diffuse large B-cell lymphoma by whole-genome and targeted amplicon sequencing

Epstein–Barr virus (EBV)-associated diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS) constitute a distinct clinicopathological entity in the current World Health Organization (WHO) classification. However, its genomic features remain sparsely characterized. Here, we combine whole-genome sequencing (WGS), targeted amplicon sequencing (tNGS), and fluorescence in situ hybridization (FISH) from 47 EBV + DLBCL (NOS) cases to delineate the genomic landscape of this rare disease. Integrated WGS and tNGS analysis clearly distinguished this tumor type from EBV-negative DLBCL due to frequent mutations in ARID1A (45%), KMT2A/KMT2D (32/30%), ANKRD11 (32%), or NOTCH2 (32%). WGS uncovered structural aberrations including 6q deletions (5/8 patients), which were subsequently validated by FISH (14/32 cases). Expanding on previous reports, we identified recurrent alterations in CCR6 (15%), DAPK1 (15%), TNFRSF21 (13%), CCR7 (11%), and YY1 (6%). Lastly, functional annotation of the mutational landscape by sequential gene set enrichment and network propagation predicted an effect on the nuclear factor κB (NFκB) pathway (CSNK2A2, CARD10), IL6/JAK/STAT (SOCS1/3, STAT3), and WNT signaling (FRAT1, SFRP5) alongside aberrations in immunological processes, such as interferon response. This first comprehensive description of EBV + DLBCL (NOS) tumors substantiates the evidence of its pathobiological independence and helps stratify the molecular taxonomy of aggressive lymphomas in the effort for future therapeutic strategies.

Leveraging gene expression subgroups to classify DLBCL patients and select for clinical benefit from a novel agent

AbstractDiffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, commonly described by cell-of-origin (COO) molecular subtypes. We sought to identify novel patient subgroups through an unsupervised analysis of a large public dataset of gene expression profiles from newly diagnosed de novo DLBCL patients, yielding 2 biologically distinct subgroups characterized by differences in the tumor microenvironment. Pathway analysis and immune deconvolution algorithms identified higher B-cell content and a strong proliferative signal in subgroup A and enriched T-cell, macrophage, and immune/inflammatory signals in subgroup B, reflecting similar biology to published DLBCL stratification research. A gene expression classifier, featuring 26 gene expression scores, was derived from the public dataset to discriminate subgroup A (classifier-negative, immune-low) and subgroup B (classifier-positive, immune-high) patients. Subsequent application to an independent series of diagnostic biopsies replicated the subgroups, with immune cell composition confirmed via immunohistochemistry. Avadomide, a CRL4CRBN E3 ubiquitin ligase modulator, demonstrated clinical activity in relapsed/refractory DLBCL patients, independent of COO subtypes. Given the immunomodulatory activity of avadomide and the need for a patient-selection strategy, we applied the gene expression classifier to pretreatment biopsies from relapsed/refractory DLBCL patients receiving avadomide (NCT01421524). Classifier-positive patients exhibited an enrichment in response rate and progression-free survival of 44% and 6.2 months vs 19% and 1.6 months for classifier-negative patients (hazard ratio, 0.49; 95% confidence interval, 0.280-0.86; P = .0096). The classifier was not prognostic for rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone or salvage immunochemotherapy. The classifier described here discriminates DLBCL tumors based on tumor and nontumor composition and has potential utility to enrich for clinical response to immunomodulatory agents, including avadomide.

Abstract PO-48: Cytotoxic mechanism of a novel transferrin receptor-targeting chemotherapeutic nanocarrier for use in diffuse large B-cell lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL), the most common hematologic malignancy, is an aggressive form of non-Hodgkin lymphoma. Approximately 60% of DLBCL patients achieve long-term disease-free survival from frontline R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), but patients with relapsed or refractory disease have poor prognosis. Doxorubicin (Dox) remains the most active anti-lymphoma agent, but its use is limited by well-characterized toxicities, including potentially irreversible cardiac damage. Targeted and specific delivery of Dox to tumors to overcome these limitations has been an area of active research. Targeted delivery of Dox via the transferrin receptor (TFR1) has preclinical efficacy in solid malignancies but has yet to be explored in lymphoma. We have uncovered for the first time a significant association between TFR1 expression and poor prognosis in DLBCL. Carbon-nitride dots (CNDs) are third-generation nanocarriers with inherent photoluminescence, which are easily conjugated with a wide variety of substrates. We conjugated Dox and transferrin (TF), the ligand for TFR1, to CNDs to develop CND-Dox-TF (CDT). CDT is uniquely designed to enhance Dox delivery to TFR1-expressing DLBCL tumors while limiting effects on nonmalignant tissues. BJAB and Farage cell lines treated with CDT for 24 hours underwent apoptosis at a significantly lower concentration when compared to Dox (50 nM vs. 1000 nM). Overall, CDT was 1-2 Log10 more potent than Dox against DLBCL cell lines. Co-incubation with 250 uM of antagonist holo-transferrin significantly decreased BJAB and Farage sensitivity to CDT, confirming activity through TFR1. Confocal microscopy of BJAB cells incubated up to 24 hours with 250 nM Dox and 30 nM CDT reveled enhanced nuclear colocalization of Dox at a significantly lower concentration. Prior dose-finding experiments in non-tumor bearing mice identified a CDT safe working dose of 33.0 mg/kg containing 16% moles of Dox in comparison to Dox maximum-tolerated dose (MTD) 3.3 mg/kg. We engrafted 2 groups of 10 NSG mice with a Dox-sensitive high-TFR1 expressing patient-derived xenograft (PDX) tumor. Mice were dosed with CDT and Dox on day 0, 14, and 24. CDT demonstrated similar anti-lymphoma efficacy with reduced toxicity: CDT-treated mice displayed no significant decline in body weight, which is characteristic and was observed following each single-agent Dox treatment. Histology analyses revealed little to no obvious damage to CDT-treated nonmalignant tissues, including myocardium. With a working dose of CDT identified, we now have under way clinically relevant R-CHOP vs. R-nanoCHOP (CDT substituted for Dox) assessments in our PDX mouse model. Capitalizing on the unique design, we expect to observe improved anti-lymphoma efficacy, with decreased toxicities and an improvement in overall survival. In sum, we provide mechanistic insight for novel DLBCL nano-chemotherapy and illustrate preclinical efficacy for a promising new therapeutic approach. Citation Format: Artavazd Arumov, Piumi Y. Liyanage, Asaad Trabolsi, Evan R. Roberts, Braulio Ferreira, Daniel Bilbao, Roger M. LeBlanc, Jonathan H. Schatz. Cytotoxic mechanism of a novel transferrin receptor-targeting chemotherapeutic nanocarrier for use in diffuse large B-cell 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-48.

Activation-induced cytidine deaminase localizes to G-quadruplex motifs at mutation hotspots in lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis.

Abstract 2657: Sphingosine-1-phosphate receptor 1 signalling modulates macrophage recruitment to diffuse large B cell lymphomas and the phagocytosis of rituximab-opsonized tumor cells

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma. Currently, approximately 40% of DLBCL patients treated with standard of care therapies, which include a combination of immunotherapy (rituximab; R) and chemotherapy (CHOP), will have disease that is refractory or will relapse. Tumor-associated macrophages can phagocytose opsonised DLBCL tumor cells and are therefore centrally important in determining therapeutic outcomes for patients treated with R-CHOP. Recent data from our lab and from others suggests that the modulation of sphingosine-1-phosphate (S1P) signalling may therapeutically benefit some patients with this tumor. Here, we have investigated the effects of S1P on macrophage functions relevant to DLBCL. We show that S1P signalling through the major receptor, S1P-receptor 1 (S1PR1), suppresses the phagocytosis of rituximab-opsonized DLBCL cells. However, we also show that chemotherapy potently induces monocyte recruitment to DLBCL tumors in vivo and that S1PR1 is a primary mediator of monocyte migration both in vitro and in vivo. According to our data, S1PR1 signalling inhibitors could improve the therapeutic efficacy of rituximab-based therapies for DLBCL patients. However, our data suggests that these drugs should be given only after chemotherapy and before rituximab administration so as to maximize the S1P-mediated recruitment of therapeutic macrophages to the tumor site. Citation Format: Tracey Adams Perry, Lauren Lupino, Katerina Vrzalikova, Navta Masand, Pamela R. Kearns, Paul G. Murray. Sphingosine-1-phosphate receptor 1 signalling modulates macrophage recruitment to diffuse large B cell lymphomas and the phagocytosis of rituximab-opsonized tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2657.

Targeting BCL10 by small peptides for the treatment of B cell lymphoma

Rationale: Constitutive activation of the NF-κB signalling pathway plays a pivotal role in the pathogenesis of activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCLs), the most aggressive and chemoresistant form of DLBCL. In ABC-DLBCLs, the CARMA1-BCL10 (CB) complex forms a filamentous structure and functions as a supramolecular organizing centre (CB-SMOC) that is required for constitutive NF-κB activation, making it an attractive drug target for ABC-DLBCL treatment. However, a pharmaceutical approach targeting CB-SMOC has been lacking. Here, we developed Bcl10 peptide inhibitors (BPIs) that specifically target the BCL10 filamentation process.Methods: Electron microscopy and immunofluorescence imaging were used to visualize the effect of the BPIs on the BCL10 filamentation process. The cytotoxicity of the tested BPIs was evaluated in DLBCL cell lines according to cell proliferation assays. Different in vitro experiments (pharmacokinetics, immunoprecipitation, western blotting, annexin V and PI staining) were conducted to determine the functional mechanisms of the BPIs. The in vivo therapeutic effect of the BPIs was examined in different xenograft DLBCL mouse models. Finally, Ki67 and TUNEL staining and histopathology analysis were used to evaluate the antineoplastic mechanisms and systemic toxicity of the BPIs.Results: We showed that these BPIs can effectively disrupt the BCL10 filamentation process, destabilize BCL10 and suppress NF-κB signalling in ABC-DLBCL cells. By examining a panel of DLBCL cell lines, we found that these BPIs selectively repressed the growth of CB-SMOC-dependent DLBCL cells by inducing apoptosis and cell cycle arrest. Moreover, by converting the BPIs to acquire a D-retro inverso (DRI) configuration, we developed DRI-BPIs with significantly improved intracellular stability and unimpaired BPI activity. These DRI-BPIs selectively repressed the growth of CB-SMOC-dependent DLBCL tumors in mouse xenograft models without eliciting discernible adverse effects.Conclusion: We developed novel BPIs to target the BCL10 filamentation process and demonstrated that targeting BCL10 by BPIs is a potentially safe and effective pharmaceutical approach for the treatment of ABC-DLBCL and other CB-SMOC-dependent malignancies.

Combined EZH2 and BCL2 Inhibitors As Precision Therapy for Genetically Defined DLBCL Subtypes

DLCBL has recently been classified into genetically defined subtypes based on groupings of particular genetic lesions (Chapuy et al, Nat Med 2018; Schmitz et al, NEJM 2018). One predominant cluster, C3 or EZB, is defined by mutations in the chromatin modifying genes EZH2, KMT2D, and CREBBP as well as alterations in BCL2 including mutations and/or translocation of BCL2 to the IgH enhancer. Since tumors in this cluster are likely dependent on both EZH2 and BCL2, and these oncogenes carry out their effects through distinct mechanisms and pathways, targeting both of these oncogenes is a rational therapeutic approach. We hypothesized that EZH2 inhibition and BCL2 inhibition would be synergistic in DLBCL with characteristics of the C3/EZB cluster. To test this, we evaluated the EZH2 inhibitor tazemetostat and the BCL2 inhibitor venetoclax in DLBCL cells, 3D lymphoma organoids, and patient derived xenografts. To assess the effect of combination therapy with tazemetostat and venetoclax, we administered each drug alone and the combination in a panel of DLBCL cell lines, including cells with and without EZH2 mutation and BCL2 translocation. In DLBCL cells with both a BCL2 translocation and EZH2 mutation, the combination resulted in increased killing compared to either drug alone (Figure 1, SUDHL6 (p&amp;lt;0.005), WSU-DLCL2 (p&amp;lt;0.005), and OCI-LY1 (p&amp;lt;0.005)). In contrast, in cells with WT EZH2 and no BLC2 translocation, the effect of the combination was not different than either drug alone. To evaluate for synergy, cells were exposed to increasing doses of each drug alone and the combination. The combined response was evaluated using the Chou-Talalay method. Synergy between tazemetostat and venetoclax was observed in SUDLH-6 (CI value 0.03), WSU-DLCL2 (CI 0.26) and OCI-LY1 (CI 0.06) but not in Farage and LY7 both of which have WT EZH2 and no BCL2 translocation. Since cell lines in suspension do not reflect lymph node architecture, we developed a 3D lymphoma "organoid" culture system that consists of extracellular matrix, lymphoma cells, and stromal cells (Tian et al, Biomaterials 2015; Beguelin et al, Nat Commun 2017). GCB-DLBCLs express integrin αvβ3 that may bind to RGD peptides or vitronectin in tumor extracellular matrix. Based on this, we developed matrix metalloproteinase (MMP)-degradable 3D lymphoma hydrogels functionalized with RGD peptides. To generate organoids, we homogeneously encapsulated DLBCL cells (40,000/gel) in 10 µL hydrogel droplets fabricated in individual wells of a 96-well plate. We established two organoid systems to evaluate response to tazemetostat/venetoclax combination therapy: 1) OCI-LY1 organoids; 2) patient derived xenograft (PDX) organoids. The PDX organoids were generated from a patient tumor after propagation in NSG mice. The PDX tumor harbors both a BCL2 translocation and EZH2 mutation. Organoid viability was evaluated using immunofluorescence for calcein AM (live cells) and ethidium homodimer (dead cells) as well as flow cytometry. In both OCI-Ly1 organoids and PDX organoids, tazemetostat and venetoclax had minimal activity as single agents, however the combination resulted in significant cell killing (Figure 2). To investigate potential mechanisms of synergy, we evaluated RNA-seq profiles of a panel of DLBCL cell lines (n=26) treated with vehicle vs. EZH2 inhibitor (Brach et al, Mol Cancer Ther 2017). Preliminary data suggest that EZH2 inhibition induces expression of pro-apoptotic proteins genes including BCL2L11 (Wilcoxon p=0.01), BAD (p=0.02), BMF (p&amp;lt;0.01), BCL2L13 (p=0.02), and BCL2L14 (p&amp;lt;0.01). BCL2 inhibition with venetoclax may be further enhancing pro-apoptotic signals and lymphoma cell death, especially in C3/EZB DLBCL tumors with dependence on BCL2. In summary, using novel model systems, we have demonstrated that BCL2 inhibition combined with EZH2 inhibition results in synergistic anti-tumor effect that is anticipated to be especially effective as precision therapy for the newly identified cluster 3/EZB DLBCL subtype. A clinical trial of this combination is currently in development. Disclosures Melnick: Janssen: Research Funding; Constellation: Consultancy; Epizyme: Consultancy. Roth:Janssen: Consultancy; ADC Therapuetics: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees.

Dual Inhibition of MCL1 and BCL2 in Lymphoma Using Tumor Targeted Nanoparticles

Cancer and normal cell utilize multiple members of the anti-apoptotic BCL2 proteins to protect them from cell death. Selective targeting of the BCL2 protein has a limited therapeutic value in the majority of cancer patients, including lymphoma, mainly due to the ability of cancer cells to utilize other members of the BCL2 family that are functionally redundant. While therapeutic targeting of multiple proteins will enhance tumor cell killing, such strategies will also lead to increased toxicity of vital organs. Here, we examined the single agent activity of the novel MCL1 inhibitor S63845 in a panel of lymphoma cell lines representing different cell of origin and differentiation states. Cell lines that were sensitive to S63845 expressed lower levels of BCL2. Consequently, we demonstrated that dual inhibition of BCL2 and MCL1, using the small molecule inhibitors Venetoclax and S63845, can lead to sustained and durable remissions of upto 250 days in mice harboring human DLBCL tumors after only 10 treatments (100 mg/kg Venetoclax oral and 25 mg/kg S63845 iv 5x/week). Monotherapy with S63845 or Venetoclax had a modest effect on tumor volume (41% and 36% tumor volume decrease upon S63845 or Venetoclax monotherapy, respectively). Immunohistochemistry of tumors from different treatment groups revealed that treatment with each drug alone resulted in minimal cleavage of caspase 3. In contrast, the combination of Venetoclax and S63845 resulted in diffuse caspase 3 cleavage, and cleavage of the caspase 3 target Poly (ADP-ribose) polymerase (PARP), indicative of apoptosis. However, unlike single agent therapy, combination treatment was also associated with significant weight loss of more than 15% pretreatment body weight, and hematological toxicity manifested in platelets, RBCs, WBCs and hemoglobin. Reduced dosing regimens induced complete remissions of tumors in mice but these were not durable, as tumor regrowth was observed around day 75 (~50 days after treatment cessation). To reduce the toxicity, we encapsulated either S63845 or Venetoclax into tumor targeted nanoparticles. Targeting of the drug loaded nanoparticle to the tumor is achieved by fucoidan, a fucosylated polysaccharide with nanomolar affinity to P-selectin, an inflammatory marker that is enriched in tumor sites. Fucoidan-decorated nanoparticles were synthesized by coencapsulating S63845 and a near infrared fluorophore IR783 that enables imaging. In vivo and ex vivo imaging studies demonstrated preferential targeting of the nanoparticles to the lymphoma while sparing vital organs, including the liver, heart, and kidneys (5 to 8-fold enrichment in the tumor). When MCL1 or BCL2 inhibitors were encapsulated into lymphoma-targeting nanoparticles we could lower the administered dose of these drugs by 3.5 and 6.5-fold, respectively. Combining either a nanoparticle encapsulated S63845 or Venetoclax with the other drug systemically administered induced durable remissions. When combination therapy was administered with one drug encapsulated as a nanoparticle, we did not observe significant weight loss or hematological toxicity, demonstrating that this nanoparticle delivery platform can deliver therapeutic drug concentration to tumor cells while sparing normal cells from treatment toxicity. This is the first study to demonstrate the potential therapeutic value of targeted BH3 mimetics nanoparticles in lymphoma. Disclosures Heller: Goldilocks Therapeutics Inc.: Equity Ownership, Other: Cofounder; Lipidsense Inc.: Other: Cofounder; Concarlo Holdings LLC: Consultancy. Younes:Merck: Honoraria, Research Funding; Takeda: Honoraria; Pharmacyclics: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding; Biopath: Consultancy; Xynomics: Consultancy; Epizyme: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; HCM: Consultancy; BMS: Research Funding; Syndax: Research Funding; Abbvie: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Curis: Honoraria, Research Funding.