Kendrick, Samantha

Abstract Introduction: Diffuse Large B cell Lymphoma (DLBCL) is up to 80-fold more likely to occur and follows a more aggressive clinical course in HIV positive (+) individuals compared to HIV negative (-) individuals. Moreover, the vast majority frequently present at advanced stages with high tumor burdens with poor prognosis. The molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood; however, it is suspected to depend on decreased host immune surveillance and alternative mechanisms of tumorogenesis. In this study, we sought to identify the differences in gene expression between HIV(+) and HIV(-) DLBCL cohorts that may contribute to the unique features of HIV(+) DLBCL. Methods: We performed a retrospective gene expression profiling (GEP) study using a total of 70 DLBCL cases, 31 HIV(+) obtained from AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 39 HIV(-) institutional cases. The H&Es for all cases were reviewed by a hematopathologist and tumor content determined. Cases that did not meet the minimum tumor content threshold of 70% were macro-dissected. RNA was extracted from 4x 5µm formalin fixed, paraffin embedded (FFPE) sections using the Qiagen AllPrep DNA/RNA FFPE Kit and quantified using a NanoDrop. The Lymph2Cx assay, which accurately subtypes DLBCL in FFPE tissues (Scott et al 2014), was used to determine the cell of origin (COO) subtypes: Germinal Center B cell (GCB), Activated B cell (ABC) and Unclassifiable (UNC). Digital GEP was performed using the NanoString PanCancer Pathways and Immunology panels, targeting a total of over 1300 genes with known roles in cancer or immune signaling. Results: Of the 31 HIV(+) cases, Lymph2Cx subtyping showed 74% (23/31) were GCB, 13% (4/31) were ABC and 13% (4/31) were UNC. In a similar distribution to the HIV(+), and comparable to prior literature, 54% (21/39) of HIV(-) cases were GCB, 31% (12/39) were ABC and 15% (6/39) were UNC. Differential gene expression was observed between HIV(+) and HIV(-) cases, but no association with COO subtype was found. When the data was re-analyzed within the GCB subtype alone, stratification by HIV status was observed. Particularly, reduced expression of genes associated with immune regulation was observed in HIV(+) GCB cases compared to HIV(-) GCB cases. In contrast, expression of genes associated with cell cycle regulation and homologous recombination (the predominate double stranded DNA repair pathway employed during the cell cycle), were higher in HIV(+) cases compared to HIV(-) GCB cases. Conclusions: The results show, that within the GCB subtype, marked differences in gene expression are associated with HIV status. Our results suggest that the aggressive nature of DLBCL in HIV(+) patients may, in part, be mediated by enhanced cell proliferation potentiated by aberrant DNA repair. Disclosures Fogel: Natural Selection Inc: Employment. Liu: Natural Selection Inc: Employment. Lamers: Bioinfoexperts LLC: Employment.

Abstract 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.

Objective: The increased risk for persons living with HIV to develop diffuse large B-cell lymphoma (DLBCL) even in the post-antiretroviral therapy eras suggests a role beyond immunosuppression in lymphoma development. However, the mechanisms leading to lymphoma in the HIV setting are not fully understood. HIV is known to induce activation-induced cytidine deaminase (AID) levels in nonneoplastic B cells in vitro and chronic AID expression may play an important role in lymphomagenesis. Although AID expression is observed in B-cell lymphoma, studies in HIV-associated DLBCL are limited. Design: In this study, we conducted a retrospective review of DLBCL tissues from patients with and without HIV infection to compare expression of AID and B-cell receptors potentially involved in HIV and B-cell interaction. Methods: We evaluated DLBCL formalin-fixed paraffin-embedded tissues from 72 HIV-seropositive and 58 HIV-seronegative patients for AID, DC-SIGN, and CD40 protein expression. BCL2 and MYC, two well established prognostically significant oncoproteins in DLBCL, were also assessed at the protein and mRNA levels. Subset analysis was performed according to DLBCL subtype and EBV status. Results: Of note, AID expression was more frequent in HIV-associated DLBCL compared with non-HIV-associated DLBCL regardless of cell-of-origin subtype, and also displayed significantly less BCL2 expression. Despite no direct correlation with AID expression, the HIV-DLBCL tissues also exhibited high levels of the DC-SIGN receptor. Conclusion: Collectively, these findings support a potential role for AID in the pathogenesis of HIV-associated lymphomas and suggest the need of further investigations into the involvement of the DC-SIGN receptor-signaling pathway.

Abstract To date, there are no effective strategies to treat patients with a particularly lethal form of diffuse large B-cell lymphoma (DLBCL) that over-express both B-cell lymphoma gene-2 (BCL2) and cellular-myelocytomatosis (MYC) oncogenes. Here, we provide a novel approach for simultaneously targeting BCL2 and MYC directly through secondary DNA structures. These structures act as molecular switches, turning gene expression on or off, and serve as scaffolds for protein and small molecule interaction. Previously identified compounds IMC-76 and GQC-05, recognize the BCL2 or MYC promoter region DNA structures, respectively, and independently lower mRNA and protein expression of each oncogene.For utility in sensitizing aggressive lymphoid tumors that concurrently dual express BCL2 and MYC, we examined the efficacy ofIMC-76 and GQC-05 in the presence and absence of a standard chemotherapy, cyclophosphamide (CPA)to inhibit tumor growth in a severe combinedimmunodeficient U2932 DLBCLxenograft mouse model. Unlike IMC-76, effects of GQC-05 in a xenograftmouse model are unknown; therefore, our initial in vivo studies involved GQC-05 alone and with IMC-76 in combination with CPA to determine the concentration for MYCdown-regulation and a maximum tolerated dose (MTD). At 2.5 and 5 mg/kg GQC-05 with 50 mg/kg CPA, we observed no effect on mean mouse weight and while the 5 mg/kg GQC-05-CPA combination group exhibited some mouse death, LD50 was not reached. Tumors from mice treated with GQC-05 and CPA were compared to diluent or CPA alone treated mice for MYC mRNA level and found to have significantly less MYC expression. As expected, there was no effect on BCL2 mRNA levels. Next, we carried over the 5 mg/kg GQC-05 to a subsequent in vivo experiment where we also tested a 10 mg/kg GQC-05 dose and included IMC-76 at 10 mg/kg, a previously established efficacious dose, to determine if we would reach an MTD, but still retain the repression of MYC. While the mice were able to tolerate the 5 mg/kg GQC-05 dose when IMC-76 and/or CPA was added to the treatment regimen, the LD50 was reached and surpassed in mice treated with the 10 mg/kg of GQC-05 combinations. However, both concentrations of GQC-05 when co-treated with IMC-76 and CPA resulted in a notable decrease of tumor MYC expression relative to untreated and CPA only treated mice as well as a detectable inhibition of BCL2. We then conducted a follow-up study to evaluate effects on tumor burden using concentrations of GQC-05 below the LD50, 5 mg/kg and 7.5 mg/kg, and also reduced the dose of CPA to 30 mg/kg. The co-treatment of mice with IMC-76 (10 mg/kg) and GQC-05 (7.5 mg/kg) in combination with CPA delayed tumor growth and decreased tumor size by 50%, 46%, and 37% at 9, 12, and 15 days post-first-day drug administration compared to the diluent and 50%, 67%, and 44% smaller tumors relative to CPA only treated mice (Figure 1). Overall, there was a prominent decrease in tumor burden in these mice as determined by area under the curveand the tumors displayed a lower expression of BCL2 and MYC at both the mRNA and protein levels. Interestingly, tumors of mice that received IMC-76 and GQC-05 also showed knock-down of BCL2 and MYC, but no effect on tumor burden unless CPA was present indicating these transcriptional inhibitors act as chemo-sensitizers. None of the various drug regimens resulted in a significant loss of mouse weight and while survival for GQC-05 7.5 mg/kg treated mice was reduced, LD50 was not reached. These are the first studies to demonstrate two differentpromoterDNA secondary structures can be targeted at the same time for direct transcriptional inhibition that leads tochemo-sensitization and slowed tumor growth in mice.Our findings demonstrate the potential utility of a dual-targeted, precision medicine-based strategy to improve the response of DLBCL patients to current chemotherapy and overcome resistant disease. Figure Tumor burden of DLBCL xenograft mice Figure. Tumor burden of DLBCL xenograft mice Disclosures Gokhale: Tetragene: Equity Ownership. Hurley:Tetragene: Consultancy, Equity Ownership. Rimsza:NCI/NIH: Patents & Royalties: L.M. Rimsza is a co-inventor on a provisional patent, owned by the NCI of the NIH, using Nanostring technology for determining cell of origin in DLBCL..

Diffuse large B‐cell lymphoma (DLBCL) is up to 17‐fold more likely to occur, follows a more aggressive clinical course and frequently presents at advanced stages in HIV infected (+) individuals compared to HIV negative (−) individuals. However, the molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood. We performed a retrospective study examining the transcriptional, genomic and protein expression differences between HIV(+) and HIV(−) germinal center B‐cell (GCB) DLBCL cases using digital gene expression analysis, array comparative genomic hybridization (CGH) and immunohistochemistry (IHC). Genes associated with cell cycle progression ( CCNA2 , CCNB1 , CDC25A , E2F1 ), DNA replication ( MCM2 , MCM4 , MCM7 ) and DNA damage repair, including eight Fanconi anemia genes ( FANCA , FANCD1/BRCA2 , FANCE , FANCG , FANCR/RAD51 , FANCS/BRCA1 , FANCT/UBE2T , FANCV/MAD2L2 ), were significantly increased in HIV(+) GCB‐DLBCL tumors compared to HIV(−) tumors. In contrast, genes associated with cell cycle inhibition ( CDKN1A , CDKN1B ) as well as apoptosis regulating BCL2 family members ( BCL2 , BAX , BIM , BMF , PUMA ) were significantly decreased in the HIV(+) cohort. BCL2 IHC confirmed this expression. Array CGH data revealed that HIV(+) GCB‐DLBCL tumors have fewer copy number variations than their HIV(−) counterparts, indicating enhanced genomic stability. Together, the results show that HIV(+) GCB‐DLBCL is a distinct molecular malignancy from HIV(−) GCB‐DLBCL; with an increased proliferative capacity, confirmed by Ki67 IHC staining, and enhanced genomic stability, the latter of which is likely related to the enhanced expression of DNA repair genes.