Citations

Abstract Lymphoma is the sixth most commonly diagnosed cancer in the US. Genome-wide association studies have identified common variants associated with risk of specific lymphoma subtypes, but less is known about the contribution of rare inherited variants in the genetic architecture of lymphoma risk. CHEK2 is important to DNA repair and 2 small studies have found evidence of an association between CHEK2 variants and risk of lymphoma. Here, we investigated loss of function (LoF) variants in CHEK2 with risk of lymphoma (overall and subtypes). The study population included newly diagnosed lymphoma cases from Mayo who were enrolled in the Lymphoma SPORE Molecular Epidemiology Resource (MER). Controls were from the Mayo Clinic Biobank, from which we excluded a prior hematologic malignancy. Whole exome sequencing was performed by Regeneron on an Illumina NovaSeq panel (mean coverage of 48X). Variants were called using GATK v4 and variant annotation was performed using BioR. All LoF variants (nonsense, frameshift and consensus splice sites) in CHEK2 with a minor allele frequency < 0.5% were included in the analyses. Logistic regression was used to estimate odds ratio (OR) and 95% confidence intervals (CI) for the association between CHEK2mutation status and risk of lymphoma overall, and by lymphoma subtypes that had more than 5 mutation carriers, which included diffuse large B-cell (DLBCL), follicular (FL), and T-cell lymphoma (TCL). All analyses were adjusted for age (at diagnosis for cases and at enrollment for controls) and sex. A total of 4,852 lymphoma cases and 49,724 controls were included in this analysis. Median age for both cases and controls was 62 years (range 18-99 years). Males accounted for 57.5% (n=2,789) of the cases and 40.6% (n=20,186) of the controls. The DLBCL (23.6% of cases) and FL (23.3% of cases) subtypes were the most common; 7.6% of cases had TCL. A total of 407 (0.7%) individuals had a LoF variant. The frequency of LoF variants was 1.2% in lymphoma cases and 0.7% in controls. LoF variants were associated with increased risk of lymphoma overall (OR=1.77; 95%CI: 1.32-2.33), DLBCL (OR=2.07; 95%CI: 1.20-3.13), and TCL (OR=2.78; 95%CI: 1.178-5.50), but not FL (OR=1.31; 95%CI: 0.65-2.34). CHEK2 c.1100delC was the most frequently mutated variant in this population, accounting for 76% of all LoF variants. Individuals with a c.1100delC variant had an increased risk of lymphoma overall (OR=1.90; 95%CI: 1.37-2.58), DLBCL (OR=2.06; 95%CI: 1.09-3.54), and TCL (OR=3.17; 95%CI:1.24-6.58), but not FL (OR=1.05; 95%CI: 0.41-2.15). When restricted to cases, there was no significant difference in age at diagnosis (P=0.62) or sex (P=0.53) between LoF variant carriers and non-carriers. In this large lymphoma case-control study, CHEK2 LoF variants were associated with increased risk of lymphoma, demonstrating that rare inherited variants may play an important role in the etiology of lymphoma. Additional work is needed to investigate missense variants in CHEK2 and risk of lymphoma. Citation Format: Nicholas J. Boddicker, Raphael Mwangi, Dennis P. Robinson, Allison C. Rosenthal, Thomas M. Habermann, Andrew L. Feldman, Lisa M. Rimsza, Rebecca L. King, Melissa C. Larson, Brianna J. Gysbers, Stephen M. Ansell, Jithma P. Abeykoon, Grzegorz S. Nowakowski, Thomas E. Witzig, Anne J. Novak, Susan L. Slager, James R. Cerhan. Germline CHEK2 variants and risk of lymphoma. [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 5233.

Abstract Introduction: Cytokines are protein factors that regulate diverse cellular processes. The malignant progression of immunoglobulin M monoclonal gammopathy of undetermined significance (IgM-MGUS) to Waldenstrom macroglobulinemia (WM) is in part cytokine mediated, and we have previously shown IL-6 mediates IgM secretion in WM via the JAK/STAT pathway. Cytokine signaling can be epigenetically modulated by micro-RNAs (miRs). We aimed to identify the role of miRs in the regulation of inflammatory cytokine pathways between these diseases. Methods: Bone marrow samples of patients with IgM-MGUS (n=7) and WM (n=25) were prospectively collected and sorted for CD19+ and/or CD138+ malignant cells. Total RNA extraction and sequencing were performed, miR and mRNA profiling was conducted, and differential expression was calculated between WM and IgM-MGUS using a log2 fold change (FC)>0.5 and ←0.5, false discovery rate<0.05. Experimental differentially expressed miR-mRNA pairing was performed, and pairs were filtered for correlated expression (i.e., upregulated miR/downregulated mRNA, and vice-versa). Filtered pairs were analyzed using the Ingenuity Pathway Analysis to identify pathways between WM and IgM-MGUS with absolute activation Z-score ≥1. Results: We identified 34 differentially expressed miRs experimentally targeting 1132 mRNAs. Pathway analysis showed overall miR-based inactivation of inflammatory cytokine signaling pathways in WM compared to IgM-MGUS, especially of the interleukin (IL) family: IL-2, IL-6, IL-8, IL-9, IL-15, and IL-17. Underlying multiple pathways was upregulation of miRs-146a, 150, 194, all targets of Signal Transducer and Activator of Transcription 1 (STAT1), observed to be downregulated. Of relevance, miR-146a has been previously shown to be an inhibitor of inflammatory cytokines, including IL-6. Next, we observed upregulation of miR-142 which targets phosphoinositide-3-kinase regulatory subunit 6 (PIK3R6), observed to be downregulated. Both the STAT and PIK3 families are important cytokine signal transducers, and we have previously shown the PI3k-AKT-IkB-p65 pathway induces expression of IL-6 via a GLI2-mediated mechanism. We additionally observed granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling to be inactivated in WM. Here, miR-19b and miR-30e were upregulated, both target CSF 2 receptor subunit beta (CSF2RB), found to be downregulated. CSF2RB is the common receptor subunit of GM-CSF, IL-3, and IL-5. Conclusion: Our study identifies multiple cytokines pathways involved in inflammation and the tumor microenvironment to be potentially inactivated by differentially expressed miRs in WM compared to IgM-MGUS. Downregulation of these pathways and targeting of the STAT and PIK3 family indicates a possible miR-based negative feedback loop to down-modulate inflammatory cytokine signaling in WM. Citation Format: Karan Chohan, Jonas Paludo, Surendra Dasari, Jithma P. Abeykoon, Prashant Kapoor, Esteban Braggio, Michelle K. Manske, Aneel Paulus, Craig B. Reeder, Sikander Ailawadhi, Asher A. Chanan-Khan, Robert A. Kyle, Morie A. Gertz, Anne J. Novak, Stephen M. Ansell. Micro-RNA induced inactivation of inflammatory cytokine signaling pathways in Waldenstrom macroglobulinemia compared to IgM-MGUS. [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 3811.

Abstract Objectives Understanding of histiocytic disorders has been revolutionized by demonstration of mitogen-activated protein kinase (MAPK) pathway mutations, most commonly BRAFV600E. The optimal testing strategy to assess BRAFV600E is unknown. We aimed to compare performance of testing modalities, to propose a framework for evaluation of BRAFV600E mutation status in histiocytic disorders. Methods We retrospectively reviewed patients with histiocytic disorders and BRAF mutation testing on a lesional tissue specimen. Results In 120 patients, BRAF assessment included immunohistochemistry (IHC) in 97 (80.2%), polymerase chain reaction (PCR) in 35 (28.9%), and next-generation sequencing (NGS) in 62 (51.2%). Forty-five underwent both NGS and IHC. With NGS as the gold standard, the sensitivity and specificity of IHC were 82.4% and 96.4%. Three false negatives were observed in biopsy specimens with low BRAFV600E variant allele frequency or decalcified tissue. One false-positive IHC was observed in a lung biopsy specimen, likely due to antibody cross-reactivity with respiratory cilia. Among 14 with successful NGS and PCR, a single discordance was observed. Two PCR-to-IHC discrepancies were observed, including one other false-positive IHC. Conclusions Immunohistochemistry was highly specific for detection of BRAFV600E. Main caveats were false negatives and lack of detection of non-BRAFV600E mutations. We propose the use of IHC as initial screening in general practice with reflex molecular testing if negative.

Abstract Introduction: Rapid cell proliferation requires intact and faithful DNA damage repair mechanisms. DNA polymerase theta (POLQ) plays a key role in repairing DNA double-strand breaks through the microhomology-mediated end-joining (MMEJ), which is one of the three main pathways involved in repairing replication-induced double-strand breaks. Limited data have suggested that concurrent depletion of POLQ and ataxia-telangiectasia mutated (ATM) could be embryonic lethal. Hence, this phenomenon has the potential to be exploited for therapeutic benefit in cancers where ATM mutations are commonly seen. Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma marked by (11;14) translocation with ATM alterations seen in 40-50% of patients. There is an unmet need to find novel therapeutic strategies, especially in relapsed and/or refractory (R/R) MCL. Here, we investigated whether targeting POLQ and ATM could be a potential therapeutic strategy in MCL. Methods: In vitro studies were conducted by using MCL cell lines. CRISPR-Cas9 system was used to genetically deplete POLQ and ATM genes and sgRNAs co-expressing fluorescence markers were used to track the cell population with respective genotypes over time. Cell viability was assessed by CellTiter-Glo assay and flow cytometry. All cell lines were profiled for ATM expression and activity. A p-value of < 0.05 was considered statistically significant. The combination index of <1 was defined as synergistic. Results: CRISPR-Cas9-mediated depletion of POLQ significantly decreased cell proliferation in multiple MCL cell lines. In particular, Granta-519, which possesses a single copy of kinase-dead ATM that is reduced in expression, was most sensitive to POLQ depletion. Concurrent genetic depletion of ATM and POLQ resulted in a synergistic antiproliferative effect in ATM-proficient MCL cell lines. Subsequently, this cellular phenotype caused by the genetic intervention was recapitulated by using two POLQ inhibitors (novobiocin and ART558) and an ATM inhibitor (AZD0156). In vitro, single-agent treatment with novobiocin or ART558 caused a significant cytotoxic effect at physiologically relevant concentrations in ATM-deficient cells and co-treatment of novobiocin or ART558 with AZD0156 was synergistic in killing ATM-proficient MCL cells. Importantly, POLQ inhibitors significantly decreased the cell viability of MCIR1, which is an ibrutinib-resistant MCL cell line. Mechanistically, novobiocin or ART558 treatment induced gH2AX and cleaved PARP upregulation, which was further enhanced by ATM depletion, suggesting that co-inhibition of POLQ and ATM caused apoptosis due to the accumulation of unrepaired DNA damage. Conclusion: POLQ is a promising target in MCL, especially in ATM-deficient setting. In ATM-proficient MCL, targeting ATM and POLQ is synergistic. Our data has the potential to uncover novel biomarker-driven drug therapy of POLQ inhibitors in R/R MCL. Citation Format: Jithma Prasad Abeykoon, Shuhei Asada, Kalindi Parmar, Xiaosheng Wu, Thomas Witzig, Geoffrey Shapiro, Alan D. D'Andrea. Targeting DNA polymerase theta and ATM leads to synergistic killing of mantle cell lymphoma cells. [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 6208.

Abstract Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can result in a cytokine storm that is associated with poor outcomes in patients. The activation of a NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome following SARS-CoV-2 infection has been shown to play a major role in inflammatory immune responses. In this study, we show that open reading frame 8 (ORF8) protein is abundantly secreted as a glycoprotein in vitro. The glycosylated ORF8 stimulates human CD14+/CD16+ monocytes to cause upregulation of proinflammatory cytokine production and cell surface marker expression within 24 hours. The data suggests that stimulation of human monocytes by ORF8 is not receptor mediated. Rather, ORF8 is likely phagocytosed by human monocytes. ORF8 then stimulates the monocytes by independently binding to the NACHT and LRR domains of the NLRP3 protein. Pharmacologic inhibition of NLRP3 significantly diminished the production of pro-inflammatory cytokines from ORF8-stimulated human monocytes. Finally, this study shows that the ORF8 protein is also secreted in patients with newly diagnosed coronavirus disease 2019 (COVID-19). Levels of ORF8 in the blood of patients diagnosed with COVID-19 correlated with disease mortality and trajectory of disease. ORF8 stimulation of monocytes causes pro-inflammatory cytokine production that leads to the development of severe COVID-19.

Abstract Purpose: IgM monoclonal gammopathy of undetermined significance (MGUS) and Waldenström macroglobulinemia (WM) represent a disease spectrum with highly varied therapeutic management, ranging from observation to chemoimmunotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. Further investigation is warranted to shed light on the biology that may account for the clinical differences. Experimental Design: We used bone marrow (BM) clonal CD19+ and/or CD138+ sorted cells, matched BM supernatant, and peripheral blood serum from 32 patients (7 MGUS, 25 WM) to perform the first multi-omics approach including whole-exome sequencing, RNA sequencing, proteomics, metabolomics, and mass cytometry. Results: We identified three clusters with distinct pathway activation, immune content, metabolomic, and clinical features. Cluster 1 included only patients with WM and was characterized by transcriptional silencing of genes involved in cell cycle and immune response, enrichment of mitochondrial metabolism, infiltration of senescent T effector memory cells, and aggressive clinical behavior. Genetic/structural alterations of TNFAIP3 were distinct events of this cluster. Cluster 2 comprised both MGUS and WM patients with upregulation of inflammatory response, senescence and glycolysis signatures, increased activated T follicular helper and T regulatory cells, and indolent clinical behavior. Cluster 3 also included both MGUS and WM patients and exhibited intermediate features, including proliferative and inflammatory signaling, as well as glycolysis and mitochondrial metabolism. Conclusions: We have identified three distinct molecular clusters, suggesting a potential biologic classification that may have therapeutic implications.