Introduction: The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by activation of NF-kB signaling and an increased risk of mortality. Recurrent somatic mutations affecting genes such as MYD88, CD79A/B and TNFAIP3 have been shown to constitutively activate the NF-kB pathway through B-cell receptor signaling in ABC DLBCL; however, there still remain cases with no known genetic basis for this pathway activation (Arthur et al. Nat Com 2018). We recently published a meta-analysis of DLBCL genome and targeted sequencing data identifying non-coding mutations. We described novel mutations affection the 3′ untranslated region (UTR) of NFKBIZ in 18% of ABC DLBCLs. Overall, NFKBIZ is mutated (amplifications and UTR mutations) in 34% of ABC DLBCLs. These NFKBIZ mutations are mutually exclusive with MYD88 mutations, implicating them in activation of the NF-kB signaling pathway. NFKBIZ encodes the IkB-ζ protein, which interacts with NF-kB transcription factors and is thought to regulate canonical NF-kB signaling. We hypothesized that these mutations affect the ability of regulatory mechanisms to target this transcript for degradation through disruption of UTR secondary structures. This leads to enhanced mRNA stability and elevated protein levels and represents a novel mechanism of promoting NF-kB signaling in ABC DLBCL. Methods: NFKBIZ 3′ UTR mutations were introduced in a DLBCL cell line using CRISPR-Cas9. NFKBIZ mRNA and protein levels were evaluated using custom designed droplet digital PCR assays and western blot. RNA-sequencing was performed on mutant and wild-type (WT) cell lines to identify genes up-regulated by IkB-ζ. A competitive growth assay with WT and CRISPR mutant lines was set up to assess whether UTR mutations provide a growth advantage in culture. The pool composition was determined by DNA sequencing and comparison of WT and mutant DNA sequences. Results: Introduction of NFKBIZ mutations into DLBCL cell lines confirmed that UTR deletions lead to increased levels of mRNA and protein. Stimulation with LPS revealed that mRNA levels stay elevated for longer in mutant lines. NFKBIZ UTR deletions also give DLBCL cells a selective growth advantage over WT when grown together in culture. RNA-sequencing of mutant and WT lines revealed possible transcriptional targets of IkB-ζ, including TNFRSF14B, HCK, GNAZ, BATF and CD274. These targets are either involved in activation of NF-kB signaling, associated with decreased survival in other lymphomas or potential new drug targets in NFKBIZ mutant DLBCL. Conclusions: This work highlights the role of NFKBIZ and 3′ UTR mutations in driving ABC DLBCL. We demonstrate that these UTR mutations can lead to over-expression of NFKBIZ and activate potentially novel drug targets in ABC DLBCL. These findings contribute to a better understanding of the genetic basis of DLBCL, which is necessary to guide personalized therapeutic strategies. Keywords: activated B-cell-like (ABC); molecular genetics; NF-kB.
Diffuse large B-cell lymphoma (DLBCL) patients are typically treated with immunochemotherapy containing rituximab (rituximab, cyclophosphamide, hydroxydaunorubicin-vincristine (Oncovin), and prednisone [R-CHOP]); however, prognosis is extremely poor if R-CHOP fails. To identify genetic mechanisms contributing to primary or acquired R-CHOP resistance, we performed target-panel sequencing of 135 relapsed/refractory DLBCLs (rrDLBCLs), primarily comprising circulating tumor DNA from patients on clinical trials. Comparison with a metacohort of 1670 diagnostic DLBCLs identified 6 genes significantly enriched for mutations upon relapse. TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations remained clonally persistent throughout treatment in paired diagnostic-relapse samples, suggesting a role in primary treatment resistance. Nonsense and missense mutations affecting MS4A1, which encodes CD20, are exceedingly rare in diagnostic samples but show recurrent patterns of clonal expansion following rituximab-based therapy. MS4A1 missense mutations within the transmembrane domains lead to loss of CD20 in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. In a time series from a patient treated with multiple rounds of therapy, tumor heterogeneity and minor MS4A1-harboring subclones contributed to rapid disease recurrence, with MS4A1 mutations as founding events for these subclones. TP53 and KMT2D mutation status, in combination with other prognostic factors, may be used to identify high-risk patients prior to R-CHOP for posttreatment monitoring. Using liquid biopsies, we show the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens.
Summary The term diffuse large B‐cell lymphoma (DLBCL) includes a heterogeneous collection of biologically distinct tumours. This heterogeneity currently presents a barrier to the successful deployment of novel, biologically targeted therapies. Molecular profiling studies have recently proposed new molecular classification systems. These have the potential to resolve the biological heterogeneity of DLBCL into manageable subgroups of tumours that rely on shared oncogenic programmes. In many cases these biological programmes straddle the boundaries of our existing systems for classifying B‐cell lymphomas. Here we review the findings from these major molecular profiling studies with a specific focus on those that propose new genetic subgroups of DLBCL. We highlight the areas of consensus and discordance between these studies and discuss the implications for current clinical practice and for clinical trials. Finally, we address the outstanding challenges and solutions to the introduction of genomic subtyping and precision medicine in DLBCL.
Abstract Introduction: The activated B cell-like (ABC) molecular subgroup of diffuse large B-cell lymphoma (DLBCL) is characterized by activation of NF-κB signaling. Recurrent mutations affecting genes such as MYD88, CD79A/B, and TNFAIP3 contribute to this in some cases, but there remain tumors with no known genetic basis for this pathway activation. This suggests that our understanding of ABC DLBCL drivers remains incomplete. Previously, NFKBIZ was shown to be amplified in 10% of ABC DLBCLs and to contribute to activation of NF-κB signaling. We recently described a novel pattern of mutations affecting the 3′ UTR of NFKBIZ resulting in an overall mutation rate of 30% (UTR or AMP) in ABC DLBCL. These NFKBIZ UTR mutations are mutually exclusive with MYD88 mutations, thus suggesting they may also lead to activation of NF-κB signaling. The NFKBIZ protein interacts with NF-κB transcription factors and is thought to regulate canonical NF-κB signaling. We hypothesized that NFKBIZ UTR mutations affect the normally rapid degradation of this mRNA by disrupting secondary structures recognized by RNA-binding proteins such as ribonucleases. The resulting elevated NFKBIZ mRNA levels would lead to accumulation of protein and may be a novel mechanism to promote cell growth and survival in ABC DLBCL. Methods: NFKBIZ 3′ UTR mutations were introduced into a DLBCL cell line using the CRISPR-Cas9 system. A competitive growth assay with wild-type (WT) and CRISPR-mutant lines was performed to assess whether UTR mutations provide a growth advantage in culture (in vitro) and in mouse xenografts (in vivo). RNA-sequencing was then performed on WT and a subset of CRISPR-mutant lines and analyses were performed to identify genes upregulated by IκB-ζ in mutant lines. The IC50 of relevant drugs was determined by WST-1 assays after drug treatment on WT and mutant lines. Results: Introduction of NFKBIZ mutations into a DLBCL cell line confirmed that UTR deletions lead to increased mRNA and protein levels. NFKBIZ UTR deletions give DLBCL cells a selective growth advantage over WT both in vitro and in vivo. RNA-sequencing of mutant and WT lines revealed possible transcriptional targets of NFKBIZ, including NF-κB targets and genes commonly overexpressed in ABC DLBCL. Novel candidate NFKBIZ targets were also discovered through this analysis, including CD274, the gene encoding PD-L1. Mutant cell lines had significantly higher IC50 compared to WT for the drugs ibrutinib, idelalisib, and masitinib, but not bortezomib, suggesting that NKFBIZ UTR mutations make cell lines more resistant to specific NF-κB pathway-targeted drugs. Conclusions: This work directly establishes a role for NFKBIZ amplifications and 3′ UTR mutations in driving ABC DLBCL through NF-κB signaling. We demonstrate that these mutations can lead to overexpression of NFKBIZ and provide a selective growth advantage to cells both in vitro and in vivo. In addition, we found that these mutant lines were more resistant to some targeted lymphoma drugs but not others. Citation Format: Sarah E. Arthur, Nicole Thomas, Jeffrey Tang, Christopher K. Rushton, Miguel Alcaide, Adèle Telenius, Shannon Healy, Anja Mottok, David W. Scott, Christian Steidl, Ryan D. Morin. NFKBIZ 3′ UTR mutations confer selective growth advantage and affect drug response 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-32.
ABSTRACT Animal models of human cancers are an important tool for the development and preclinical evaluation of therapeutics. Canine B-cell lymphoma (cBCL) is an appealing model for human mature B-cell neoplasms due to the high sequence similarity in cancer genes to humans and inactive telomerase in adult tissues. We performed targeted sequencing on 86 canine patients from the Canine Comparative Oncology Genomic Consortium, with 61 confirmed as B-cell lymphomas. We confirmed a high frequency of mutations in TRAF3 (45%) and FBXW7 (20%) as has been reported by our group and others. We also note a higher frequency of DDX3X (20%) and MYC (13%) mutations in our canine cohort. We compared the pattern and incidence of mutations in cBCL to human diffuse large B-cell lymphoma (hDLBCL) and human Burkitt lymphoma (hBL). Canine MYC mutations displayed a focal pattern with 80% of mutations affecting the conserved phosphodegron sequence in MYC box 1, which are known to stabilize MYC protein. We also note that MYC and FBXW7 mutations do not co-occur in our cBCL cohort, leading to the hypothesis that these mutations represent alternative approaches to stabilize MYC in canine lymphoma. We observed striking differences in the pattern of DDX3X mutations in canine lymphoma as compared to hBL and uncovered a sex-specific pattern of DDX3X mutations in hBL that is not consistent with those identified in canine lymphomas. In sum, we describe key differences between cBCL and human mature B-cell lymphomas which may indicate differences in the biology of these cancers. This should be considered in future studies of cBCL as a model of human lymphomas.
