DDX3X Mutations Research Articles

Deficiency of DDX3X results in neurogenesis defects and abnormal behaviors via dysfunction of the Notch signaling

The molecular mechanisms underlying the neurodevelopmental disorders (NDDs) caused by DDX3X variants remain poorly understood. In this study, we validated that de novo DDX3X variants are enriched in female developmental delay (DD) patients and mainly affect the evolutionarily conserved amino acids based on a meta-analysis of 46,612 NDD trios. We generated a ddx3x deficient zebrafish allele, which exhibited reduced survival rate, DD, microcephaly, adaptation defects, anxiolytic behaviors, social interaction deficits, and impaired spatial recognitive memory. As revealed by single-nucleus RNA sequencing and biological validations, ddx3x deficiency leads to reduced neural stem cell pool, decreased total neuron number, and imbalanced differentiation of excitatory and inhibitory neurons, which are responsible for the behavioral defects. Indeed, the supplementation of L-glutamate or glutamate receptor agonist ly404039 could partly rescue the adaptation and social deficits. Mechanistically, we reveal that the ddx3x deficiency attenuates the stability of the crebbp mRNA, which in turn causes downregulation of Notch signaling and defects in neurogenesis. Our study sheds light on the molecular pathology underlying the abnormal neurodevelopment and behavior of NDD patients with DDX3X mutations, as well as providing potential therapeutic targets for the precision treatment.

The Role of the RNA Helicase DDX3X in Medulloblastoma Progression.

Medulloblastoma is the most common pediatric brain cancer, with about five cases per million in the pediatric population. Current treatment strategies have a 5-year survival rate of 70% or more but frequently lead to long-term neurocognitive defects, and recurrence is relatively high. Genomic sequencing of medulloblastoma patients has shown that DDX3X, which encodes an RNA helicase involved in the process of translation initiation, is among the most commonly mutated genes in medulloblastoma. The identified mutations are 42 single-point amino acid substitutions and are mostly not complete loss-of-function mutations. The pathological mechanism of DDX3X mutations in the causation of medulloblastoma is poorly understood, but several studies have examined their role in promoting cancer progression. This review first discusses the known roles of DDX3X and its yeast ortholog Ded1 in translation initiation, cellular stress responses, viral replication, innate immunity, inflammatory programmed cell death, Wnt signaling, and brain development. It then examines our current understanding of the oncogenic mechanism of the DDX3X mutations in medulloblastoma, including the effect of these DDX3X mutations on growth, biochemical functions, translation, and stress responses. Further research on DDX3X's mechanism and targets is required to therapeutically target DDX3X and/or its downstream effects in medulloblastoma progression.

Case report: Mutation evolution in a patient with TdT positive high grade B cell lymphoma with MYC and BCL2 rearrangements following the treatment of concurrent follicular lymphoma and diffuse large B-cell lymphoma

BackgroundConcurrent follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL)was reported in some studies, while the diagnosis of TdT (terminal deoxynucleotydil transferase) positive high grade B cell lymphoma (HGBL) with MYC and BCL2 rearrangements (“double hit”) transformed from FL/DLBCL has been rarely reported. Herein, we described the clinical features and mutation profiles of a case diagnosed with TdT positive “double hit” HGBL following the treatment of FL/DLBCL.Case presentationThis is a 43-year-old Chinese man who was diagnosed with low grade FL (account for 80%) combined with DLBCL (20%) at a stage of IVB. The patient presented with BCL2/IGH translocation without MYC rearrangement, as well as the expressions of CD20, CD19, CD10 and BCL2 at the initial diagnosis of FL/DLBCL. MYC rearrangement and TdT expression occurred after the treatment. The targeted sequencing revealed mutations in KMT2D, FOXO1, CREBBP, ATM, STAT6, BCL7A, DDX3X, MUC4, FGFR3, ARID5B, DDX11 and PRKCSH genes were the co-mutations shared by the FL/DLBCL and TdT positive “double hit” HGBL, while CCND3, BIRC6, ROBO1 and CHEK2 mutations specifically occurred after the treatment. The overall survival time was 37.8 and 17.8 months after the initial diagnosis of FL/DLBCL and TdT positive “double hit” HGBL, respectively.ConclusionThis study reports a rare case of TdT positive “double hit” HGBL following the treatment of concurrent FL/DLBCL and highlights the mutation characteristics. Collectively, this study will help enrich the knowledge of TdT positive “double hit” HGBL transformed from FL/DLBCL.

Abstract P33: Novel Reporter Cell Models for Efficient Interrogation of Helicase Activity in Cancer-associated DDX3X Mutations

Abstract DDX3X is an ATP-dependent RNA helicase involved in diverse cellular processes. Somatic mutations in the DDX3X gene exhibit oncogenic or tumor suppressive activities depending on cancer types. However, helicase modulating roles of specific DDX3X mutations remain elusive mainly due to lack of an appropriate cell culture model system. Here, we report our development of novel transgenic cell lines capable of interrogating DDX3X helicase activity. To evaluate the helicase activity of the DDX3X mutations in question, we designed a reporter plasmid that measures the luciferase signal corresponding to the helicase activity. Using 8 different point mutations in the DDX3X that we identified earlier in relapsed/refractory patients with diffuse large B cell lymphoma (DLBCL), we show that these DDX3X mutants displayed defective helicase activity. DDX3X mutant knock-in DLBCL cells also displayed abolished helicase unwinding capabilities, validating our reporter cell models. We were able to extend the application of our reporter cells to interrogate Y chromosome-linked male paralog DDX3Y. In male lymphoma patients, ectopic DDX3Y expression can compensate for DDX3X loss to restore global protein synthesis. We narrowed down DDX3Y helicase and ATPase domains to be critical for cellular translation, ruling out DDX3Y involvement in ribosomal elongation. Unexpectedly, DDX3Y helicase is less efficient in RNA unwinding as compared to DDX3X revealing subtle differences despite having a high level of homology. The developed DDX3X reporter cells can be applied widely to evaluate the effects of DDX3X mutations in diseases as well as study the biology of DDX3X and DDX3Y. Citation Format: Zhi Sheng Poh, James Tan Chia Wei, Brandon Han Siang Wong, Kottaiswamy Amuthavalli, Suat Hoon Tan, Nicholas Francis Grigoropoulos, Navin Kumar Verma. Novel Reporter Cell Models for Efficient Interrogation of Helicase Activity in Cancer-associated DDX3X Mutations [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P33.

Mutation landscape in Chinese nodal diffuse large B-cell lymphoma by targeted next generation sequencing and their relationship with clinicopathological characteristics

BackgroundDiffuse large B-cell lymphoma (DLBCL), an aggressive and heterogenic malignant entity, is still a challenging clinical problem, since around one-third of patients are not cured with primary treatment. Next-generation sequencing (NGS) technologies have revealed common genetic mutations in DLBCL. We devised an NGS multi-gene panel to discover genetic features of Chinese nodal DLBCL patients and provide reference information for panel-based NGS detection in clinical laboratories.MethodsA panel of 116 DLBCL genes was designed based on the literature and related databases. We analyzed 96 Chinese nodal DLBCL biopsy specimens through targeted sequencing.ResultsThe most frequently mutated genes were KMT2D (30%), PIM1 (26%), SOCS1 (24%), MYD88 (21%), BTG1 (20%), HIST1H1E (18%), CD79B (18%), SPEN (17%), and KMT2C (16%). SPEN (17%) and DDX3X (6%) mutations were highly prevalent in our study than in Western studies. Thirty-three patients (34%) were assigned as genetic classification by the LymphGen algorithm, including 12 cases MCD, five BN2, seven EZB, seven ST2, and two EZB/ST2 complex. MYD88 L265P mutation, TP53 and BCL2 pathogenic mutations were unfavorable prognostic biomarkers in DLBCL.ConclusionsThis study presents the mutation landscape in Chinese nodal DLBCL, highlights the genetic heterogeneity of DLBCL and shows the role of panel-based NGS to prediction of prognosis and potential molecular targeted therapy in DLBCL. More precise genetic classification needs further investigations.

Loss of DDX3X Function Promotes CLL Progression By Facilitating NOTCH1 mRNA Translation

A variety of driver genes are present in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), and those with high mutation frequencies include SF3B1, NOTCH1, and TP53, which are involved in the development of CLL/SLL and are associated with poor prognosis. The biological significance of some driver genes, such as DDX3X, has not yet been elucidated. DDX3X is located on the X chromosome. As one of the members of the DEAD-box helicase family, it is expressed ubiquitously in human tissues and participates in many biological processes. Here, we described the mapping of the DDX3X abnormalities and demonstrated its clinical significance in CLL/SLL. We also explored the effect and mechanism of DDX3X dysregulation on tumorigenesis and development of CLL by in vitro experiments. To examine the mutational landscape of DDX3X, we performed next-generation sequencing on 402 CLL/SLL patients, and 17 patients (4.2%) had DDX3X mutations. Six patients were frameshift mutations and 2 were nonsense mutation, which caused truncation of DDX3X. Among the 7 cases of missense mutations, 4 cases involved the ATP-binding or C-terminal domain, which may affect the function of DDX3X. The remaining two cases are deletion mutation and splicing mutation. Next, we evaluated the clinical significance and prognostic role of DDX3X mutation. The patients with DDX3X mutations are mostly male (94.1% vs 66.5%, P=0.017), and had more adverse prognostic indicators: unmutated IGHV (88.2% vs 48.6%, P=0.002), 17p deletion (42.9% vs 11.4%, P=0.041), and 11q deletion (46.7% vs 15.7%, P=0.006). DDX3X mutation was an independent risk factor for disease specific survival of CLL/SLL in the competing-risk regression. Also, we found all (14/14) the mutated patients and 15.2% (5/33) of the non-mutated patients had low expression of DDX3X in bone marrow samples by immunohistochemistry. Overall, these results indicates that dysregulation of DDX3X are common in CLL/SLL and may contribute to its progression. To delineate the functional consequences of DDX3X loss in CLL, we used CRISPR/cas9 system to construct stable DDX3X knockout (DDX3X-KO) MEC-1 and JVM-3 cells. DDX3X-KO cells showed significantly increased proliferation and reduced spontaneous apoptosis compared to vector control. Additionally, DDX3X-KO cells showed higher rates of cell viability and reduced apoptosis after treatment of ibrutinib or venetoclax. We further found that DDX3X-KO cells had higher expression level of phosphorylated BTK and MCL-1, which may explain the resistance to ibrutinib and venetoclax, respectively. Moreover, DDX3X-KO cells showed increased expression level of CXCR4 and enhanced chemotaxis in response to CXCL-12. In summary, loss of DDX3X function promotes progression of CLL by mediating more invasive biological behaviors of CLL cells. To evaluate the mechanisms by which DDX3X deletion affects CLL, we detected key signaling pathways in the tumorigenesis and development of CLL in DDX3X-KO and control cells. We found increased expression of NOTCH1 and its downstream target proteins in DDX3X-KO cells (Figure 1), indicating that loss of DDX3X activated the NOTCH1 pathway. Considering that DDX3X is an RNA-binding protein and mainly regulates translation, we speculated that DDX3X may regulate the translation of NOTCH1. We performed RNA binding protein immunoprecipitation assay and found that DDX3X directly interacted with NOTCH1 mRNA (Figure 2A, B). Dual-luciferase reporter assay showed enhanced activity of NOTCH1 5‘ UTR in DDX3X-KO cells (Figure 2C), further suggesting that DDX3X may regulate translation of NOTCH1 mRNA through binding to the 5‘ UTR. Finally, polysome profiling demonstrated that DDX3X knockout resulted in increased NOTCH1 mRNAs in polysome fractions (Figure 2D), confirming that loss of DDX3X function facilitates NOTCH1 mRNA translation. In conclusion, DDX3X abnormalities are common in CLL and associated with poor prognosis. DDX3X dysregulation is involved in the progression of CLL by facilitating NOTCH1 mRNA translation through binding to the 5' UTR.

Decoding the molecular heterogeneity of pediatric monomorphic post–solid organ transplant lymphoproliferative disorders

AbstractPosttransplant lymphoproliferative disorders (PTLDs) represent a broad spectrum of lymphoid proliferations, frequently associated with Epstein-Barr virus (EBV) infection. The molecular profile of pediatric monomorphic PTLDs (mPTLDs) has not been elucidated, and it is unknown whether they display similar genetic features as their counterpart in adult and immunocompetent (IMC) pediatric patients. In this study, we investigated 31 cases of pediatric mPTLD after solid organ transplantation, including 24 diffuse large B-cell lymphomas (DLBCLs), mostly classified as activated B cell, and 7 cases of Burkitt lymphoma (BL), 93% of which were EBV positive. We performed an integrated molecular approach, including fluorescence in situ hybridization, targeted gene sequencing, and copy number (CN) arrays. Overall, PTLD-BL carried mutations in MYC, ID3, DDX3X, ARID1A, or CCND3 resembling IMC-BL, higher mutational burden than PTLD-DLBCL, and lesser CN alterations than IMC-BL. PTLD-DLBCL showed a very heterogeneous genomic profile with fewer mutations and CN alterations than IMC-DLBCL. Epigenetic modifiers and genes of the Notch pathway were the most recurrently mutated in PTLD-DLBCL (both 28%). Mutations in cell cycle and Notch pathways correlated with a worse outcome. All 7 patients with PTLD-BL were alive after treatment with pediatric B-cell non-Hodgkin lymphoma protocols, whereas 54% of patients with DLBCL were cured with immunosuppression reduction, rituximab, and/or low-dose chemotherapy. These findings highlight the low complexity of pediatric PTLD-DLBCL, their good response to low-intensity treatment, and the shared pathogenesis between PTLD-BL and EBV-positive IMC-BL. We also suggest new potential parameters that could help in the diagnosis and the design of better therapeutic strategies for these patients.

The RNA helicase DDX3 and its role in c-MYC driven germinal center-derived B-cell lymphoma.

DDX3X is an RNA helicase with many functions in RNA metabolism such as mRNA translation, alternative pre-mRNA splicing and mRNA stability, but also plays a role as a regulator of transcription as well as in the Wnt/beta-catenin- and Nf-κB signaling pathways. The gene encoding DDX3X is located on the X-chromosome, but escapes X-inactivation. Hence females have two active copies and males only one. However, the Y chromosome contains the gene for the male DDX3 homologue, called DDX3Y, which has a very high sequence similarity and functional redundancy with DDX3X, but shows a more restricted protein expression pattern than DDX3X. High throughput sequencing of germinal center (GC)-derived B-cell malignancies such as Burkitt Lymphoma (BL) and Diffuse large B-cell lymphoma (DLBCL) samples showed a high frequency of loss-of-function (LOF) mutations in the DDX3X gene revealing several features that distinguish this gene from others. First, DDX3X mutations occur with high frequency particularly in those GC-derived B-cell lymphomas that also show translocations of the c-MYC proto-oncogene, which occurs in almost all BL and a subset of DLBCL. Second, DDX3X LOF mutations occur almost exclusively in males and is very rarely found in females. Third, mutations in the male homologue DDX3Y have never been found in any type of malignancy. Studies with human primary GC B cells from male donors showed that a loss of DDX3X function helps the initial process of B-cell lymphomagenesis by buffering the proteotoxic stress induced by c-MYC activation. However, full lymphomagenesis requires DDX3 activity since an upregulation of DDX3Y expression is invariably found in GC derived B-cell lymphoma with DDX3X LOF mutation. Other studies with male transgenic mice that lack Ddx3x, but constitutively express activated c-Myc transgenes in B cells and are therefore prone to develop B-cell malignancies, also showed upregulation of the DDX3Y protein expression during the process of lymphomagenesis. Since DDX3Y is not expressed in normal human cells, these data suggest that DDX3Y may represent a new cancer cell specific target to develop adjuvant therapies for male patients with BL and DLBCL and LOF mutations in the DDX3X gene.

Indolent T-cell lymphoproliferative disorder of gastrointestinal tract with unusual clinical courses: report of 6 cases and literature review.

Indolent T-cell lymphoproliferative disorder of the gastrointestinal tract (iTLPD-GI) is a rare neoplasm usually having an indolent clinical course and easily misdiagnosed as inflammatory bowel disease or other T-cell lymphomas. A subset of the disorders that progressed to overt peripheral T-cell lymphoma have been reported, and the etiology and pathogenesis are poorly understood. The current study retrospectively examined the pathological, molecular, and clinical features of 6 cases of iTLPD-GI. Hematoxylin and eosin staining, immunohistochemistry, in situ hybridization, T-cell receptor gene rearrangement, and next-generation sequencing (NGS) were performed with the diseased tissues. All the 6 patients were immunocompetent Chinese men, who presented with recurrent abdominal pain and diarrhea for 4 to 13years. Histologically, the intestinal tissue was expanded by lymphoid infiltration, composed of small-to-medium-sized lymphocytes with gland intact. The neoplastic cells were CD4 - /CD8 + with expression of TIA1 and variable granzyme B in five cases, and the other one was CD4 + /CD8 - . Two of the 5 patients progressed to more aggressive T-cell lymphoma and died of disease with complications. NGS identified TET2 and DDX3X mutations in patient 1, and BIRC6 and REV3L mutations in patient 2. Literature review indicated that iTLPD-GI with CD4 - /CD8 + immunophenotype was more commonly reported in Chinese cases. Our limited data indicated CD4-/CD8 + iTLPD-GI have similar potential to progress to more aggressive T-cell lymphoma as that of CD4 + /CD8 - , and gradually increased expression of granzyme B and Ki-67 may be early signs of the disease progression. Gain of novel gene mutations may be indicators of the pathogenesis.

Potential Drivers of Acquired Resistance to Idelalisib in CLL Patients

Idelalisib is an isoform-specific PI3-kinase-δ inhibitor approved for the treatment of chronic lymphocytic leukemia (CLL). We seek to understand the biologic basis of the variability in depth of response among patients. We have previously reported that intrinsic resistance to idelalisib is associated with RAS/RAF/MAP2K1 mutations and constitutive ERK activation [Blood 2021; 138: 44]. Unlike these non-responder patients, some patients with initial response will acquire resistance to therapy over time, but molecular mechanisms of this acquired resistance to idelalisib remain elusive. To identify somatic mutations that fuel acquired resistance, we performed whole exome sequencing of serial PBMC samples from three CLL patients who responded to idelalisib, but later acquired resistance (average 4.1 yrs from start). We observed that all three patients acquired distinct somatic mutations during idelalisib treatment. Patient 1 harbored TP53 and BIRC3 at baseline, but at progression (5.3 yrs later) had acquired somatic mutations in RAF1, PDIA3, RB1, and ATM. Patient 2 had baseline mutations in SF3B1, BRCA2, and HIST1HIE but acquired additional mutations in PIK3R1 and IKZF3 at progression (4.4 yrs later). Patient 3 carried somatic mutations in TP53 and SF3B1 at baseline and acquired somatic mutations in DDX3X, NOTCH1, FBXW7, CD79B, NFATC1 and NFATC3 at progression (2.7 yrs later). To identify novel drivers of resistance and validate our findings from the initial three CLL patients, we sequenced progression samples from eight additional CLL patients following resistance to idelalisib treatment. In addition to patient 1, we detected mutations in BIRC3 in two additional CLL cases, and mutations in KRASand NRAS (both of which are known to aberrantly activate the MAPK pathway) in two and one CLL patients, respectively, separate from the cases with BIRC3 mutation. In addition to the PIK3R1 acquired mutation in patient 2, we also identified mutations in other PI3K pathway-related genes, such as PIK3CA, PIK3CG, andPLCG2. Since we only sequenced progression samples from these 8 patients, we cannot confirm whether the mutations were acquired or were present in the baseline samples. Overall, these results suggest that mutations in the RAS/RAF/MAP2K1 pathway, as well as in the PI3K pathway itself, and BIRC3 (NF-kB pathway), are associated with PI3K inhibitor resistance. To identify expression programs that may lead to idelalisib resistance, we performed comparative transcriptomic profiling of responding and resistant time point samples from the three initially studied CLL patients (patient 1, 2 and 3). In this analysis, we detected 214 differentially expressed genes with adjusted p-value < 0.05 and fold change of 1.5. GSEA analysis revealed enrichment of cancer related cell signaling pathways, most notably an enrichment for the MAPK pathway (FDR q-value- 0.04). We found that AICDA, LTK, and MAPK12 are overexpressed in resistant samples from the three original CLL patients with acquired resistance as compared to the matched responding time points. In a separate cohort of frontline patients treated with idelalisib, we also found that these same 3 genes were consistently overexpressed before, during and after treatment in early progressors after idelalisib discontinuation. Higher expression of AICDA in progression samples may drive resistance by inducing somatic mutations and/or altering the epigenome of the tumor cells in CLL patients. LTK receptor tyrosine kinase activates PI3K and MAPK pathways in hematopoietic cells. Previously we have reported that MAPK pathway activation plays a key role in primary resistance to idelalisib in CLL, hence overexpression of LTK is a possible mechanism of MAPK activation. We checked the phosphorylation/activation level of AKT and ERK1/2 at the responding and progression time points in the three original CLL patients with acquired resistance and observed that pERK levels are inhibited by idelalisib at baseline, but not at progression, in 2 patients (patient 1 and 2). In conclusion, we have identified potential drivers of acquired resistance to idelalisib in CLL patients, including MAPK pathway activation as in our prior study, and we continue with ongoing work evaluating the potential role of PI3K pathway mutations, BIRC3, AICDA and LTK in treatment resistance, since insights from these studies will help us guide therapy for CLL patients with refractory disease.

Comprehensive Analysis of TP53 and CD58 Mutations and Identification of Patients with Inferior Prognosis and Enhanced Immune Escape in Diffuse Large B Cell Lymphoma

Background: Studies on TP53 mutations as prognostic markers in diffuse large B-cell lymphoma (DLBCL) have historically been controversial and these results have not been consistent across different studies. Considering the complex pathophysiological mechanisms involved in DLBCL, we assumed that the interaction of TP53 with other genetic variants, which shape the discrepant immune landscape, could further promote the development of DLBCL, and thus be more prognostically predictive. Materials and methods: Comprehensive analysis of the genomic characteristics of TP53 and CD58 was performed through high-throughput DNA (n=176) and relevant RNA (n=152) sequencing in patients with de novo DLBCL. Mutation data of 1,001 DLBCL patients from Duke University's cohort were downloaded for validation. Another validation cohort was from REMoDL-B trail (N=400). Results: A total of 227 significantly mutated genes were identified, of which TP53 was the second most frequently mutated gene, with a rate of 30% (53 of 176) and 62 sequence variants detected. Among these variants, 74% were missense mutations, and the remaining were inactivating frameshift indels, nonsense mutations, coding sequencing indels and splicing mutations. Importantly, most mutations (87.5%) occurred in exons 5-8, which encoded the DBD region of TP53. Codons 175, 273, and 248 of the p53 protein had the highest mutation frequency. However, TP53 mutations alone are insufficient to effectively differentiate the risk of DLBCL, even when only considering mutations in the DBD region. We further found that DDX3X, MYLK2, and FUT6 mutations co-occurred with TP53 mutations, and CD58 mutations were mutually exclusive with TP53 mutations. No significant difference was observed in survival among those groups according to the combination of co-occurring mutation genes with TP53 (p=0.37 for DDX3X; p=0.11 for MYLK2; p=0.54 for FUT6). However, the combination of TP53 and CD58 mutations could significantly distinguish the prognosis of patients with DLBCL (p=0.033). Patients with both wild-type TP53 and CD58 had a better prognosis than patients with either of the two mutually exclusive modes of CD58 and TP53 mutations. Unexpectedly, patients with TP53 wild-type and CD58 mutations (TP53WT&CD58MUT) had worse survival than those with TP53 mutations and CD58 wild-type (TP53MUT&CD58WT). The relationship of a mutually exclusive mutant between CD58 and TP53 and the prognostic significance of this interaction was validated using publicly available data from 1,001 patients with DLBCL from the Duke university's cohort. We then explored whether the cooperation of the mutually exclusive mutations between TP53 and CD58 may profoundly influence the microenvironment in DLBCL. We found that the overall TMB was significantly higher in the TP53WT&CD58MUT group than in the TP53MUT&CD58WT group (p=0.0177), while there was no difference in TMB when dividing patients only according to TP53 mutation status (p=0.5348). In addition, the ESTIMATE immune scores in the TP53WT&CD58MUT group were significantly higher than that in the TP53MUT&CD58WT groups (p=0.0047). Moreover, the exhausted T cell, macrophage cell, NK cell, and Th1 cell enriched in the TP53WT&CD58MUT group. The difference between the two groups was mainly due to the combined influence of the mutation pattern 'TP53WT&CD58MUT', but rather only affected by the CD58 mutations, given the immune cell infiltration was similar when dividing patients just according to CD58 mutation status. Furthermore, the co-inhibitory receptors such as PD-1, TIM3, and LAG3 were preferentially expressed in the TP53WT&CD58MUT group. Inhibitory immunomodulators were also significantly upregulated in the TP53WT&CD58MUT group when comparing with the TP53WT&CD58WT group, suggesting the unique immune phenotype in the TP53WT&CD58MUT group. The findings that high immune scores and abundant infiltrating exhausted T cells in the TP53WT&CD58MUT group were validated in an independent external cohort from the REMoDL-B trail (N=400). GSEA showed significantly activated interferon-α and -γ responses and IL-6/JAK/STAT3 signaling in the TP53WT&CD58MUT group. Conclusions: Our findings suggest that TP53 mutation alone is insufficient to effectively differentiate the risk of DLBCL. The mutually exclusive patterns between TP53 and CD58 mutations accurately stratified patients with DLBCL to permit the optional immunotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Laboratory Mutation Analysis Performed on Tumor Biopsies from Patients with Newly-Diagnosed Germinal Center Aggressive B Cell Lymphoma

Background Comprehensive genomic analyses of tumor biopsies (bx) from patients (pts) with newly-diagnosed germinal center B cell (GCB) diffuse large B cell lymphoma (DLBCL) and high grade B cell lymphoma (HGBL) have identified molecular subtypes predictive of inferior survival outcomes following receipt of first-line immunochemotherapy (PMID: 29713087, 29641966, 30523719, 30523716). These experimentally-defined molecular subtypes are characterized by somatic mutations (mut), which can be detected through clinical laboratory mutation analysis (CLMA). We sought to identify the frequency and predictive value of these mut as detected by CLMA performed at our center on tumor bx from pts with newly-diagnosed GCB DLBCL/HGBL. Methods Pts diagnosed with de novo or previously-untreated transformed indolent GCB DLBCL/HGBL defined by Hans algorithm from 2015-21 who received R-CHOP or R-EPOCH in the first-line setting whose initial tumor bx underwent CLMA performed at the University of Pennsylvania with either the lymphoma sequencing panel (LSP) or PennSeq Lymphoma panel (PSLP) were retrospectively analyzed. Immunohistochemical staining (IHC) for MYC and BCL2 expression as well as fluorescence in situ hybridization (FISH) for MYC, BCL2 and BCL6 rearrangements were performed as per institutional standard. Therapy was given at the discretion of the treating physician. Disease free survival (DFS) was defined as the interval between diagnosis of DLBCL/HGBL to DLBCL/HGBL relapse or last follow-up in remission. Overall survival (OS) was defined as the interval between diagnosis and time of death or last follow-up while alive. Data were censored on 7/1/22. Results CLMA was performed on 59 pt tumor bx with 58 successful assays (98% success rate) and a median result turnaround time (TAT) of 16 days. 5 bx were excluded due to specific request for CLMA by treating clinician (3) or diagnosing pathologist (2) and 2 additional bx were excluded due to lack of clinical follow-up, resulting in analysis of 51 bx performed at random from pts evaluable for survival (n=32 LSP, n=19 PSLP). Baseline characteristics, including mut in genes of interest (GOI) identified in the above references, are listed in the Table. Of note, BCL2, MYC, DDX3X and PTEN mutations were not included the LSP and therefore not included in statistical analyses due to small sample size. R-EPOCH was received by 15 pts (29%) and R-CHOP by 36 pts (71%). With a median follow-up of 25.2 months, the Kaplan Meier estimate of 2 year (y) DFS was 60% (95% confidence interval [CI] 41-74%) and 2y OS 80% (95% CI 64-89%). As shown in the Figure, 2y DFS was significantly lower for pts with CREBBP mut vs not (45% [95% CI 18-68%] vs 67% [95% CI 44-83%], P=0.045), but not for pts based on other mut analyzed. All progression events for pts with CREBBP mut occurred ≤12 months (mo) from diagnosis. Of 16 bx with CREBBP mut, 15 mut were predicted to result in loss of function (LOF) (PMID: 28890946, 21390126). CREBBP mut were not significantly associated with baseline characteristics except for EZH2 mut (50% with CREBBP mut vs 11% no CREBBP mut, P=0.005 by Fisher's exact). Characteristics listed in the Table which were predictive of disease relapse by 2y with P<0.10 on univariate Cox regression analysis were International Prognostic Index Score ≥3 vs <3 (hazard ratio [HR] 2.3, 95% CI 0.86-6.5, P=0.098), HGBL vs DLBCL histology (HR 5.4, 95% CI 1.8-15.7, P=0.002), double hit lymphoma vs not (HR 9.0, 95% CI 2.6-30.8, P<0.001) and CREBBP mut vs not (HR 2.6, 95% CI 0.98-7.0, P=0.054). Conclusions In this cohort of pts with newly-diagnosed GCB DLBCL/HGBL treated with first-line immunochemotherapy whose tumor bx underwent CLMA at random, mut in CREBBP resulting in predicted LOF were frequently detected, not associated with routinely-analyzed baseline clinicopathologic characteristics and predicted for significantly lower 2y DFS with progression events occurring early. These findings may be of interest in light of availability of agents which may overcome loss of histone acetyltransferase function. Given the high success rate and rapid TAT of CLMA, results from this assay could inform the management of pts with newly-diagnosed DLBCL/HGBL in real-time, and additional efforts to incorporate findings from experimental molecular assays into CLMA-based studies as well as those to standardize CLMA gene panels may ultimately benefit this pt population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The human DEAD-box helicase DDX3X as a regulator of mRNA translation.

The human DEAD-box protein DDX3X is an RNA remodelling enzyme that has been implicated in various aspects of RNA metabolism. In addition, like many DEAD-box proteins, it has non-conventional functions that are independent of its enzymatic activity, e.g., DDX3X acts as an adaptor molecule in innate immune signalling pathways. DDX3X has been linked to several human diseases. For example, somatic mutations in DDX3X were identified in various human cancers, and de novo germline mutations cause a neurodevelopmental condition now termed 'DDX3X syndrome'. DDX3X is also an important host factor in many different viral infections, where it can have pro-or anti-viral effects depending on the specific virus. The regulation of translation initiation for specific mRNA transcripts is likely a central cellular function of DDX3X, yet many questions regarding its exact targets and mechanisms of action remain unanswered. In this review, we explore the current knowledge about DDX3X's physiological RNA targets and summarise its interactions with the translation machinery. A role for DDX3X in translational reprogramming during cellular stress is emerging, where it may be involved in the regulation of stress granule formation and in mediating non-canonical translation initiation. Finally, we also discuss the role of DDX3X-mediated translation regulation during viral infections. Dysregulation of DDX3X's function in mRNA translation likely contributes to its involvement in disease pathophysiology. Thus, a better understanding of its exact mechanisms for regulating translation of specific mRNA targets is important, so that we can potentially develop therapeutic strategies for overcoming the negative effects of its dysregulation.

Clinical relevance of molecular characteristics in Burkitt lymphoma differs according to age

While survival has improved for Burkitt lymphoma patients, potential differences in outcome between pediatric and adult patients remain unclear. In both age groups, survival remains poor at relapse. Therefore, we conducted a comparative study in a large pediatric cohort, including 191 cases and 97 samples from adults. While TP53 and CCND3 mutation frequencies are not age related, samples from pediatric patients showed a higher frequency of mutations in ID3, DDX3X, ARID1A and SMARCA4, while several genes such as BCL2 and YY1AP1 are almost exclusively mutated in adult patients. An unbiased analysis reveals a transition of the mutational profile between 25 and 40 years of age. Survival analysis in the pediatric cohort confirms that TP53 mutations are significantly associated with higher incidence of relapse (25 ± 4% versus 6 ± 2%, p-value 0.0002). This identifies a promising molecular marker for relapse incidence in pediatric BL which will be used in future clinical trials.

Shared and Distinct Genetic Features in Human and Canine B-Cell Lymphomas

IntroductionAnimal models of human cancers are an important tool for the development and preclinical evaluation of new treatments. Canine B-cell lymphoma (cBCL) is an appealing alternative to murine preclinical models due to its frequent, spontaneous incidence and its clinical and histological similarity to human B-cell non-Hodgkin lymphoma (NHL). The potential utility of cBCL as a veterinary model of human B-cell lymphomas would be bolstered by a more complete understanding of the genetic features found in cBCL.MethodsTo study the genetics of cBCL, we obtained fresh frozen and matched plasma/serum from 86 patients from the Canine Comparative Oncology Genomic Consortium(CCOGC) with 65 confirmed as B-cell lymphomas by immunophenotyping. Tumor DNA was prepared into libraries using the QIAseq FX DNA Library Kit (Qiagen). Plasma and serum DNA was prepared into libraries using the NebNext Ultra II DNA Library Prep Kit. Targeted hybridization enrichment was performed on the libraries using our custom baits and sequencing reads were aligned to canFam3.1 using Geneious and each mutation was visually confirmed. Variants were annotated with Variant Effect Predictor and human-dog pairwise alignments were extracted from Ensembl to identify the orthologous human amino acid for all canine variants.ResultsOur analysis confirmed the previously reported high frequency of mutations in TRAF3 and FBXW7. We also observed mutations in POT1, TP53, and SETD2 at similar frequencies to those reported in previous studies. DDX3X was mutated in 20% of cases, which is substantially higher than previously reported. MYC mutations were also more frequent (13%) than has been previously described in cBCL.In human lymphomas, MYC is commonly deregulated by translocation to a potent enhancer and these events are often associated with point mutations in MYC that are induced by aberrant somatic hypermutation (aSHM). Interestingly, we identified a more focal pattern of MYC mutations in cBCL that implies they do not result from aSHM and are likely functional. This finding implicates the conserved MYC phosphodegron sequence, a motif commonly mutated among additional aSHM-associated mutations, as the target of bona fide driver mutations in both human and cBCLs. Mutations in FBXW7 primarily affected the substrate recognition domain responsible for MYC degradation. The observation that MYC and FBXW7 mutations did not co-occur in any canine patient is consistent with the notion that FBXW7 mutations operate as an alternative path to MYC stabilization which is not frequently observed in human NHL.DDX3X was one of the most frequently mutated genes in our cohort (20%). DDX3X mutations are common in human Burkitt lymphoma and, though less abundant in hDLBCL, tend to be observed in samples with MYC translocations. In Burkitt lymphoma, these mutations display a sex-specific pattern, wherein females show mainly missense mutations, while males are affected by loss-of-function mutations. Interestingly, all DDX3X mutations in cBCL are missense variants and are presumed to be dominant acting. This lack of sex difference in DDX3X mutations is an important distinction between human and canine B-cell lymphomas that warrants further exploration.ConclusionsOur study has revealed key differences in the mutational profiles of canine and human B-cell lymphomas and provides an impetus for enhanced genomic characterization of canine lymphomas as a model for human NHL, particularly in clinical trial settings. DisclosuresGrande: Sage Bionetworks: Current Employment. Alcaide: GA Diagnostics AB: Current Employment. Morin: Celgene: Consultancy; Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees; Epizyme: Patents & Royalties. Coyle: Allakos, Inc.: Consultancy.

Novel Genetic Subgroups Inform on Shared Pathobiology within Adult and Pediatric Burkitt Lymphoma

Novel Genetic Subgroups Inform on Shared Pathobiology within Adult and Pediatric Burkitt Lymphoma

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven lymphomagenesis

DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

Molecular and cellular basis of hyperassembly and protein aggregation driven by a rare pathogenic mutation in DDX3X

SummaryCurrent studies estimate that 1–3% of females with unexplained intellectual disability (ID) present de novo splice site, nonsense, frameshift, or missense mutations in the DDX3X protein (DEAD-Box Helicase 3 X-Linked). However, the cellular and molecular mechanisms by which DDX3X mutations impair brain development are not fully comprehended. Here, we show that the ID-linked missense mutation L556S renders DDX3X prone to aggregation. By using a combination of biophysical assays and imaging approaches, we demonstrate that this mutant assembles solid-like condensates and amyloid-like fibrils. Although we observed greatly reduced expression of the mutant allele in a patient who exhibits skewed X inactivation, this appears to be enough to sequestrate healthy proteins into solid-like ectopic granules, compromising cell function. Therefore, our data suggest ID-linked DDX3X L556S mutation as a disorder arising from protein misfolding and aggregation.

Genetic Background of Chronic Active Epstein-Barr Virus Disease

IntroductionEpstein-Barr virus (EBV) is associated with various benign and malignant diseases. Chronic active EBV disease (CAEBV) is a rare EBV-associated disease characterized by persistent EBV infection in natural killer (NK) and/or T cells, which is mainly prevalent in East Asia. While patients with CAEBV initially present with persistent or recurrent symptoms similar to ones present in infectious mononucleosis (IM), they often develop peripheral NK/T-cell lymphoma or multiple organ failure in the long run, and prognosis is dismal.Numerous aspects of the molecular pathogenesis of CAEBV are unresolved; it is unclear whether this disease is a clonal disorder. Although several case reports indicate the presence of primary immunodeficiency (PID) in patients with persistent EBV disease, the prevalence of PID or other inherited diseases in patients with CAEBV remains unclear. To reveal the genetic background of CAEBV, we performed whole-exome and targeted sequencing.Patients and MethodsWe studied 83 patients with CAEBV. The diagnosis of CAEBV was based on the fulfillment of the following criteria: (1) persistent or recurrent symptoms similar to those present in IM, (2) increased quantities of EBV viral load in either affected tissues or peripheral blood, and (3) exclusion of other possible diagnoses. The Institutional Review Board of Nagoya University Graduate School of Medicine approved this study.We performed whole-exome sequencing (WES) of T-, B-, and NK-cell subsets. Subsequently, we performed targeted gene sequencing of 605 genes associated with PID, inherited blood diseases, and hematological malignancies.ResultsRegarding germline mutations, we identified two patients who carried nonsense mutations in EVC2, which led to the diagnosis of Weyers acrofacial dysostosis (WAD). We also identified a patient who carried POLH mutations, which led to the diagnosis of the variant type of xeroderma pigmentosum, and a patient who carried an APC mutation, which led to the diagnosis of familial adenomatous polyposis. Mutations leading to the diagnosis of PID were not identified in any patient.WES led to the identification of at least one somatic mutation in 58% of patients. We identified DDX3X (n= 14; 16%), KMT2D (n= 4; 4.8%), BCOR / BCORL1 (n= 3; 3.6%), KDM6A (n= 3; 3.6%), and TET2 (n= 2; 2.4%) as undergoing recurrent mutations.We analyzed multiple EBV-infected cell subsets within a patient. One patient (UPN125) developed CAEBV in which both T and NK cells were positive for EBV DNA. The patient's CD4+ and CD56+ cells carried 21 and 27 mutations, respectively, of which 8 were shared including a DDX3X p.Arg534Cys driver mutation. Similar to UPN125, CD3+ and CD56+ cells of UPN404 carried a shared DDX3X p.Thr198Pro mutation and private mutations. CD19+ cells of UPN404 also carried the identical DDX3X mutation.In the survival analysis, patients carrying DDX3X mutations at diagnosis demonstrated a significantly worse OS compared with others (5-year OS: 40.6% vs. 78.6%; P= 0.007). Patients carrying KMT2D mutations also demonstrated poor prognosis (5-year OS: 50.0% vs. 73.9%; P= 0.02).DiscussionIn the present study, we found that CAEBV was a malignant disease carrying somatic driver mutations, the spectrum of which showed similarity with that of extranodal NK/T-cell lymphoma (ENKTL) and Burkitt lymphoma. DDX3X mutations and other shared mutations, which were present in multiple cell subsets in a patient, strongly suggest the presence of common ancestry, possibly lymphoid progenitor cells, which acquired driver mutations.We identified the presence of somatic DDX3X mutations as a marker of poor prognosis. A prospective study can be designed using genetic testing for DDX3X mutations and subsequent interventions, possibly chemotherapies used for ENKTL and early hematopoietic stem cell transplantation, to improve patient outcome.Furthermore, we identified several germline mutations. WAD caused by EVC / EVC2 mutations is characterized by the malformation of mesoderm and ectoderm caused by decreased hedgehog signaling mediated via EVC/EVC2 . Together with POLH and APC mutations, EVC / EVC2 mutations may be a genetic predisposition to CAEBV.In conclusion, we revealed the genetic background of CAEBV, which will lead to further clarification of its molecular pathogenesis and the development of therapeutic strategy. Clinical trials utilizing genetic diagnosis and stratified therapy are warranted. DisclosuresNo relevant conflicts of interest to declare.