Bhardwaj, Vaishali

Abstract Background: Waldenström Macroglobulinemia (WM) is an indolent B-cell lymphoplasmacytic lymphoma characterized by heterogeneous clinical behavior and variable progression patterns. While MYD88L265Pand CXCR4 mutations are frequently encountered, the broader genomic landscape underpinning disease evolution from smoldering WM (SWM) to active treatment naïve WM (TNWM), post-treatment remission WM (PTWM) and relapsed/refractory WM (RRWM) remains incompletely understood. To address this gap, we studied the mutational profile across the WM disease spectrum. Methods We performed whole exome sequencing (WES) on sorted (CD19 and/or CD138) bone marrow aspirates of patients across the spectrum of WM. Paired germline sequencing was performed using peripheral blood mononuclear cells (PBMCs). Unique molecular identifier-tagged reads were deduplicated using fgbio. WES data were processed through the Sarek pipeline. Any valid mutation from either caller, present on the index or in COSMIC was considered. Copy number variations (CNVs) were inferred via CNVkit and summarized at the gene and arm level (≥50% directional change). Mean tumor and germline coverages were 174X and 74X, respectively. Fisher's exact test was used for enrichment and hypergeometric test for overlap analyses. Results A total of 78 high-quality WM samples [15 SWM, 44 TNWM, 13 RRWM, 6 PTWM in remission (all with paired TNWM samples)] were sequenced. The mutational burden varied across disease states (median SWM=1.48 m/Mb, TNWM= 1.12 m/Mb, RRWM = 1.59 m/Mb; p=0.04). Density plots revealed bimodal VAF distributions, consistent with clonal and subclonal architectures in WM. Overall, 77% harbored a MYD88L265P mutation by WES. Among MYD88^WT^ patients, AS-PCR identified MYD88L265P in 4 (25%) not detected by WES, raising the overall mutation rate to 83%, plus one with a non-L265P variant. Low VAF (<10%) MYD88L265P mutations were seen in 6% of matched PBMCs. MYD88 copy number gain was seen in 2 (17%) MYD88WT TNWM patients. CXCR4 mutations were detected in 38% by WES (18% each frameshift and nonsense). Two (4%) CXCR4WT samples were positive by PCR, giving an overall rate of 40%. CXCR4 mutations were more frequent in RRWM (54%) vs TNWM (41%) and SWM (27%), though not statistically significant. Del(6q) and Del(17p) were noted in 16% (n=12) and 7% (n=5) of patient samples. All del(17p) events were noted in TNWM samples and in one paired post-treatment sample. Overall rate of del(17p) and TP53 mutation was 14% (n=11). RRWM was enriched in ARID1A alterations, a gene implicated in chromatin remodeling and plasma cell identity formation [38% (n=5) in RRWM vs. 11% (n=6) in TNWM and SWM], compared to TNWM and SWM p=0.03]. Alterations in NF-kB signaling gene MAP3K14 were more frequent in RRWM (31%, n=4) compared to the SWM/TNWM cohort [(7%), n=4; p=0.036). We noted an enrichment in the TP53 mutation rate in RRMW [n=3, 23%, OR 7.7] compared to TNWM and SWM cohorts [n=2, 4%), p=0.04]. Mutations in FUBP1 (p=0.03), an upstream regulator of MYC, and CARD11 (p=.043), a B-cell signaling scaffold protein were also more frequent in RRWM. Notably, none of the SWM patients harbored an ARID1A alteration (p=0.057), suggesting acquisition during progression. CNV analysis revealed that MAP3K14 amplifications were frequent in RRWM (n=3, 23%) compared to SWM/TNWM [n=2 (4%), p=0.043]. RRWM samples showed increased CD79B mutations (15%) and significant overlap with the C5/ABC diffuse large B-cell lymphoma (DLBCL) genomic cluster (Chapuy et al., Nat. Med. 2018; p=0.007). This overlap remained significant after excluding MYD88L265P (p=0.019), driven by CARD11, CD79B, and IGLL5, suggesting that a subset of RRWM may acquire an ABC DLBCL-like mutational profile. These findings warrant further investigation using transcriptional profiling. Conclusions This study provides a genomic map of WM evolution from precursor to relapse. Increasing somatic mutations and CNVs support stepwise progression. Enrichment of genes involved in chromatin remodeling and plasma cell identity formation (ARID1A), NF-kB or BCR signaling (CARD11, MAP3K14), and DNA damage repair (TP53) were noted at relapse, all of which have been shown to drive aggressive disease biology. Emergence of an ABC-like genotype in a proportion of RRWM indicates convergent evolution with aggressive lymphomas. Transcriptional studies are warranted to explore this further.

Abstract Description A subset of follicular lymphoma patients has an early relapse with a poor outcome. We showed that this unfavorable group displays increased expression of IRF4, dysregulated immune signaling and an immunosuppressive microenvironment. However, little is known on the molecular mechanisms whereby IRF4 controls the crosstalk between B and T cells. To this aim, we leveraged Cγ1cre;irf4fl/fl mice that yield conditional deletion of irf4 (cKO) in germinal center B cells (GCB). After 10 days of immunization, cKO GCB showed significant increase of HLA-DR and CD40 expression, a skewed LZ:DZ ratio towards increased DZ, and suppression of plasma cells compared to WT. Upon irf4 deletion, we also found a significant increase of total CD4 and TFH cells and a decrease in Treg cells. To test if this effect was antigen-dependent, we cocultured sorted GCB from NP-OVA-immunized cKO and WT mice with CSFE-labeled OVA-specific OT-II CD4 T cells at a ratio of 2:1 for 5 days. We observed increased antigen specific proliferation of CD4 T cells with cKO GCB compared to WT. We then performed seahorse assay and mass spectrometry of the same GCB to define if IRF4-dependent B cell metabolic reprogramming influences immune responses. GCB cKO had lower glycolytic ECAR and increased intracellular glucose and non-essential amino acids than WT cells, suggesting a metabolic shift from glycolysis to the TCA cycle. Thus, IRF4 controls immune and metabolic signaling with functional effects on the B-T cell crosstalk. Funding Sources NCI K08CA279652, LRF Clinical Investigator Award (CDA 1020588), the Gerstner Family Career Development Award, the ASH Junior Faculty Scholar Award. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Signal regulatory protein-α (SIRPα) is a key member of the “do-not-eat-me” signaling pathway and is expressed on intratumoral monocytes/macrophages in lymphoma. In this study, we found that SIRPα is also expressed by a subset of malignant B cells in lymphoma. SIRPα+ B cells have a memory phenotype with germinal center (GC) dark-zone features, and RNA velocity analysis showed that SIRPα+ B cells exhibit higher spliced transcripts confirming a more mature phenotype. SIRPα expression is substantially upregulated by treating lymphoma B cells with LPS or CpG and SIRPα+ B cells showed increased proliferation when compared to SIRPα- B cells, which was associated with reduced cellular autophagy. SIRPα+ B cells exhibited increased expression of BAFF signaling pathway molecules as the likely mechanism of increased proliferation and survival. Furthermore, in co-culture, SIRPα+ lymphoma B cells suppressed T-cell function and blocking SIRPa/CD47 signaling reversed T cell inhibition. Clinically, we found that a greater number of SIRPα+ B cells present in the diagnostic tumor biopsies of FL patients was associated with a significantly poorer overall survival. We conclude that SIRPα+ lymphoma B cells are a proliferative component of indolent lymphomas, are associated with T-cell suppression, and increased prevalence of these cells is associated with a poor prognosis. Targeting SIRPα+ B cells in lymphoma may therefore be a promising therapeutic strategy. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description The efficacy of immunotherapy for splenic marginal zone lymphoma (SMZL) remains limited due to the strong immune suppression present in the tumor microenvironment (TME). Tregs play a key role in maintaining TME homeostasis by suppressing effector T cells and malignant B cells. Previously, we identified two Treg subsets CD26+ and CD161+ in SMZL. CD161+ Tregs were more effective at suppressing both CD8+T cells and malignant B cells, whereas CD26+Tregs primarily inhibited CD8+T cells. Patients with higher levels of CD26+ Tregs and fewer CD161+ Tregs tend to have worse outcomes. In this study, we found that cytokine-inducible SH2-containing protein (CISH), a known negative regulator of immune activation, is highly expressed in CD26+Tregs compared to CD161+ Tregs. Co-culturing B cells enhanced the expression of CISH and CD26 in CD4+CD25- Treg cells, knockdown of CISH in Tregs reduced CD26 expression and their immune suppression activity of CD8+T cells, while CRISPR knockout of CISH in Karpas 299 cells led to higher expression CD70, a molecule associated with less immunesuppression. Our findings suggest that increased CISH expression in Tregs could result in increased immune suppression and unfavorable outcome in SMZL, suggesting CISH is a potential therapeutic tagrget in SMZL by dempening immunosuppressive property of the Treg cells hence boosting the antitumor immunity. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Twenty percent of patients with follicular lymphoma relapse early with poor outcomes; however, the molecular mechanisms underlying this aggressive behavior are unknown. Using a multiomic approach, we show that patients with follicular lymphoma with elevated IRF4 expression (IRF4hi) have increased transformation risk, dysregulated immune signaling, and a suppressive tumor microenvironment. Loss- and gain-of-function experiments in IRF4hi lymphoma cells, along with chromatin profiling, demonstrate that IRF4 impairs their interaction with T cells by repressing antigen presentation and co-receptor gene modules while promoting the expression of cytokines that antagonize T-follicular helper cell and regulatory T-cell functions. Additionally, IRF4 rewires tumor metabolism and restricts glucose availability to immune cells. Silencing of IRF4 inhibits tumor cell growth and restores immune surveillance mechanisms, thus representing a promising target for therapy. Our data suggest that IRF4hi lymphoma cells co-opt a developmental mechanism used to exit the germinal center response in promoting a more aggressive cancer via engagement of multiple immune-evasive mechanisms. Significance: We show that IRF4 controls immune and metabolic signaling with functional effects on the cross-talk between B and T cells, resulting in a suppressive immune microenvironment when IRF4 is overexpressed in lymphoma. This underscores the importance of IRF4 as a pivotal therapeutic target to restore antitumor immunosurveillance for IRF4-associated lymphomas.

Abstract Intracellular protein transport (ICT) is a tightly regulated process that orchestrates protein localization and expression, ensuring proper cellular function. Dysregulated ICT can lead to aberrant expression of surface proteins involved in cell-cell communication, adhesion, and immune responses, contributing to disease progression and therapeutic resistance. Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) enables the simultaneous measurement of mRNA and surface protein levels in the same cell, providing a powerful opportunity to investigate the molecular mechanisms underlying surface protein regulation. In this study, we introduce a novel computational frame for Modeling Protein Expression and Transport (MPET) that evaluates the contribution of ICT activity to differential surface protein expression using CITE-seq data. MPET comprises three modules for identification of ICT– surface protein regulatory circuits across biological scales and their contributions to phenotypic variation. We applied MPET to analyze single-cell data from COVID-19 patients with varying disease severity. Our analysis revealed context-dependent recruitment of ICT genes and pervasive rewiring of ICT pathways throughout the course of disease progression. Notably, we found that even when the transcriptional levels of key immune response proteins remained stable, their expression on cell surface were significantly altered due to dysregulated ICT. MPET provides a valuable new tool for dissecting complex regulatory networks and offers mechanistic insight into post-transcriptional regulation of cell surface proteins in diseases.

Splenic marginal zone lymphoma (SMZL) is a B-cell malignancy with a typically indolent course. However, some patients have more aggressive disease and initial treatment can range from observation to chemoimmunotherapy. The reason for this spectrum of disease behavior is incompletely understood but the tumor microenvironment (TME) likely plays a role as recent studies have shown that an optimized antitumor immune response in SMZL patients would allow the immune system to better target malignant B cells and irradicate the disease. Regulatory CD4+T cells (Tregs) are crucial in maintaining TME homeostasis by suppressing effector T cells and malignant B cells and in this study, we therefore explored the key factors influencing the function and development of intratumoral Tregs in SMZL patients. We identified two distinct Treg subsets in the TME of SMZL patients: CD26+Tregs and CD161+Tregs, each with unique phenotypic profiles, functions, and prognostic significance. CD161+Tregs suppress CD8+T cells and malignant B cells more effectively than CD26+Tregs, which only inhibit CD8+T cells. Clinically, patients with inferior outcomes have significantly higher numbers of CD26+Tregs and fewer CD161+Tregs. To delineate differences between these subsets, we performed Cellular Indexing of Transcriptomes and Epitopes Sequencing (CITE-seq) on biopsies from 7 SMZL patients. We found that cytokine-inducible SH2-containing protein (CISH) is the most highly expressed gene in CD26+Tregs compared to CD161+Tregs, aside from FOXP3. CISH has been reported to be a negative regulator of immune effector cell activation in tumor models. Although CISH deficiency leads to a loss of Foxp3 expression and reduced Treg suppressive function, the mechanism by which CISH modulates Treg function, especially immune suppression in hematologic malignancies, remains largely unknown. To further investigate the mechanism of CISH in Treg function in MZL patients, we conducted CITE-seq analysis on a larger cohort of MZL patients (n=12), divided into groups based on outcomes (EFS24 failed, n=4; EFS24 achieved, n=8). The CITE-seq analysis results showed significantly higher CISH expression in Tregs from patients with inferior outcomes (P=1.07E-06). We then explored factors influencing CISH expression and found that TCR engagement and treatment with cytokines (IL-15, IL-2, and IL-7) elevated CISH expression in Tregs. While we previously observed that lymphoma B cells could enhance Foxp3 expression in CD4+CD25-T cells, this study we found that B cells could also enhance CISH and CD26 expression in CD4+CD25-T cells in vitro, indicating that a high frequency of B cells in the environment may lead to increased CISH expression in Treg cells leading to an unfavorable outcome in patients. We further investigated the role of CISH in the function of Treg cells. Following CISH knockdown in Treg cells, we observed decreased CD26 expression and reduced suppressive function on CD8+ T cell proliferation. Using CRISPR-Cas9, we knocked out CISH in the Karpas 299 cell line, a human T cell lymphoma line that constitutively expresses CD25, Foxp3, and CISH. CISH-KO Karpas 299 cells showed significantly less apoptosis and Foxp3 expression compared to CISH-WT cells. Bulk RNA-seq analysis of CISH-KO and CISH-WT Karpas 299 cells revealed that CISH-KO cells showed enhanced DNA replication and cell cycle pathway activity. Taken together, our results suggest that high expression of CISH in Tregs may predict an unfavorable outcome in SMZL patients. Malignant B-cells in the TME of SMZL patients may promote expression of CD26 and CISH in Tregs, thereby modulating Treg function. Targeting CISH could decrease the immunosuppressive activity of Tregs in the TME, potentially optimizing antitumor immunity in SMZL patients.