Waldenstrom Macroglobulinemia Cell Research Articles

Bruton Tyrosine Kinase Inhibition: an Effective Strategy to Manage Waldenström Macroglobulinemia.

The treatment of Waldenström macroglobulinemia (WM) has evolved over the past decade. With the seminal discoveries of MYD88 and CXCR warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) mutations in WM cells, our understanding of the disease biology and treatment has improved. The development of a new class of agents, Bruton tyrosine kinase inhibitors (BTKi), has substantially impacted the treatment paradigm of WM. Herein, we review the current and emerging BTKi and the evidence for their use in WM. Clinical trials have established the role of covalent BTKi in the treatment of WM. Their efficacy is compromised among patients who harbor CXCR4WHIM mutation or MYD88WT genotype. The development of BTKC481 mutation-mediated resistance to covalent BTKi may lead to disease refractoriness. Novel, non-covalent, next-generation BTKi are emerging, and preliminary results of the early phase clinical trials show promising activity in WM, even among patients refractory to a covalent BTKi. Covalent BTK inhibitors have demonstrated meaningful outcomes in treatment-naïve (TN) and relapsed refractory (R/R) WM, particularly among those harboring the MYD88L265P mutation. The next-generation BTKi demonstrate improved selectivity, resulting in a more favorable toxicity profile. In WM, BTKi are administered until progression or the development of intolerable toxicity. Consequently, the potential for acquired resistance, the emergence of cumulative toxicities, and treatment-related financial burden are critical challenges associated with the continuous therapy approach. By circumventing BTK C481 mutations that alter the binding site to covalent BTKi, the non-covalent BTKi serve as alternative agents in the event of acquired resistance. Head-to-head comparative trials with the conventional chemoimmunotherapies are lacking. The findings of the RAINBOW trial (NCT046152), comparing the dexamethasone, rituximab, and cyclophosphamide (DRC) regimen to the first-generation, ibrutinib are awaited, but more studies are needed to draw definitive conclusions on the comparative efficacy of chemoimmunotherapy and BTKi. Complete response is elusive with BTKi, and combination regimens to improve upon the efficacy and limit the treatment duration are also under evaluation in WM.

Deciphering the Immune Microenvironment in Waldenstrom's Macroglobulinemia

Recent studies have demonstrated the occurrence of somatic mutations of MYD88 and CXCR4 as key players in Waldenstrom's Macroglobulinemia (WM) pathogenesis and disease progression. Despite the significant improvement in the knowledge of the molecular mechanisms supporting WM biology whether immunosuppressive mechanisms could contribute to WM pathogenesis remains unexplored. We interrogated the transcriptome signatures of the bone marrow (BM) microenvironment of WM patients (n:22) as compared to healthy individuals (n:10), we found a significant enrichment for a Treg-signature, and for CD40/CD40L signaling-related genes (FDR <0.001; P <0.01). These findings were confirmed using a transgenic murine lymphoplasmacytic/WM model, where the BM immune microenvironment showed an increased number of infiltrating CD4+ T lymphocytes over CD8+ T cells. Further characterization of the T-cell compartment revealed the presence of abundant CD4+CD25+FoxP3+ Treg cells, with respect to control mice. Murine WM cells recruited a significantly higher number of more abundant Ki67+Treg cells, as compared to B lymphocytes from healthy mice. Collectively, these results suggest that immunosuppressive Treg cells may play in supporting WM disease biology. These findings prompted us to focus on Tregs and their role in supporting WM pathogenesis. We performed bulk RNA sequencing of Tregs isolated from both WM patients (n:14) and healthy donors/HD (n:8); showing a peculiar transcriptome signature characterizing WM-derived Tregs as compared to their normal cellular counterpart. WM patient-derived Tregs presented with transcriptome profiling enriched for FOXP3-target genes and to Treg induction, as compared to healthy donor-derived Tregs; with WM-Tregs showing a significant enrichment for genes related to interferon- and TNFα-related genes via NF-kB. The functional impact of Treg in WM biology was next studied. We observed a significantly higher ability of WM cells to favor both induction and expansion of CD4+CD25+FoxP3+ Tregs, as compared to HD-derived CD19+ B-cells; paralleled by a significantly higher increase of Ki67-positive Treg induced by WM cells as compared to HD-derived CD19+ B-cells. Same results were obtained using WM primary cells. Data were further corroborated by demonstrating significant enrichment of several pro-proliferative- and pro-survival pathways in WM-Tregs, versus HD-counterpart, including MAPK- and PI3K/AKT-related genes. To gain insight into the potential molecular mechanisms responsible for the observed Treg induction in WM, we performed scRNAseq using the whole cell population harvested at the end of the induction assay. We evaluated the B-T-cell interactions at single cell level, adopting a B->T cross-talk model; and identified a set of four high-priority genes (CD40, TNF, TNFSF14, ICAM2): although all of them were expressed in B cells, only the expression of CD40 was maximal and virtually restricted to the B clusters, with the other three genes showing a more diffuse pattern. Importantly, CD40-ligand was shown to be the interactor expressed within the Treg cell compartment. Given the identified CD40/CD40L axis as a potential regulator of the WM cell/Treg cross-talk, we used the CD40/CD40L inhibitor DRI-C21045; and found a significant decrease in Treg induction and Treg proliferation. Halting CD40/CD40L interaction inhibited Treg induction and growth, also within the context of CXCR4-mutated WM, supported by inhibition of p-AKT and p-ERK in Treg cells. CD40/CD40L blockade led to inhibition of WM cell growth. Overall our studies have demonstrated the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

Establishment of the Bcwm.2 Cell Line As a BTK-Inhibitor Resistant, BCL2 Inhibitor Sensitive in Vitro and In Vivo Study Model for Waldenström's Macroglobulinemia

Background: Waldenström's macroglobulinemia (WM) is an indolent B-cell lymphoproliferative disorder characterized by bone marrow (BM) infiltration with lymphoplasmacytic lymphoma and monoclonal immunoglobulin M (IgM) production. Mutations in MYD88 are present in 95-97% of WM patients. Deletions involving chromosome 6q (del6q) are found in up to 50% of WM patients, and include genes that regulate MYD88, BCL2 and apoptotic signaling. Cell lines can play a pivotal role as disease models, contributing to a comprehensive understanding of WM biology and advancing therapeutic strategies. The availability of cell lines for the study of WM remains limited, and none demonstrate del6q. Patient and Methods: We developed and characterized a novel cell line (BCWM.2) from long-term cultures of CD19 + selected BM lymphoplasmacytic cells from a symptomatic, treatment naive WM patient who then received ibrutinib monotherapy on a clinical trial. The patient attained a major response to treatment, and after 2 years progressed. The patient underwent serial bone marrow biopsies, and extensive genomic and transcriptome analysis of his tumor during the study, and the corresponding cell line established at baseline. An in vivo xenograft mouse model of BCWM.2 was also established in NOD SCID mice, and the origin of the xenografted tumor was confirmed by genomic sequencing and immunohistochemistry. Results: BCWM.2 cells exhibited morphologic and immunophenotypic characteristics resembling lymphoplasmacytic cells and demonstrated robust propagation when co-cultured with HS-5 stromal cells in IMDM medium supplemented with 20% FBS. Flow cytometric analysis revealed that BCWM.2 exhibited an immunophenotype consistent with the source WM patient. Whole genome sequencing demonstrated that both BCWM.2 cells and original patient WM cells carried somatic activating mutation in MYD88 (S243N) and shared trisomy in chromosomes 3 and 12, heterozygous deletion of 6q, and amplification of 6p. Notably, a novel mutation in the SRC family member LYN (I297N; T>A), a component of BCR, was identified in BCWM.2 but not the original patient tumor. The I297N mutation found in the regulatory hinge region is predicted to cause activation of LYN. At time of progression on ibrutinib, the dominant clone that emerged in the patient's tumor also carried the I297N mutation. Additionally, mutated genes in BCWM.2 included histone deacetylase HDAC5, tumor suppressor RUNX3, B-cell transcriptional activator SPI1, and AKAP9 (A-Kinase Anchoring Protein 9). Transcriptome analysis showed shared expression patterns between BCWM.2 and patient tumor samples, including diminished gene expression of chromosome 6q. Drug response testing revealed that BCWM.2 cells did not respond to the BTK-inhibitors ibrutinib, zanubrutinib or pirtobrutinib but were sensitive to the BCL2 inhibitor venetoclax. Furthermore, BCWM.2 cells readily engrafted in NOD-SCID mice by direct subcutaneous injection of 2 x 10^6 cells/mouse. Tumor growth (~500 mm^3) was observed in the mice four weeks post-inoculation. Importantly, follow-up evaluations demonstrated serial increases in human IgM levels in the mouse sera post-engraftment. Moreover, BCWM.2 engraftment was confirmed by immunohistochemistry with tumors exhibiting intermediate lymphoid cell morphology with plasmacytoid differentiation, CD20 and MUM1 expression, clonality for lambda light chain and IgM heavy chain, 60% KI67 staining. Whole exome sequencing of engrafted tumors further confirmed the presence of the MYD88 S243N and LYN I297N mutations detected in the cell line and the patient at the time of ibrutinib progression. Conclusions: BCWM.2 represents a novel, BTK-inhibitor resistant, BCL2 inhibitor sensitive WM cell line that demonstrates MYD88 (S243N) and LYN (I297N) somatic activating mutations, and deletions of 6q. BCWM.2 thereby provides an important new disease appropriate cell model for the in vitro and in vivo study of WM, including the development of targeted therapies for this disease.

Aberrant Expression of Spliced WNK2 Is an Early Event in MYD88 Mutated WM That Activates ERK1/2 and Supports Tumor Growth

Background: Waldenström's Macroglobulinemia (WM) patients carry MYD88 (MYD88 MUT) and CXCR4 (CXCR4 MUT) mutations in 95-97% and 30-40% of cases, respectively. The mechanisms of MYD88-driven lymphomagenesis remain to be clarified. Our previous transcriptome analysis highlighted WNK2 as a highly dysregulated gene in MYD88 MUT WM (Guerrera ML et al, Haematologica 2018). WNK2 is a serine/threonine protein kinase that negatively regulates ERK1/2 activation in a kinase-dependent manner; is a known tumor suppressor in certain solid cancers, where it is primarily silenced by promoter methylation. ERK1/2 is a pro-survival signal in WM; its activation is accompanied by the release of inflammatory cytokines and can mediate ibrutinib resistance. We previously demonstrated WNK2 transcriptional and protein upregulation in CXCR4 WT and silencing in CXCR4 MUT WM and identified several mechanisms of WNK2 transcriptional dysregulation including i) the preferential expression of short isoforms lacking the kinase domain; ii) the recurrence of an identical, aberrant, exon skipping event in all gene isoforms; and iii) aberrant methylation and chromatin accessibility in CXCR4 WT vs CXCR4 MUT WM. We therefore hypothesized that spliced WNK2 may contribute to MYD88 MUT WM oncogenesis. Methods: We investigated WNK2 regulation in 253 untreated WM patients by combined transcriptome and PacBio IsoSeq analysis in a subgroup of 12 WM and 3 healthy donors (HD). Lentiviral transduction was used to overexpress and functionally characterize WNK2 isoforms in MYD88 MUT BCWM1 and MWCL1 WM and control lymphoma cell lines. Results: We identified that tumors from patients with MYD88 MUTCXCR4 WT WM preferentially expressed the short Ensembl WNK2-207 transcript, whose annotation is incomplete. Via PacBio analysis, we identified 8 previously undocumented short isoforms of WNK2 mapping to the original WNK2-207 annotation, all of which lack the kinase domain and bear the same mRNA skipping of two exons near the 3' end. RNA-Seq analysis of 253 WM patents confirmed the IsoSeq predictions and showed that the novel PacBio isoform WNK2-PB4 was among the most expressed transcripts in WM. We next sought to functionally characterize WNK2-PB4 overexpression in MYD88 mutated lymphoma cells. We successfully cloned WNK2-PB4 from patient CD19+ BM cells and obtained an artificial construct of WNK2-PB4 that included the 2 skipped exons, WNK2-SKEX, through the Genewiz gene synthesis service. Stable cell lines overexpressing either WNK2-PB4 or WNK2-SKEX or the empty vector were successfully generated by lentiviral transduction. Overexpression of WNK2-PB4 showed increased phosphorylation of ERK1/2 and SRC family members in the BCWM1 and MWCL1 cells; in contrast, no activation of those targets was seen in MYD88 MUT DLBCL TMD8 and MYD88 WT Burkitt Ramos cell lines ( Figure 1). Increased activation of ERK1/2 was also observed in WM cell lines overexpressing WNK2-SKEX. Study of the downstream signaling of ERK1/2 by WB revealed increased phosphorylation of p90RSK in BCWM1 WNK2-PB4 cells. BCWM1 and MWCL1 overexpressing WNK2-PB4 showed greater proliferation compared to the empty vector control by CellTrace Violet, while no differences in apoptosis were noticed. By co-immunoprecipitation, we observed WNK2 complexed with HCK. Upon treatment with the highly potent dual HCK/BTK inhibitor KIN-8194, we observed dose-dependent decreases in phosphorylation levels of HCK, BTK, and ERK and total levels of WNK2 in WNK2-PB4 overexpressing BCWM.1 and MWCL-1 cells versus vector control transduced cells. Transcriptome analysis of WM samples based on a novel subtype and early/late evolutionary staging classification for WM (Hunter et al, ASH 2022) revealed that WNK2 was aberrantly upregulated from an early stage of WM and was either silenced or further upregulated with disease progression in the B-cell like or Plasma cell like WM subgroups, respectively. Conclusions: Taken together, our findings show a high selection pressure to aberrantly regulate WNK2 expression at an early stage of MYD88 MUT WM with different evolution in CXCR4 WT vs CXCR4 MUT WM, thus suggesting a role for aberrantly spliced WNK2 in the oncogenesis of MYD88 MUT WM. Overexpression of the aberrantly spliced WNK2-PB4 isoform in stable WM cell lines provided a proliferation advantage and led to ERK1/2 activation, which may be induced by the WNK2/HCK interaction.

Genome-Wide DNA Methylation Analysis Reveals Epigenetic Influence on Intracellular and Cytokine Signaling Pathways in Waldenström Macroglobulinemia Compared to IgM-MGUS

Introduction: Waldenström macroglobulinemia (WM) is a rare B-cell lymphoma that may be preceded by an asymptomatic IgM-monoclonal gammopathy of undetermined significance (MGUS). The mechanisms underlying the progression from IgM-MGUS to WM remain to be fully elucidated; however, the stepwise accumulation of genomic abnormalities has been suggested as a potential driver. Currently, the role of epigenetic regulation of DNA remains underexplored in this disease spectrum. Aberrant DNA methylation is a hallmark of oncogenesis that plays an important role in hematologic malignancies. The aim of this study was to assess differential DNA methylation in WM compared to IgM-MGUS and identify hyper- and hypomethylated pathways that may alter key cellular functions. Methods: This study included 15 patients with WM and 6 with IgM-MGUS. Bone marrow samples were prospectively collected and CD19+ and/or CD138+ B cells were positively selected. Subsequently, genome-wide DNA methylation analysis was performed. CpG methylation ratios were segmented into 200-bp regions and differentially methylated regions (DMRs) comparing WM to IgM-MGUS were identified. DMRs with a Q-value <0.05 and absolute methylation difference of ≥20% were considered significant and utilized for analysis. DMRs were annotated according to genomic regions of interest (promotors, 3'UTR, 5'UTR, introns, exons, CpG islands, CpG shores). Next, genes with significant promotor hyper- or hypomethylation were assessed using the Ingenuity Pathway Analysis. Pathways with a corrected p-value<0.05 were considered statistically significant and a Z-value was calculated based on the degree of differential methylation. Results: Genome-wide DNA methylation analysis revealed a significant number of DMRs (n= 15691, hyper: 8248; hypo: 7443) in WM compared to IgM-MGUS. Assessing genomic regions, promotors (hyper: 1379; hypo: 2403), introns (hyper: 1548; hypo: 2292), CpG shores (hyper: 587; hypo: 849) and 3'UTR (hyper: 66; hypo: 193) regions were observed to have a significantly greater number of hypomethylated DMRs (all comparisons, p<0.001) in WM compared to IgM-MGUS (Figure A). While exons (hyper: 1686; hypo: 1378), CpG islands (hyper: 2128; hypo: 82), and 5'UTR (hyper: 854; hypo: 246) regions had a significantly greater number of hypermethylated DMRs (all comparisons, p<0.001) (Figure A). Next, promotor methylation-based pathway analysis revealed differential methylation of multiple cellular pathways important in WM pathogenesis (Figure B). These included pathways involved in intracellular signaling and the regulation of cellular proliferation, such as the STAT3 and CXCR4 pathways which were hypomethylated, and the PTEN signaling pathway which was hypermethylated. Of interest, we have previously demonstrated the transcription factor, STAT3, to have an increased expression in WM and to be implicated in IgM secretion (Elsawa, 2011). Next, multiple cytokine signaling pathways were observed to be hypomethylated in WM compared to IgM-MGUS including members of the interleukin (IL) family (IL-1, IL-8, IL-15, IL-23), VEGF, and TGF-β. Of relevance, previous studies have demonstrated the elevation of IL-8, IL-15, and increased expression of angiogenic cytokines, including VEGF, in patients with WM (Lewicki, 2010; Anagnostopoulos, 2007). Furthermore, our group has also shown miRNA-based epigenetic regulation of IL-8 and IL-15 in WM compared to IgM-MGUS (Chohan, 2023). Lastly, several pathways critical in the regulation of the cell structure and the extracellular matrix (ECM) were found to be hypomethylated including the FGF, actin cytoskeleton, PAK, and FAK signaling pathways. Of note, the FGF/FGFR axis has been demonstrated to be dysregulated in WM, and its targeting has been shown to halt WM cell growth (Sacco, 2021). Conclusions: This study demonstrates differential DNA methylation of key genomic regions in WM compared to IgM-MGUS. Promotor methylation-based analysis reveals hypomethylation of several important cellular pathways involved in cytokine signaling, cell structure/ECM, and intracellular signaling. These results underscore the influence of epigenetics in the IgM-gammopathy disease spectrum and suggest the potential role of aberrant DNA methylation underlying the malignant progression from IgM-MGUS to WM.

A High-Throughput Screen Identifies New Drugs for the Treatment of Lymphoma

Waldenström Macroglobulinemia (WM) is an incurable lymphoma. Despite the advances in molecular characterisation of WM and the development of novel drugs such as Bruton tyrosine kinase inhibitors, rates of deep response with complete remission remain very low at ~ 30%. Therefore, newer and more effective treatments are needed for WM. Novel drug development is hampered by high costs and failure rates, and long development times. These limitations and the unmet need for anti-cancer therapies have created a growing interest in repurposing drugs that are already approved for other indications to manage cancer. To identify safe and effective therapeutic options for the treatment of WM, we performed an unbiased high-throughput in vitro drug screening at the Australian National University Centre for Therapeutic Discovery. We used a cell line derived from a patient with WM called MWCL-1 to discover drug candidates with cytotoxicity potential. We treated these cells with 2,393 compounds approved by the Food and Drug Administrator (FDA) using five different concentrations and measured the cell viability using Cell Titre Glo assay which quantifies the amount of ATP released from the viable cells. In addition, we performed live cell imaging using Incucyte to confirm the cytotoxic potential of lead compounds. Furthermore, immunoblotting was used to identify the programmed cell death pathway induced by the lead compounds. Our high-throughput screen of 2,393 compounds from the FDA library identified 33 drug candidates which substantially reduced the viability of MWCL-1 cells as compared to vehicle control (dimethyl sulfoxide). This screen yielded a hit rate of 1.3%, based on the cut-off score of IC 50 2 μM or less. Of these hits, we identified five highly potent compounds with an IC 50 of less than 0.1 μM ( Fig. 1A). We confirmed the cytotoxicity potential of these lead compounds and found that all five compounds reduced the viability of MWCL-1 cells as early as 12 hours after treatment, with the highest reduction in viability observed at 72 hours. Furthermore, these compounds substantially reduced the viability of another WM cell line called BCWM.1 which morphologically and phenotypically resembles lymphoplasmacytic cells of WM. Importantly, we observed that these compounds reduced the viability of bone marrow-derived primary lymphocytes from a patient with WM as compared to vehicle control ( Fig. 1B). The compound SN01005979 was most effective and exerted a similar cytotoxic potential as standard treatment Doxorubicin ( Fig. 1B). To characterize the mechanisms of action by which these lead compounds kill MWCL-1 and BCWM.1 cells, we performed immunoblotting and tested the activation of programmed cell death pathways including caspase-1, gasdermin (GSDM)-D, GSDME (pyroptosis), caspase-8 (extrinsic apoptosis), caspase-9 (intrinsic apoptosis), caspase-3, -6, and -7 (executioners of apoptosis), and phosphorylation of MLKL (necroptosis). We found that these compounds selectively induce caspase-3-dependent cleavage of GSDME. In summary, using a high throughput screen, we identified novel drug candidates with potent cytotoxic activity against WM. Our identification of key molecules targeted by these drug compounds could pave the way for the treatment of tumours expressing caspase-3 and GSDME. Moreover, our results offer new insights into the mechanism of certain FDA-approved drugs and could help in the development of safer and more effective cancer chemotherapy against WM and potentially other cancers. Future studies are required to test the tumour-killing potential of these drugs in vivo using mouse and humanised mouse models of WM.

Changes in Methylation and Chromatin Accessibility Underlie Subtype Classification and Disease Evolution in Waldenström's Macroglobulinemia

Background: We previously identified three subtypes of Waldenstrom's Macroglobulinemia (WM): B-cell like (BCL), Plasma cell like (PCL), and an intermediate group enriched for early/smoldering WM from which the other two subtypes evolved (Hunter et al, ASH 2022). Diffusion pseudo-time (DPT) analysis suggested all WM samples could be placed on a shared evolutionary path independent of subtype, and when samples were separated into Early and Late DPT values, it mirrored disease progression. The role of epigenetic dysregulation that underlies subtype classification and disease evolution remains poorly understood in WM. Methods: We therefore performed chromatin availability analysis using ATACSeq and bisulfite sequencing of WM patients. ATAC-Seq libraries were constructed from 37 CD19+ BM cells from treatment-naive WM patients along with 6 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood (PB) controls and 3 HD CD138+ plasma cell (PC) samples. Enhanced reduced representational bisulfite sequencing (ERRBS) was also performed on 65 MYD88 mutated and 13 MYD88 wild type (WT) WM patients using CD19-selected BM lymphoplasmacytic cells. For 11 WM MYD88 mutated patients, CD138+ cells were also selected. In addition ERRBS was also performed on paired CD19+ and CD138+ selected samples from 5 IgM multiple myeloma (MM) patients, 6 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood cells; 8 HD CD138+ selected PC; as well as MYD88 mutated BCWM.1, BCWM.2, MWCL-1, RPCI-WM1, OCI-Ly3, TMD-8, HBL-1 and SU-DHL2 cells; MYD88 wild-type OCI-Ly7, OCI-Ly19, Ramos B-cells. To infer 5-hydroxymethylcytosine (5HMC) levels, oxidative bisulfite sequencing was also performed for 73 samples including the HD, MM, WT and WM CD19/138 pairs. All ATAC-Seq and 37 of the WM ERRBS samples were part of our multi-omic study that included 253 treatment naïve patients who underwent whole exome and RNASeq sequencing. Results: A UMAP projection of the CpG ERRBS data clustered WM patients into two distinct groups ( Figure 1). One group clustered directly with HD Memory B-cells (MB) and was greatly enriched for CXCR4 mutated WM patients. The other WM group clustered with IgM MM patients and was largely CXCR4 WT. The findings are consistent with the BCL and PCL WM subtypes, which were confirmed by a perfect assignment correlation with our larger multi-omic data set wherein these subtypes were defined. Notably, BCWM.1, BCWM.2 and MWCL-1 WM cell lines clustered with the WM samples. The 5HMC data displayed no clear clustering of the patient samples but clearly separated all HD samples from those of WM and IgM MM samples, consistent with a role for 5HMC dysregulation in oncogenesis. Differential methylation analysis showed differences by MYD88 and CXCR4 mutation status, deletions in Chr6q, familial history for WM, WM subtype, and stratification by early and late DPT values. Notably, the only comparison that did not yield any differences was WM CD19+ to WM CD138+, suggesting that this may not be a meaningful epigenetic distinction in WM. Similar comparisons were performed on the ATAC peaks. Differential chromatin accessibility was noted near many of the corresponding differentially expressed genes. ATAC peak coverage significantly correlated with the expression of top genes. This was particularly true for WM subtype and DPT comparisons ( Table 1). DNA sequences from differentially accessible peaks between BCL and PCL subtypes were also evaluated for relative TF motif enrichment in open vs. closed chromatin revealing PAX5, FUBP1, and ZNF121 enrichment in BCL subtypes using PCL subtype peaks as a background control. TFs REL, SP1, SPIB, BC11A and JUN were enriched PCL subtype compared with BCL subtype. Correlation between DPT associated genes revealed two clear groups, one associated with stem cell and pre-B-cell genes (Group 1) and the other with inflammatory, myeloid, and T-cell genes (Group 2). Motif analysis of differential peaks correlating with genes in each group demonstrated comparative enrichment for MYC, MXI1, and E2F2 in group 1 while RUNX1, NFKB1, and ATF1 were in enriched in group 2. Conclusions: This is the first independent validation of our previously reported multi-omic driven WM subtype classification. The studies underscore that epigenetic differences underlie the biology of WM subclassification and support a strong role for epigenetic changes driving WM evolution.

Abstract 5866: Mavorixafor enhances apoptosis of tumor cells treated with ibrutinib or venetoclax in diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia

Abstract Despite advances in therapies for B-cell lymphoma, patients may still develop resistance and often relapse. Contributing factors may include interactions between lymphoma cells (LCs) or leukemia cells and bone marrow microenvironment (BMM). Overexpression of CXCR4WT and its ligand CXCL12 promote LC-BMM interactions, contributing to disease severity in lymphomas or leukemia such as diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). Mavorixafor is an investigational oral CXCR4 antagonist being evaluated in combination with the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in patients with Waldenström’s macroglobulinemia (WM). In preclinical models of MYD88L265P CXCR4WT WM, mavorixafor restored sensitivity of WM cells to B-cell-targeted therapies in coculture of WM cells and bone marrow stromal cells (BMSCs); however, the effects of mavorixafor on other lymphomas with CXCR4WT have not been evaluated. We report the effects of mavorixafor alone or in combination with B-cell─targeted therapies on LCs in an in vitro preclinical model and assess the role of BMM in the pathogenesis of B-cell lymphomas in an in vitro coculture model of LCs and BMSCs. Bioinformatic analysis of publicly available gene expression datasets GSE11318 and GSE32918 showed patients with DLBCL and CXCR4hi have significantly inferior survival probability compared with those with CXCR4low (P<.001 and P<.05, respectively). In vitro studies assessing effects of targeting CXCR4 with mavorixafor showed apoptosis of CXCR4+ LCs OCILY19 (DLBCL), MEC1 (CLL), MINO (MCL), and DOHH2 (FL) cells increased by ≈10%─60%. The apoptosis of LCs was further enhanced by ≈5%─60% when treated with mavorixafor in combination with ibrutinib or venetoclax compared to ibrutinib or venetoclax alone. Coculture of LCs with BMSCs reduced the sensitivity of LCs to apoptosis-inducing effects of ibrutinib and venetoclax. Combining mavorixafor with ibrutinib or venetoclax restored sensitivity of LCs to apoptosis-inducing effects of the tested B-cell─targeted inhibitors. Mavorixafor also inhibited migration of LCs toward CXCL12, suggesting prevention of the homing of LCs to protective niche. Overall, our findings suggest the contribution of CXCR4WT to pathogenicity of LCs. This is the first in vitro study to show that reduced sensitivity of LCs against B-cell-targeted therapies conferred by BMSCs can be overcome by inhibition of the CXCL12-CXCR4 axis with mavorixafor. Our study provides supporting evidence for further exploration of mavorixafor alone or in combination with other B-cell-targeted therapies in the treatment of lymphomas and leukemias. Further studies using additional LC lines and/or primary patient cells are warranted to support these findings. Citation Format: Tom Kruitwagen, Thalia Martins Rebelo, Barbara Maierhofer, Gerwin Heller, Halenya Monticelli, Arthur G. Taveras, Chi Nguyen. Mavorixafor enhances apoptosis of tumor cells treated with ibrutinib or venetoclax in diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia [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 5866.

Targeting the immune microenvironment in Waldenström macroglobulinemia via halting the CD40/CD40-ligand axis

AbstractRecent investigations have improved our understanding of the molecular aberrations supporting Waldenström macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. First, we showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T cells (Tregs) relative to control mice. These findings were translated into the WM clinical setting, in which the transcriptomic profiling of Tregs derived from patients with WM unveiled a peculiar WM-devoted messenger RNA signature, with significant enrichment for genes related to nuclear factor κB–mediated tumor necrosis factor α signaling, MAPK, and PI3K/AKT, which was paralleled by a different Treg functional phenotype. We demonstrated significantly higher Treg induction, expansion, and proliferation triggered by WM cells, compared with their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-cell–to–T-cell cross talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis that supports WM cell–Tregs interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

Dysfunctions of innate and adaptive immune tumor microenvironment in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a rare subtype of non-Hodgkin lymphoma characterized by malignant lymphoplasmacytic cells in the bone marrow (BM). To dissect the pathophysiology of WM, we evaluated clonal cells by mapping of B cell lymphomagenesis with adaptive and innate immune tumor microenvironment (TME) in the BM of WM patients using mass cytometry (CyTOF). In-depth immunophenotypic profiling of WM cells exhibited profound expansion of clonal cells in both unswitched and switched memory B cells and also plasma cells with aberrant expression variations. WM B lymphomagenesis was associated with reduction of most B cell precursors assessed with the same clonally restricted light chain and phenotypic changes. The immune TME was infiltrated by mature monocytes, neutrophils and adaptive T cells, preferentially subsets of effector T helper, effector CTL and effector memory CTL cells that were associated with superior overall survival (OS), in contrast to progenitors of T cells and myeloid/monocytic lineage subsets that were suppressed in WM cohort. Moreover, decrease in immature B and NKT cells was related to worse OS in WM patients. Innate and adaptive immune subsets of WM TME were modulated by immune checkpoints, including PD-1/PD-L1&PD-L2, TIGIT/PVR, CD137/CD137-L, CTLA-4, BTLA and KIR expression. The response of ibrutinib treatment to the reduction of clonal memory B cell was associated with high levels of immature B cells and effector memory CTL cells. Our study demonstrates that CyTOF technology is a powerful approach for characterizing the pathophysiology of WM at various stages, predicting patient risk and monitoring the effectiveness of treatment strategies.

Gli3 Is Required for M2 Macrophage Polarization and M2-Mediated Waldenström Macroglobulinemia Growth and Survival

The macrophage polarization paradigm has become a forefront in cancer research in recent years, but the effects of this phenomenon have yet to be investigated in Waldenström Macroglobulinemia (WM). WM is an indolent, B-cell lymphoma, characterized by increased IgM production and infiltration of the bone marrow niche by malignant cells. While macrophage polarization is typically investigated as a singular point in time, it is important to understand that M2-type macrophages can switch to an M1 phenotype, or vice versa, based on environmental changes. M1 macrophages are typically pro-inflammatory and anti-tumor (although some cancers have reported an increased presence of M1 macrophages leading to worse clinical outcomes), and M2 macrophages are typically anti-inflammatory and pro-tumorigenic. Understanding which phenotype is prevalent in WM, as well as the mechanism behind increased tumorigenesis is important for guiding WM research and treatment. To assess the effects of macrophages on WM cell proliferation, we examined the effects of macrophage polarization using THP-1 cells, CD14+-derived macrophages from PBMCs and bone marrow-derived macrophages from C57BL/6 mice on WM cell growth and viability. WM cells grown in direct co-culture with macrophages exhibited increased proliferation compared to WM cells grown alone. WM cell viability was also enhanced when cells were directly co-cultured with macrophages. To investigate whether M1 or M2 macrophages were responsible for this increased proliferation and viability, we performed a qPCR analysis of macrophages in indirect and direct co-culture with WM cells. We found that WM cells induce a M2 phenotype upon direct co-culture, but this effect was not seen in indirect co-cultures. In previous work, we found that the transcription factor GLI3 plays a role in regulating cytokine expression and secretion in response to LPS stimulation. We performed RNA-seq on macrophages derived from mice lacking Gli3 in myeloid cells (M-Gli3-/-). Using a generalized linear model in edgeR, we identified 495 genes with significant interaction effects between genotype and LPS treatment. Ingenuity Pathway Analysis of the interaction genes revealed "Inflammatory Response” and "Immune Cell Trafficking” pathways as most significantly enriched. The 25 significant interaction genes on these pathways included 9 with a positive interaction and 16 with a negative interaction. Analysis also suggested Gli3 may play a role in M2 macrophage polarization. Bone marrow-derived macrophages were isolated from M-Gli3-/- and WT mice. We found that M-Gli3-/- macrophages were unable to polarize into the M2 phenotype and both proliferation and viability of WM cells cocultured with these macrophages did not increase. Our findings identify a novel role for Gli3 in regulating M2 polarization and subsequently a role for M2 macrophages in promoting WM growth and survival. This suggests therapeutic targeting of Gli3 in the tumor microenvironment may be beneficial in the treatment of WM.

Increased Tumor-Associated CD66b+ Myeloid-Derived Suppressor Cells in Waldenstrom Macroglobulinemia Inhibit T-Cell Immune Function

Waldenstrom macroglobulinemia (WM) is a discrete clinicopathologic entity resulting from the accumulation of lymphoplasmacytic lymphoma (LPL) cells in the bone marrow (BM) that secrete a monoclonal immunoglobulin (Ig)M protein. MYD88 (>90%) and CXCR4 (30-35%) mutations are the most commonly identified mutations in this disease. While much is known about the genomics of WM, far less is known about the role of the BM microenvironment in this disease. Emerging evidence has attested to the role of myeloid-derived suppressor cells (MDSCs) and their potent immunosuppressive activity, however, there is limited information about these cells in WM and monoclonal gammopathy of undetermined significance (MGUS). This study was therefore designed to explore the phenotype of MDSCs and their interaction with other cells in the BM in WM. A multiparametric approach was combined with single-cell genomic and proteomic tools to better understand MDSC immunology and the antitumor immune response in WM. In the present study, using flow cytometry, we first analyzed the number of MDSCs (by gating on Lin- HLA-DR- CD11b+ CD33+) in normal BM (NBM; n=11) and WM symptomatic /MGUS (n=18) and found an increased number of MDSCs in WM specimens compared to controls (p=0.005). We also found that most MDSCs in WM specimens had a granulocytic phenotype (Lin- HLA-DR- CD11b+ CD33+ CD15+). A detailed phenotypic study using CyTOF (mass spectrometry) was performed on MDSCs from NBM (n=4) and WM (n=8). The CyTOF analysis identified 3 subtypes of MDSCs including CD66b+ MDSCs with a granulocytic phenotype (G-MDSCs) and confirmed the expansion of CD66b+ MDSCs in WM symptomatic patients when compared to smoldering WM and normal controls. Our previous studies have shown that granulocyte colony-stimulating factor (G-CSF) expression is increased in the bone marrow of WM (Jalali et al., 2018). Therefore, to determine whether G-CSF accounted for the expansion of CD66b+ G-MDSCs, we treated WM-BM MDSCs with G-CSF and found that the CD66b+ G-MDSC population substantially increased (p=0.0017). Further, to establish a better understanding of the transcriptome profile and a unique phenotype of CD66b+ G-MDSCs, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) on WM (n=3) and NBM (n=2) specimens. A significantly high expression of NLRP12, IGFBP7, CXCR4, IL2, CD55, SOX4, and FoxP1 genes was observed in CD66b+ MDSCs as compared to other MDSC populations. This gene expression signature suggested upregulation of inflammatory pathways potentially affecting immune function. Therefore, to evaluate the effect of CD66b+ G-MDSCs on the immune function of other cells in the BM, CD66b+ and CD66b- MDSCs were co-cultured with activated autologous T-cells. We found that while all populations of MDSCs suppressed T-cell activation, CD66b+ G-MDSCs had the greatest suppressive effect. In conclusion, this study has shown that CD66b+ G-MDSCs are expanded in WM patients, exhibit a unique inflammatory transcriptome, and substantially inhibit T-cell activation and proliferation. Furthermore, CD66b+ G-MDSCs are expanded due to increased G-CSF in the BM microenvironment in WM. Clinical strategies to inhibit the expansion of CD66b+ G-MDSCs may therefore enhance the efficacy of immunological therapies in patients with WM.

Multi-Omic Analysis of 253 Untreated Patients with Waldenström's Macroglobulinemia Reveals Clinically and Genomically Distinct Disease Subtypes and a Model for Disease Progression

Background: Waldenström's Macroglobulinemia (WM) is characterized by recurring mutations in MYD88 (95-97%) and CXCR4 (40%). While these genomic insights have heralded new insights into disease pathogenesis and treatment, a multi-omic approach in a large population of WM patients may further clarify underlying disease biology. Methods: Stranded 150bp paired end RNA sequencing was performed on CD19+ bone marrow (BM) biopsies from 253 treatment-naive WM patients along with paired CD19+CD27- and CD19+CD27+ peripheral blood (PB) cells from 13 healthy donors. For 216 patients, whole exome sequencing of CD19+ BM WM cells with CD19- PB controls was also performed. All p-values were subjected to correction using the false discovery rate. Results: Baseline characteristics were typical of treatment-naive patients. Their median age was 66 years; BM disease involvement 50% ; and serum IgM 3,108 mg/dL. The median follow-up was 7.5 years. MYD88 mutations were identified in 250 (98.8%) patients. Exploratory analysis of MYD88 mutated patients revealed three distinct groups of patients that were observed in a hierarchical clustering analysis shown in Fig 1A. Gene set enrichment analysis revealed distinct B-cell Like (BCL) and Plasma Cell Like (PCL) patient groupings and a third non-BCL/PCL patient group that clustered independently. Non-BCL/PCL patients were more likely to be asymptomatic (36/84; 43%) versus those in the BCL (62/96; 64.6%) and PCL (48/68; 70.6%) groups (p<0.005). Among BCL and PCL grouped patients, important differences in disease presentation were noted including BM involvement (50% vs. 70%; p<0.001), adenopathy (18.6% vs. 48.4%; p<0.002), CD10 (1.1% vs. 13.4%; p=0.012), CD23 (39.4% vs. 22.4%; p=0.09) and CD5 (19.1% vs. 6%; p=0.09) expression, respectively. CXCR4 mutations were identified in 76/97 (78.4%) of BCL versus 6/69 (8.7%) of PCL patients (p<0.001). Amplifications in chr18q amplifications were also more prevalent in BCL (14/83; 16.9%) versus PCL (1/64; 1.6%) grouped patients (p=0.035). chr6q deletions were observed in 24/83 (29%) of BCL versus 30/64 (47%) PCL patients (p=0.11). BCL patients also exhibited fewer chr6p amplifications, i.e. 3/83 (3.6%) versus 12/64 (18.8%) of PCL patients (p=0.036). Differences in deletions in chr17p (0/83; 0%) vs (6/64; 9.4% (p=0.036); as well as NOTCH1 (0/82; 0%) vs. (6/64; 9.4%) (p=0.084) and EP300 3/82; 3.7%) vs. (11/64; 17.2%) (p=0.084) mutations were also identified in BCL vs PCL patients, respectively. NOTCH pathway mutations were also found in fewer BCL (17/82; 20.7%) versus PCL (30/64; 46.9%) patients (p=0.01). Diffusion mapping and pseudo-time analysis were performed on the top 1,000 high variance genes which bifurcated patients into two distinct populations over time as shown in Fig. 1B, with the point size scaled for BM involvement. By diffusion pseudo-time (DPT), patients were stratified into early- and late-stage WM, the former showing lower median BM involvement (30% vs. 70%; p<0.001) and serum IgM (2,666 vs. 3713; p=0.133); higher hemoglobin (12.1g/dL vs. 10.65g/dL; p<0.001), serum IgA (56.5mg/dL vs. 41mg/dL; p=0.018) and IgG (629mg/dL vs. 498mg/dL; p=0.015), and less splenomegaly (3.7% vs. 17.2%; p=0.006). A longer time to first treatment (5.3 vs 0.9 months; (p<0.001) was also observed in those with early- versus late- stage WM, respectively. DPT showed that the non-BCL/PCL patients were more likely to be early stage (68/84; 81%) versus BCL (51/97; 53%) and PCL (30/69; 43%) grouped patients (p<0.001). In addition, hierarchical clustering delineated the non-BCL/PCL group into two subgroups with CXCR4 mutations rates of 30/57 (52.6%) and 2/27 (7.4%), and gene expression similarities that aligned with either the BCL or PCL subtypes, respectively. Taken together, these findings suggest that the non-BCL/PCL group represents an earlier evolving stage of WM that may progress into BCL and PCL like WM disease. Conclusions: In this largest multi-omic investigation undertaken into WM, we identified three distinct WM subtypes with distinct clinical and genomic features. Central these findings were the identification of BCL, PCL, and an earlier non-BCL/PCL evolving stage recognized through DPT modeling. The discovery of the DPT model for WM disease progression provides insight into the biology and targeting of WM progression. Our findings provide a critical framework for multi-omic based study and targeted treatment approach to WM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pembrowm: Results of a Multi-Centre Phase II Trial Investigating the Safety and Efficacy of Rituximab and Pembrolizumab in Relapsed/Refractory Waldenström's Macroglobulinaemia

Background Waldenström's macroglobulinemia (WM) is a rare low grade B-cell NHL, with generally excellent outcomes underpinned by treatment with Rituximab combination therapies and covalent BTK inhibitors (cBTKi). However, these options are not curative and there are very little data to guide treatment options or a clear understanding of prognosis of relapsed/refractory (R/R) WM post cBTKi. PD-1 inhibition has been shown to be highly effective in classical Hodgkin lymphoma, and soluble PD-1 ligands have been implicated in T-cell regulatory function in the WM microenvironment. There is therefore a clear rationale to investigate the efficacy of PD-1 blockade in WM. Aim To determine the safety, tolerability and efficacy of pembrolizumab in combination with rituximab for the treatment of relapsed/refractory WM following prior cBTKi exposure. Methods PembroWM is a phase II, non-randomised, single arm, open label NCRI UK-wide study (NCT03630042) enrolling patients (pts) with R/R WM who had received 1 or more prior lines of treatment, who were not refractory to rituximab based therapy (progressing on or relapsing within 6 months of a rituximab containing regimen). Eligible pts accross 6 sites received rituximab 375mg/m2 weekly for 4 weeks, then 3 monthly (total of 8 doses) and pembrolizumab 200mg IV on a 3-weekly cycle, for a maximum of 18 cycles. The primary end point was the percentage of pts achieving an overall response rate (ORR) of greater than 25% reduction in serum IgM at 24 weeks post treatment. Secondary end points included safety and tolerability, response rates (CR/VGPR) , progression free survival (PFS) and overall survival (OS) at 1 year. Disease response was assessed with serum paraprotein/immunofixation, BM and CT imaging at 24 weeks and 1 year using the International Working Group for WM response criteria. The trial required recruitment of 42 patients with a 55% response rate to meet the primary end point. The trial closed early due to low recruitment rate with a total of 17 patients with a revised response requirement of 9 patients to meet primary end point. Results 17 patients were registered with median age of 70 years (range 43-84), 83% were male, 94% had a PS of 0-1, and the median number of lines of previous treatment was 3 (range 1-6). The majority of patients had a cBTKi as their last treatment prior to enrollment (only 2 patients were BTK naïve). 8 patients were positive for the MYD88 L265P mutation, and 4 patients had mutations in BTKC481. Median IgM levels were 40 g/L (range 2.5-72g/L) with 53% of patients exhibiting WM cells in bone marrow and extra nodal disease in 6%. Median follow up is 15.1 months (0.5 - 24.9). ORR at 24 weeks post treatment was 47.1% (60% CI 34.5-60%) with 6% attaining VGPR, 18% PR, 18% MR and in 6% response was unavailable but there was evidence of MR at week 12. 47% were non-responders; 35% progressed, 12% had stable disease and one patient withdrew during cycle 1 so was non-evaluable. ORR at 52 weeks post treatment was 36.4% (95% CI 10.9-69.2%) with 9% obtaining a VGPR, 27% PR and nil achieving CR. Median PFS was 12.6 months (95% CI 3-n/a) and median OS was not reached. 5 patients completed 18 cycles of treatment. 6 progressed, 3 withdrew due to AE, 2 withdrew consent and one withdrew to an unrelated clinician decision. The AE leading to withdrawal were immune thrombocytopenia (one), and infection in the other two (including one COVID-19 infection) The majority of adverse events were grade 1-2 with the commonest events involving anaemia (29%), fever (29%), infusion related reactions (35%) and raised creatinine (29%)., There were only 2 grade 3 infections. 1 patient experienced an embolic stroke and 1 patient died from respiratory failure; both events were considered unrelated to treatment. Conclusions PEMBROWM is the first study to investigate checkpoint inhibition in WM, and demonstrates the safety and tolerability of the combination of pembrolizumab and rituximab, with encouraging efficacy in a heavily pre-treated population. The recruitment challenges highlighted the combination of reduction in research activity during the COVID-19 pandemic, the difficulties of recruiting to an investigator-led study in a rare indication and the timing of availability/effectiveness of cBTKi in WM in the United Kingdom. A randomised study would be required to evaluate the additional benefit that PD-1 blockade is adding to rituximab monotherapy in a highly relapsed/refractory patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bing-Neel Syndrome: Real-Life Experience in Personalized Diagnostic Approach and Treatment.

The involvement of the central nervous system (CNS) in Waldenström’s Macroglobulinemia (WM) is a rare extramedullary manifestation of the disease known as Bing-Neel syndrome (BNS). To expand our understanding of this disease manifestation, we conducted a retrospective analysis of the incidence of BNS in 86 consecutive patients with WM [70% male, median age 65 years (range 33-86)] seen in our center during a 30-year period. Six patients (7%) from this group were diagnosed with BNS. The median period of time between WM diagnosis and BNS diagnosis was 6.8 years (range 2.3-15). They demonstrated a range of neurological deficits, including transient expressive aphasia, impaired vision, resting hand tremor, foot drop, and headache. Between the onset of symptoms and the diagnosis of BNS, the median time interval was 12.5 months (range 1-30). The diagnosis was made not on the basis of neurological symptoms or radiological evidence, but on the basis of the presence of WM cells in cerebrospinal fluid (CSF). Intrathecal chemotherapy with methotrexate, cytarabine, and dexamethasone (IT MTX, ARA-C, DEX) was used as front-line treatment, followed by intensive immunochemotherapy with rituximab, high-dose MTX, and ARA-C (R-Hi MTX/ARA-C) in three patients who were fit enough to receive this type of cytotoxic regimen, and rituximab plus bendamustine (R-Benda) in two patients who simultaneously required treatment for WM. Ibrutinib was administered to five patients (three as consolidation and two for initial treatment). All patients responded to front-line treatment, with four (67%) achieving partial response (PR) and two (33%) achieving complete response (CR). This study provides insight into the clinical presentation, diagnostic and treatment options, as well as the outcome of patients who have BNS.

P1253: MAVORIXAFOR DISRUPTS THE CROSS TALK BETWEEN WALDENSTRÖM’S MACROGLOBULINEMIA CELLS AND THE BONE MARROW MICROENVIRONMENT RESULTING IN ENHANCED SENSITIVITY TO B-CELL TARGETED THERAPIES

Background: Waldenström’s macroglobulinemia (WM) is a rare B-cell malignancy characterized by infiltration of bone marrow (BM) and lymphoid tissues with lymphoplasmacytic cells along with hypersecretion of monoclonal immunoglobulin M (IgM) by progenitor B cells. Studies have shown that more than 90% of patients with WM have an L265P mutation in the myeloid differentiation primary response gene 88 (MYD88), and 30% to 40% of those patients show mutations in the carboxyl terminus of C-X-C receptor 4 (CXCR4). The CXCR4/C-X-C motif ligand 12 (CXCL12) axis plays an important role in the homing and retention of WM cells in the BM. The interaction of WM cells and the BM microenvironment, via cell-to-cell contacts or indirectly through secreted factors (e.g. interleukin 6 [IL-6], C-C motif ligand 5 [CCL5]), is thought to be essential for the pathogenesis of WM and to confer drug resistance. A clinical trial evaluating the efficacy of the CXCR4 antagonist, mavorixafor, in combination with the BTK inhibitor, ibrutinib in patients with MYD88L265P and CXCR4WHIM WM is currently ongoing (NCT04274738). However, the effects of mavorixafor with ibrutinib and other B-cell targeted therapies on WM cells harboring only the single mutation in MYD88L265P and without a CXCR4 mutation have not been evaluated. Aims: This study was designed to elucidate the crosstalk between WM cells and the BM stromal cell (BMSC)-based microenvironment and to explore the effect of mavorixafor in combination with different B-cell targeted therapies in a WM/BMSC co-culture model. Methods: WM cells (MWCL-1 cell line, MYD88L265PCXCR4WT) pretreated with mavorixafor and B-cell targeted therapies (BTK antagonists: ibrutinib, zanubrutinib, evobrutinib, pirtobrutinib, nemtabrutinib; or BCL2 inhibitor venetoclax) were co-cultured with established BMSCs (HS27a cells). Apoptosis, IL-6 production, and IgM release were measured after 48 or 72 hours. The effects of mavorixafor on calcium mobilization, cell migration, and adhesion to BMSCs were also measured. Results: Co-culture of WM cells with established BMSCs led to increased IgM secretion and enhanced surface expression of CXCR4 in WM cells by activation of the IL-6 receptor (IL-6R)/ Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Co-culture with established BMSCs protected WM cells from the effects of B-cell targeted therapies, reducing apoptosis of tumor cells and decreasing the IgM-reducing efficacy of the targeted therapies. Mavorixafor alone reduced IgM secretion, blocked CXCL12-induced cell migration and Ca2+ mobilization and disrupted the adhesion of WM cells to established BMSCs. Mavorixafor in combination with B-cell targeted therapies synergistically enhanced antitumor activity in WM cells, inhibited BMSC-induced IgM hypersecretion, and overcame the protective effects of BMSCs on tumor cells. Summary/Conclusion: Our studies provide preliminary evidence for the potential use of mavorixafor in disrupting the interaction of WM cells with the BMSC-based microenvironment, enhancing the efficacy of B-cell targeted therapies in the treatment of WM and potentially other lymphomas. Further studies using additional WM cell lines and/or primary patient cells are warranted to support these findings.

MLL1 inhibition reduces IgM levels in Waldenström macroglobulinemia

Waldenström macroglobulinemia (WM) is a B cell lymphoma characterized by the overproduction of a monoclonal IgM antibody, a leading pathogenic feature of the disease. Current therapies are based on our knowledge at the signaling and genetic scale, but recent research has identified epigenetic dysregulation as one of the important dynamics in the biology of this disease. In this study, we found that Mixed-lineage leukemia 1 (MLL1) histone methyltransferase and its chromatin tethering partner Menin are upregulated in WM patients. KMT2A knockdown using short hairpin RNA (shRNA) and inhibition of MLL1 function using the menin-MLL1 inhibitor (MI-2) in WM cells resulted in a significant reduction in IgM levels without significantly impacting WM cell growth and survival. Further analysis identified MLL1 binding at multiple sites in the 5′ Eμ enhancer of the immunoglobulin heavy (IGH) chain. We found increased histone 3 lysine 4 trimethylation (H3K4me3) enrichment at multiple MLL1 binding sites upon LPS stimulation, a known inducer of IgM. Finally, we found that disruption of Menin-MLL1 complex using the MI-2 inhibitor in tumor-bearing mice significantly reduced human IgM levels in mice sera. Taken together, these results identify MLL1 as a regulator of IgM and define MLL1 as a new therapeutic target for WM.

A new role for the SRC family kinase HCK as a driver of SYK activation in MYD88 mutated lymphomas

AbstractThe SRC family kinase (SFK) HCK is transcriptionally upregulated and activated by mutated MYD88 (MYD88Mut), a key adaptor for Toll-receptor signaling. HCK activates BTK, AKT, and ERK in MYD88Mut lymphomas. SYK, a B-cell receptor (BCR) component, is activated in MYD88Mut lymphoma cells. Although the SFK LYN serves as a trigger for SYK activation in MYD88Mut ABC DLBCL cells, LYN activity is muted in MYD88Mut Waldenstrom macroglobulinemia (WM) cells. We therefore investigated a role for HCK in mediating SYK activation. Overexpression of wild-type (WT) (HCKWT) or gatekeeper mutated (HCKThr333Met) HCK in MYD88Mut lymphoma cells triggered SYK activation. Conversely, HCK knockdown reduced p-SYK in MYD88Mut lymphoma cells. Coimmunoprecipitation experiments showed that HCK was complexed with p-SYK in MYD88Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. Rescue experiments in MYD88Mut lymphoma cells expressing HCKThr333Met led to persistent HCK and SYK activation and resistance to the HCK inhibitor A419259. Treatment of primary MYD88Mut WM cells with A419259 reduced p-HCK and p-SYK expression. Taken together, our findings show that SYK is activated by HCK in MYD88Mut B-cell lymphomas cells, broaden the prosurvival signaling generated by aberrant HCK expression in response to MYD88Mut, and help define HCK as an important therapeutic target in MYD88Mut B-cell lymphomas.

Waldenstrom Macroglobulinaemia with Signet Ring Cells: A Case Report

Waldenstrom Macroglobulinemia (WM) is an indolent lymphoproliferative disorder characterised by Immunoglobulin M (IgM) production and neoplastic lymphoid infiltrate. The morphology of the neoplastic cells show spectrum of maturation characterised by small lymphocytes, lymphoplasmacytoid cells and plasma cells. The infiltrate is predominantly lymphoid with relatively small population of plasma cells. The plasma cell morphology is commonly of mature type with basophilic cytoplasm, eccentric nucleus with clumped chromatin and perinuclear halo (hof). But incidence of signet ring cells in WM is very rare and only one case has been reported till date. Here, authors report an interesting case of a 61-year-old man who presented with on and off chest pain and later diagnosed as WM with signet ring cells. On examination, the patient was found to have anaemia and lymphadenopathy. Bone marrow aspirate cytology showed hypercellular particles and scattered signet ring like plasma cells with large globular cytoplasmic inclusions. Flow Cytometry (FCM) established the diagnosis by demonstrating the clonal nature of plasma cells and B-lymphoid cells. Immunofixation confirmed the presence of monoclonal protein of IgM lambda type. The cytoplasmic content in these signet ring plasma cells is probably the accumulated IgM due to the defect in releasing. Clustering of these signet ring cells in marrow may raise a suspicion of metastatic adenocarcinoma and should not be misinterpreted.

A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas

A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas