Waldenstrom Macroglobulinemia Cell Research Articles

Glutathione Metabolism Contributes to the Pathobiology of Waldenstrom's Macroglobulinemia

Cancer cells have altered energy demand due to their increased proliferative capacity. In Waldenstrom Macroglobulinemia (WM), an indolent yet incurable B-cell lymphoma, the homeostasis of the bone marrow (BM) environment is disturbed due to the infiltration of the lymphoplamacytic cells that continuously produce monoclonal IgM. An alteration in energy demand could skew the balance of key proteins and metabolites towards a permissive niche for WM tumor cells growth, and unfavorably effect on the immune response against tumor cells. Therefore, the aim of our study was to identify how changes in certain metabolites and/or proteins could contribute to the pathobiology of WM and whether the cytokine and chemokine composition of the BM microenvironment play a role in such changes. WM patient's samples including BM plasma, peripheral blood serum and BM cells (n=101) as well as equivalent normal counterparts (n=86) were collected and used for metabolomics analysis. Comprehensive targeted metabolomics analysis was performed using Capillary Electrophoresis Time-of-Flight Mass spectrometry (CE-TOFMS), CE-triple quadrupole mass spectrometry (CE-qQqMS) and Liquid Chromatography (LC-TOFMS). Normal and WM peripheral blood serums samples were also used for untargeted proteomics analysis using a fully automated proteomic technology platform that includes an Agilent 1200 Series Quaternary HPLC system connected to a Q Exactive Plus mass spectrometer. Real-time PCR analysis was performed to detect the gene expression of the relevant metabolite transporters located on the cell membrane. BM cells from control and WM patients' samples were used for flow cytometry analysis. IHC was used to detect the proteins on the BM tissues. Principal Component Analysis (PCA) and Hierarchal Clustering Analysis (HCA) on both metabolomics and proteomics data identified two distinct clusters for disease and normal samples, indicating that there are differentially expressed proteins and metabolites in WM versus normal samples. Furthermore, pathway analysis showed that the majority of the altered metabolites were the members of the glutathione (GSH) metabolism pathway. This finding was further validated not only by data obtained from metabolomics analysis of BM cells and BM plasma, but also by proteomic data WM patients serum, implying that GSH metabolism is key to the biology of WM. Moreover, stimulation of WM cell lines by IL-6 and IL-21, cytokines involved in inducing WM cell proliferation and IgM secretion, resulted in increased gene expression of the transporters mediating uptake of metabolites required for GSH synthesis, including SLC7A11, 4F2HC and LAT1, indicating that cytokines in the WM BM could modulate GSH metabolism. In addition, IHC staining of the BM tissues as well as flow cytometry analysis of patients' lymphoplasmacytic cells identified glutathione peroxidase as one of the major proteins modulating GSH metabolism in WM. In summary, our data highlight a central role for GSH metabolism in WM disease biology and indicate that intervening with the metabolic processes could be a potential therapeutic strategy for patients with WM. Disclosures Ansell: AI Therapeutics: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Bristol Myers Squibb: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials.

CXCR4-targeted PET imaging using 64Cu-AMD3100 for detection of Waldenström Macroglobulinemia

ABSTRACTObjective: Waldenström Macroglobulinemia (WM) is a rare B-cell malignancy characterized by secretion of immunoglobulin M and cancer infiltration in the bone marrow. Chemokine receptor such as CXCR4 and hypoxic condition in the bone marrow play crucial roles in cancer cell trafficking, homing, adhesion, proliferation, survival, and drug resistance. Herein, we aimed to use CXCR4 as a potential biomarker to detect hypoxic-metastatic WM cells in the bone marrow and in the circulation by using CXCR4-detecting radiopharmaceutical.Methods: We radiolabeled a CXCR4-inhibitor (AMD3100) with 64Cu and tested its binding to WM cells with different levels of CXCR4 expression using gamma counter in vitro. The accumulation of this radiopharmaceutical tracer was tested in vivo in subcutaneous and intratibial models using PET/CT scan. In addition, PBMCs spiked with different amounts of WM cells ex vivo were detected using gamma counting.Results: In vitro, 64Cu-AMD3100 binding to WM cell lines demonstrated a direct correlation with the level of CXCR4 expression, which was increased in cells cultured in hypoxia with elevated levels of CXCR4, and decreased in cells with CXCR4 and HIF-1α knockout. Moreover, 64Cu-AMD3100 detected localized and circulating CXCR4high WM cells with high metastatic potential.Conclusions: In conclusion, we developed a molecularly targeted system, 64Cu-AMD3100, which binds to CXCR4 and specifically detects WM cells with hypoxic phenotype and metastatic potential in the subcutaneous and intratibial models. These preliminary findings using CXCR4-detecting PET radiopharmaceutical tracer indicate a potential technology to predict high-risk patients for the progression to WM due to metastatic potential.

Multicenter prospective phase II study of venetoclax in patients with previously treated Waldenstrom macroglobulinemia

The role of the oral BCL2 antagonist venetoclax in Waldenstrom macroglobulinemia (WM) is unknown. Immunophenotypic and genomic sequencing studies have shown that BCL2 is highly expressed and activated in WM cells. We initiated a phase II study to evaluate the safety and efficacy of venetoclax monotherapy in previously treated patients with WM (NCT02677324).

Cell-Free DNA as Alternative to Bone Marrow CD19+ Selection for Diagnostic MYD88 L265P in Waldenstrom’s Macroglobulinemia

Mutations in MYD88 are found in over 95% Waldenstrom's Macroglobulinemia (WM) patients, nearly all of which correspond to the c.978T>C mutation resulting in an p.Leu265Pro (L265P) substitution at the protein level. Increasingly, allele specific PCR (AS-PCR) to detect the L265P mutation is being used in clinical pathology for diagnostic and prognostic purposes. Unless B-cell enrichment by CD19+ selection is used, the sensitivity of these tests can suffer. Given the heavy involvement of WM cells in the bone marrow (BM) of most patients, BM aspirates provide a greater enrichment of WM cells compared with peripheral blood (PB), regardless of CD19+ selection.

Targeting IL-6 receptor reduces IgM levels and tumor growth in Waldenström macroglobulinemia

ABSTRACTThe tumor microenvironment (TME) plays an important role in cancer cell biology and is implicated in resistance to therapy. In Waldenström macroglobulinemia (WM), a subtype of Non-Hodgkin lymphoma, the TME modulates WM biology by secreting cytokines that promote the malignant phenotype. In previous work, we have shown that TME-IL-6 promotes WM cell growth and IgM secretion in WM. Tocilizumab/Actemra is an anti-IL-6R antibody, which can competitively block IL-6 binding to the IL-6R. We investigated the efficacy of Tocilizumab in a preclinical mouse model of WM that considers the role of the TME in disease biology. Hairless SCID mice were subcutaneously implanted with BCWM.1 or RPCI-WM1 and bone marrow stromal cells. Groups of mice were treated with Tocilizumab or control antibody three times/week for 5 weeks and the effect on tumor burden and disease biology were evaluated. Although Tocilizumab had no effect on mice survival, there was a significant reduction in tumor growth rate in mice injected with RPCI-WM1 cells treated with Tocilizumab. In mice injected with BCWM.1 cells, there was a significant reduction in human IgM secretion in mice sera with Tocilizumab treatment. There was no significant change in mice weight suggesting Tocilizumab induced no toxicities to the mice. Taken together, our data found that administration of Tocilizumab to tumor bearing mice, results in a significant reduction in tumor volume and IgM secretion. Therefore, the evaluation of the role of Tocilizumab in WM patients may provide therapeutic efficacy by reducing IgM production and slowing the rate of tumor growth.

SYK inhibition with fostamatinib in Waldenström's macroglobulinemia: In vitro cell based assays.

e19047 Background: Waldenström's macroglobulinemia (WM) is characterized by accumulation of IgM secreting neoplastic B cells in the bone marrow (BM). B-cell antigen receptor (BCR) signaling plays a pivotal role in the regulation of integrin-mediated retention of lymphoma cells in the BM. Signaling through the BCR leads to activation of spleen tyrosine kinase (SYK) and downstream kinases which play a role in lymphoma pathobiology. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib targets BCR-controlled signaling and integrin-mediated adhesion in WM cells, but patients with WM may become refractory to ibrutinib treatment. As SYK signaling is upstream of BTK, we explored the effect of the SYK inhibitor R406 (the active metabolite of fostamatinib) on BCR mediated signaling and cell adhesion in WM. Methods: MWCL-1 cells were treated with R406, CAL101 (PI3 kinase inhibitor) or ibrutinib at concentrations up to 10µM and stimulated with anti-IgM antibodies or PMA. The cells were transferred to plates coated with fibronectin to assess cell adhesion. For the proliferation assays, MWCL-1 cells or primary B cells were pretreated with kinase inhibitors, followed by stimulation of primary B cells with anti-IgM. Results: Anti-IgM or PMA alone stimulated the adhesion of MWCL-1 on fibronectin. The addition of R406 had a comparable effect to ibrutinib on inhibition of IgM mediated cell adhesion (IC50 = 0.32µM), consistent with the effect of both compounds on BCR mediated signaling. Neither compound affected PMA induced cell adhesion, indicating that the effect on IgM mediated cell adhesion was not due to toxicity. Lack of off-target toxicity was further confirmed in the MWCL-1 proliferation assays where the compounds showed less potency than in BCR-mediated assays. Comparable inhibition of adhesion and proliferation was seen with CAL101. Conclusions: Preliminary preclinical studies with R406 show that SYK is involved in BCR mediated signaling and cell adhesion in WM. CAL101 mediated inhibition of adhesion further confirmed the role of SYK, which is upstream of PI3K. R406 was as effective as ibrutinib in blocking WM cell adhesion. Further studies of SYK inhibition with fostamatinib as a potential treatment option for WM are warranted.

Targeting the tumor microenvironment with the monoclonal antibody Tocilizumab, reduces IgM secretion in Waldenström macroglobulinemia

Abstract The tumor microenvironment (TME) plays an important role in cancer and has been shown to play a role in resistance to therapy and is therefore important in cancer cell biology. In Waldenström macroglobulinemia (WM), a B cell malignancy characterized by the overproduction of a monoclonal IgM protein, the TME secrets cytokines that promote malignant phenotype. In previous work, we have shown that TME-IL-6 promotes WM cell growth and IgM secretion in WM. Tocilizumab/Actemra is an anti-IL-6R antibody, which can competitively block IL-6 binding to the IL-6R. Here we investigated the efficacy of Tocilizumab in a preclinical mouse model of WM that considers the role of the TME in disease biology. Hairless SCID mice were injected subcutaneously with BCWM.1 or RPCI-WM1 and bone marrow stromal cells (HS-5) at a ratio of 5:1. Groups of mice with treated with Tocilizumab or control antibody three times/week for 5 weeks and the effect on tumor burden and disease biology were evaluated. Although Tocilizumab had no effect on mice survival, there was a reduction in tumor growth rate in mice injected with RPCI-WM1 cells + stromal cells and treated with Tocilizumab (p=0.0394). In mice injected with BCWM.1 + stromal cells, there was a significant reduction in human IgM secretion in mice sera with Tocilizumab treatment (p=0.0099). There was no significant change in mice weight suggesting Tocilizumab induced no toxicities to the mice. Taken together, our data suggests that administration of Tocilizumab to tumor bearing mice, results in a significant reduction in tumor volume and IgM secretion. Therefore, the evaluation of the role of Tocilizumab in WM patients may provide therapeutic efficacy by reducing IgM production and slowing the rate of tumor growth.

Gene Expression Profiling Reveals Aberrant T-cell Marker Expression on Tumor Cells of Waldenström's Macroglobulinemia.

That the malignant clone of Waldenström's macroglobulinemia (WM) demonstrates significant intraclonal heterogeneity with respect to plasmacytoid differentiation indicates the mechanistic complexity of tumorigenesis and progression. Identification of WM genes by comparing different stages of B cells may provide novel druggable targets. The gene expression signatures of CD19+ B cells (BC) and CD138+ plasma cells (PC) from 19 patients with WM were compared with those of BCs from peripheral blood and tonsil and to those of PCs from the marrow of healthy (N-PC) and multiple myeloma donors (MM-PC), as well as tonsil (T-PC). Flow cytometry and immunofluorescence were used to examine T-cell marker expression on WM tumor cells. Consistent with defective differentiation, both BCs and PCs from WM cases expressed abnormal differentiation markers. Sets of 55 and 46 genes were differentially expressed in WM-BC and WM-PC, respectively; and 40 genes uniquely dysregulated in WM samples were identified. Dysregulated genes included cytokines, growth factor receptors, and oncogenes not previously implicated in WM or other plasma cell dyscrasias. Interestingly, strong upregulation of both IL6 and IL6R was confirmed. Supervised cluster analysis of PC revealed that marrow-derived WM-PC was either MM-PC-like or T-PC-like, but not N-PC-like. The aberrant expression of T-cell markers was confirmed at the protein level in WM-BC. We showed that comparative microarray profiles allowed gaining more comprehensive insights into the biology of WM. The data presented here have implications for the development of novel therapies, such as targeting aberrant T-cell markers in WM.

Activation of the Non-Canonical Estrogen Receptor Gper As a Novel Therapeutic Strategy Against Waldenström Macroglobulinemia

Although estrogens have been shown to regulate normal B cell proliferation and differentiation, the impact of estrogen receptor (ER) signaling on B cell malignancies, including Waldenström Macroglobulinemia (WM), remains unexplored. To address this issue, we have analyzed, through preclinical WM models, the druggability either of the classical estrogen receptor ERβ, an emerging target for acute leukemias and certain types of lymphomas, and of the novel, more recently deorphanized G-protein coupled ER GPER, whose therapeutic potential in solid tumors has recently emerged.Both ERβ and GPER were found expressed at mRNA and protein level in a panel of WM and IgM-secreting lymphoma cell lines. However, while treatment with ERβ agonists did not elicit any effect, selective GPER activation significantly reduced WM cell viability. On this basis, we investigated GPER expression pattern and therapeutic potential in WM.By interrogating public microarray datasets, we found GPER transcript significantly upregulated in WM as compared to normal B cells; moreover, immunohistochemical analysis showed elevated GPER expression in lymph node biopsies from newly-diagnosed WM patients as compared to healthy lymph nodes. Treatment with the selective GPER agonist G-1 ( (±) -1-[(3aR*,4S*,9bS*)-4-(6-Bromo-1,3-benzodioxol-5-yl) -3a,4,5,9b-tetrahydro-3H cyclopenta [c]quinolin-8-yl] ethanone) led to reduced viability, clonogenicity and migration of WM and IgM-secreting lymphoma cell lines (IC50 at 48h ranging between 2.5 and 5.0 mM), including MYD88 and CXCR4 mutated cell lines, and even in the presence of bone marrow-derived stromal cells, while it did not affect healthy CD19+ B cell viability; conversely, GPER antagonists G-36 and G-15 slightly enhanced cell proliferation. The growth inhibitory activity of G-1 was associated with accumulation in G2/M cell cycle phase and induction of apoptosis, the latter phenomenon assessed by Annexin V/7AAD staining, analysis of mitochondrial membrane depolarization and cleavage of caspase 3, 7 and 9; hybridization of an antibody-array also highlighted G-1-induced down-regulation of anti-apoptotic proteins, including survivin and Bcl2. A significant effect of G-1 was also observed in primary tumor cells from refractory WM patients. Moreover, G-1 synergistically enhanced bortezomib and ibrutinib cytotoxicity in vitro. Importantly, intraperitoneal injection of G-1 (2mg/kg) significantly reduced the growth of BCWM-1 xenografts in NOD/SCID mice.To gain further insight into the consequences of selective GPER activation, we performed gene expression profile and GSEA analysis of WM cell lines treated with G-1. Of note, the p53 signaling pathway was strongly induced upon GPER activation, and upregulation of p53 and of its target genes or microRNAs (p21CIP1, Bax, Bad, PUMA and miR-34a) was confirmed both in G-1 treated WM cell lines and primary WM cells; moreover, G-1 combination with bortezomib or ibrutinib led to stronger increase in p53 and p21CIP1 levels as compared to single agent treatment. Finally, p53 knock-down partially reversed G-1-dependent anti-WM effects, thus suggesting p53 as downstream mediator of GPER.In summary, we report a significant anti-tumor activity upon selective GPER activation in WM cells, with apoptosis induction and a potent synergistic anti-WM activity in combination with ibrutinib and bortezomib. Altogether, these preclinical data suggest GPER agonists as a potential therapeutic option in WM. DisclosuresMunshi:OncoPep: Other: Board of director.

Multicenter Prospective Phase II Study of Venetoclax in Patients with Previously Treated Waldenstrom Macroglobulinemia

Multicenter Prospective Phase II Study of Venetoclax in Patients with Previously Treated Waldenstrom Macroglobulinemia

Ibrutinib for the Treatment of Bing-Neel Syndrome

Ibrutinib for the Treatment of Bing-Neel Syndrome

Profiling of circulating exosomal miRNAs in patients with Waldenström Macroglobulinemia.

Waldenström Macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by disease progression from IgM MGUS to asymptomatic and then symptomatic disease states. We profiled exosomes from the peripheral blood of patients with WM at different stages (30 smoldering/asymptomatic WM, 44 symptomatic WM samples and 10 healthy controls) to define their role as potential biomarkers of disease progression. In this study, we showed that circulating exosomes and their miRNA content represent unique markers of the tumor and its microenvironment. We observed similar levels of miRNAs in exosomes from patients with asymptomatic (smoldering) and symptomatic WM, suggesting that environmental and clonal changes occur in patients at early stages of disease progression before symptoms occur. Moreover, we identified a small group of miRNAs whose expression correlated directly or inversely with the disease status of patients, notably the known tumor suppressor miRNAs let-7d and the oncogene miR-21 as well as miR-192 and miR-320b. The study of these miRNAs’ specific effect in WM cells could help us gain further insights on the mechanisms underlying WM pathogenesis and reveal their potential as novel therapeutic targets for this disease.

Soluble PD-1 ligands regulate T-cell function in Waldenstrom macroglobulinemia

AbstractAlthough immune checkpoint molecules regulate the progression of certain cancers, their significance in malignant development of Waldenstrom macroglobulinemia (WM), an incurable low-grade B-cell lymphoma, remains unknown. Recently, cytokines in the bone marrow (BM) microenvironment are shown to contribute to the pathobiology of WM. Here, we investigated the impact of cytokines, including interleukin-6 (IL-6) and IL-21, on immune regulation and particularly on the programmed death-1 (PD-1) and its ligands PD-L1 and PD-L2. We showed that IL-21, interferon γ, and IL-6 significantly induced PD-L1 and PD-L2 gene expression in WM cell lines. Increased PD-L1 and PD-L2 messenger RNA was also detected in patients' BM cells. Patients' nonmalignant BM cells, including T cells and monocytes, showed increased PD-L1, but minimal or undetectable PD-L2 surface expression. There was also very modest PD-L1 and PD-L2 surface expression by malignant WM cells, suggesting that ligands are cleaved from the cell surface. Levels of soluble ligands were higher in patients' BM plasma and blood serum than controls. Furthermore, IL-21 and IL-6 increased secreted PD-L1 in the culture media of WM cell lines, implying that elevated levels of soluble PD-1 ligands are cytokine mediated. Soluble PD-1 ligands reduced T-cell proliferation, phosphorylated extracellular signal-regulated kinase and cyclin A levels, mitochondrial adenosine triphosphate production, and spare respiratory capacity. In conclusion, we identify that soluble PD-1 ligands are elevated in WM patients and, in addition to surface-bound ligands in WM BM, could regulate T-cell function. Given the capability of secreted forms to be bioactive at distant sites, soluble PD-1 ligands have the potential to promote disease progression in WM.

Targeting CD38 with daratumumab is lethal to Waldenström macroglobulinaemia cells.

CD38 is expressed on Waldenström macroglobulinaemia (WM) cells, but its role as a therapeutic target remains undefined. With recent approval of the anti-CD38 monoclonal antibody, daratumumab (Dara), we hypothesized that blocking CD38 would be lethal to WM cells. In vitro Dara treatment of WM cells (including ibrutinib-resistant lines) elicited antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP) and direct apoptosis. In vivo, Dara treatment was well tolerated and delayed tumour growth in RPCI-WM1-xenografted mice. CD38 is reported to augment B-cell receptor (BCR) signalling; we noted that Dara significantly attenuated phosphorylated SYK, LYN, BTK, PLCγ2, ERK1/2, AKT, mTOR, and S6 levels, and this effect was augmented by cotreatment with ibrutinib. Indeed, WM cells, including ibrutinib-resistant WM cell lines treated with the ibrutinib+Dara combination, showed significantly more cell death through ADCC, CDC, ADCP and apoptosis relative to single-agent Dara or ibrutinib. In summary, we are the first to report the invitro and invivo anti-WM activity of Dara. Furthermore, we show a close connection between BCR and CD38 signalling, which can be co-targeted with ibrutinib+Dara to induce marked WM cell death, irrespective of acquired resistance to ibrutinib.

BTKCys481Ser drives ibrutinib resistance via ERK1/2 and protects BTKwild-type MYD88-mutated cells by a paracrine mechanism

AbstractAcquired ibrutinib resistance due to BTKCys481 mutations occurs in B-cell malignancies, including those with MYD88 mutations. BTKCys481 mutations are usually subclonal, and their relevance to clinical progression remains unclear. Moreover, the signaling pathways that promote ibrutinib resistance remain to be clarified. We therefore engineered BTKCys481Ser and BTKWT expressing MYD88-mutated Waldenström macroglobulinemia (WM) and activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) cells and observed reactivation of BTK-PLCγ2-ERK1/2 signaling in the presence of ibrutinib in only the former. Use of ERK1/2 inhibitors triggered apoptosis in BTKCys481Ser-expressing cells and showed synergistic cytotoxicity with ibrutinib. ERK1/2 reactivation in ibrutinib-treated BTKCys481Ser cells was accompanied by release of many prosurvival and inflammatory cytokines, including interleukin-6 (IL-6) and IL-10 that were also blocked by ERK1/2 inhibition. To clarify if cytokine release by ibrutinib-treated BTKCys481Ser cells could protect BTKWT MYD88-mutated malignant cells, we used a Transwell coculture system and showed that nontransduced BTKWT MYD88-mutated WM or ABC DLBCL cells were rescued from ibrutinib-induced killing when cocultured with BTKCys481Ser but not their BTKWT-expressing counterparts. Use of IL-6 and/or IL-10 blocking antibodies abolished the protective effect conferred on nontransduced BTKWT by coculture with BTKCys481Ser expressing WM or ABC DLBCL cell counterparts. Rebound of IL-6 and IL-10 serum levels also accompanied disease progression in WM patients with acquired BTKCys481 mutations. Our findings show that the BTKCys481Ser mutation drives ibrutinib resistance in MYD88-mutated WM and ABC DLBCL cells through reactivation of ERK1/2 and can confer a protective effect on BTKWT cells through a paracrine mechanism.

Extracellular vesicle–mediated transfer of constitutively active MyD88L265P engages MyD88wt and activates signaling

AbstractThe link between inflammation and cancer is particularly strong in Waldenström macroglobulinemia (WM), a diffuse large B-cell lymphoma wherein the majority of patients harbor a constitutively active mutation in the innate immune-signaling adaptor myeloid differentiation primary response 88 (MyD88). MyD88Leu265Pro (MyD88L265P) constitutively triggers the myddosome assembly providing a survival signal for cancer cells. Here, we report detection and a functional role of MyD88 in the extracellular vesicles (EVs) shed from WM cells. MyD88L265P was transferred via EVs into the cytoplasm of the recipient mast cells and macrophages, recruiting the endogenous MyD88 that triggered the activation of proinflammatory signaling in the absence of receptor activation. Additionally, internalization of EVs containing MyD88L265P was observed in mice with an effect on the bone marrow microenvironment. MyD88-loaded EVs were detected in the bone marrow aspirates of WM patients thus establishing the physiological role of EVs for MyD88L265P transmission and shaping of the proinflammatory microenvironment. Results establish the mechanism of transmission of signaling complexes via EVs to propagate inflammation as a new mechanism of intercellular communication.

Circulating Soluble Receptor Activator of Nuclear Factor Kappa B Ligand and C-C Motif Ligand 3 Correlate With Survival in Patients With Waldenström Macroglobulinemia

BackgroundSerum receptor activator of nuclear factor κB ligand (sRANKL) and chemokine (C-C) motif ligand 3 (CCL-3) have been reported to be elevated in Waldenström macroglobulinemia (WM) patients. However, there are no published data regarding the prognostic value of these molecules in WM regarding progression-free and overall survival. MethodsTo evaluate the effect of these markers of bone remodeling on survival parameters, we prospectively evaluated serum cytokines and biological markers in 55 patients with symptomatic WM before they received any kind of treatment. Serum levels of CCL-3 and bone remodeling markers were also evaluated in asymptomatic WM and IgM monoclonal gammopathy of undetermined significance. Furthermore, we assessed bone marrow biopsy samples from newly diagnosed WM patients for CCL-3 and RANKL expression. ResultsHigh circulating sRANKL values predicted shorter median overall survival (46 months vs. not reached, P = .025). High serum levels of CCL-3 predicted shorter median progression-free survival (27 months vs. not reached, P = .048). At bone marrow biopsy evaluation, the whole number of the neoplastic cells revealed strong cytoplasmic positivity for CCL-3, while the neoplastic clone did not express RANKL. ConclusionWe conclude that WM cells produce CCL-3 and possibly enhance the production of RANKL in the bone microenvironment. The correlation of sRANKL and CCL-3 with survival reveals the importance of these cytokines in disease biology and highlights the significance of the interactions between WM and stromal cells for the development of WM. Finally, these findings provide the rationale for the use of anti-RANKL and anti–CCL-3 drugs in animal models of WM before their clinical evaluation.

PAK4 Inhibition Impacts Growth and Survival, and Increases Sensitivity to DNA-Damaging Agents in Waldestrostom Macroglobulinemia

p21-activated kinases (PAKs) are effectors for the small GTPases Cdc42 and Rac that control a wide range of cellular processes including cytoskeleton reorganization, cell proliferation, survival and motility. Based on our initial results showing significant biological and functional role of PAK4 in plasma cell malignancies, we investigated its role in Waldenström macroglobulinemia (WM). We first confirmed high level of PAK4 expression and activity in WM cells. Both genetic depletion via stable lentiviral knock down and pharmacological inhibition of PAK4 via allosteric modulator KPT-9274 decreased tumor cell growth and survival in a dose and time dependent manner in a panel of WM cell lines. The growth inhibitory effect was associated with induction of apoptotic cell death, caspases activation and PARP cleavage as well as modulation of pro-apoptotic proteins such as PUMA. In addition, related-PAK4 biological processes such as migration, invasion and adhesion to bone marrow stromal cells (BMSCs) were significantly impacted by the inhibitor. A significant effect of KPT9274 was also observed in primary tumor cells from both newly diagnosed and relapse WM patients, including primary cells from ibrutinib resistant patients.To gain further insight into the consequences of PAK4 inhibition, we performed gene expression profile in WM cell lines treated with KPT-9274. Intriguingly we detected a significant impact on expression of Fanconi Anemia (FA)/BRCA pathway related genes, a DNA-damage response pathway essential for the repair of DNA interstrand crosslinks (ICLs) and regulation of cellular responses to replication stress. Since FA/BRCA deficient cells are defective in the formation of Rad51 foci, a crucial component of the homologous recombination (HR) repair machinery, we examined the effect of PAK4/NAMPT inhibition on Rad51 as well its impact on HR. We observed that, apart from inducing DNA damage, assessed by γH2AX expression, KPT-9274 specifically inhibited Rad51 and the double strand break repair by the homologous recombination pathway. Protein pull down analysis revealed the existence of a complex between nicotinamide phosphoribosyltransferase (NAMPT) and the conserved PAK binding domain; treatment with KPT-9274 disrupted this interaction and inhibited the enzymatic activity of NAMPT. NAD is necessary for PARP function especially with increasing DNA damage; and its depletion severely hamper DNA repair. Consistent with this, we observed that nicotinic acid supplementation rescued the effect of KPT-9274 on NAMPT inhibition as well as DNA damage and repair.As FA/BRCA pathway has been shown to affects sensitivity to alkylating agents, we next combined KPT-9274 with either melphalan or bendamustine in MM and WM cells. The combination treatment resulted in a synergistic time- and dose-dependent inhibition of cell survival and growth in cell lines and primary patient tumor cells. We observed a synergistic induction of apoptosis and activation of caspase-3/7, -8 and -9 following treatment with combined versus single-agent therapy. Similarly, in PAK4 knock-down WM cells bendamustine significantly increased the rate of early and late apoptotic cells (60%) compared to scrambled knockdown cells (37%).In summary, we report a significant anti-proliferative activity of dual PAK4-NAMPT inhibition in WM cells, with induction of apoptosis, DNA-damage response and FA/BRCA pathway disruption and a strong synergistic activity in combination with DNA damaging agents such as Bendamustine. Altogether, our data suggests dual PAK4-NAMPT inhibition represents a novel therapeutic strategies in WM. DisclosuresSenapedis:Karyopharm Therapeutics Inc: Employment, Equity Ownership. Baloglu:Karyopharm Therapeutics Inc: Employment, Equity Ownership. Oliva:Takeda: Honoraria; Celgene: Honoraria. Anderson:Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees. Treon:Pharmacyclics: Consultancy, Research Funding.

Waldenstrom macroglobulinemia cells devoid of BTKC481S or CXCR4WHIM-like mutations acquire resistance to ibrutinib through upregulation of Bcl-2 and AKT resulting in vulnerability towards venetoclax or MK2206 treatment

Although ibrutinib is highly effective in Waldenstrom macroglobulinemia (WM), no complete remissions in WM patients treated with ibrutinib have been reported to date. Moreover, ibrutinib-resistant disease is being steadily reported and is associated with dismal clinical outcome (overall survival of 2.9–3.1 months). To understand mechanisms of ibrutinib resistance in WM, we established ibrutinib-resistant in vitro models using validated WM cell lines. Characterization of these models revealed the absence of BTKC481S and CXCR4WHIM-like mutations. BTK-mediated signaling was found to be highly attenuated accompanied by a shift in PI3K/AKT and apoptosis regulation-associated genes/proteins. Cytotoxicity studies using the AKT inhibitor, MK2206±ibrutinib, and the Bcl-2-specific inhibitor, venetoclax±ibrutinib, demonstrated synergistic loss of cell viability when either MK22016 or venetoclax were used in combination with ibrutinib. Our findings demonstrate that induction of ibrutinib resistance in WM cells can arise independent of BTKC481S and CXCR4WHIM-like mutations and sustained pressure from ibrutinib appears to activate compensatory AKT signaling as well as reshuffling of Bcl-2 family proteins for maintenance of cell survival. Combination treatment demonstrated greater (and synergistic) antitumor effect and provides rationale for development of therapeutic strategies encompassing venetoclax+ibrutinib or PI3K/AKT inhibitors+ibrutinib in ibrutinib-resistant WM.

HCK Transcription Is Regulated By AP1, NF-Kb and STAT3 Transcription Factors in MYD88 Mutated WM and ABC-DLBCL Cells

Hematopoietic cell kinase (HCK) is a member of the SRC family of tyrosine kinases (SFKs). HCK transcription is aberrantly upregulated in Waldenström’s Macroglobulinemia (WM) and Activated B-cell (ABC) subtype Diffuse Large B-cell Lymphoma (DLBCL) in response to activating mutations in MYD88 (Yang et al, Blood 2016). To clarify the mechanism responsible for the aberrant upregulation of HCK transcription inMYD88 mutated cells, we analyzed the promoter sequence of HCK using PROMO and identified consensus binding sites for transcription factors (AP1, NF-kB, STAT3, and IRF1) that are regulated by mutated MYD88 (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013; Juilland et al, Blood 2016; Yang et al, Blood 2016). We performed Chromatin Immuno-precipitation (ChIP) assays using ChIP grade antibodies to JunB, c-Jun, NF-kB-p65, STAT3 and IRF1 in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1, OCI-Ly3) cells that highly express HCK transcripts, as well as wild type MYD88 expressing GCB DLBCL (OCI-Ly7, OCI-Ly19) cells that show low HCK transcription. Following ChIP, a HCK promoter specific quantitative PCR assay was used to detect HCK promoter sequences. These studies showed that JunB, NF-kB-p65 and STAT3 bound more robustly to the HCK promoter in MYD88 mutated WM and ABC-DLBCL cells versus MYD88 wild type GCB DLBCL cell lines, while c-Jun bound more abundantly to the HCK promoter sequence in all DLBCL cell lines, regardless of MYD88 mutation status. In contrast c-Jun binding was low in MYD88 mutated WM cells. IRF1 binding to the HCK promoter was similar in all cell lines, regardless of the MYD88 mutation status. To further investigate HCK regulation, we developed an HCK promoter driven luciferase reporter vector (WT) with mutated AP-1 binding (AP1-mu-1~6), NF-kB binding (NF-kB-mu-1~5), and STAT3 binding (STAT3-mu) sites and investigated their impact on HCK promoter activity in MYD88 mutated BCWM.1 cells. We observed that mutation of AP1-mu-1,4,5,6; NF-kB-mu-1,4,5, as well as STAT3-mu binding sites greatly reduced HCK promoter activity, thereby supporting a role for AP-1, NF-kB and STAT3 transcription factors in HCK gene expression in MYD88 mutated cells. To further clarify the importance of these transcription factors in aberrant HCK gene expression in MYD88 mutated cells, we treated BCWM.1, MWCL-1, TMD-8 and HBL-1 cells with the AP-1 inhibitor SR 11302; NF-kB inhibitor QNZ; and the STAT3 inhibitor STA-21. Treatment of cells for 2 hours with SR 11302, QNZ, and STA-21 at sub-EC50 concentrations resulted in decreased HCK expression in MYD88 mutated all cell lines. Lastly, we investigated the contribution of BCR signaling to HCK transcription. BCWM.1, MWCL-1, TMD-8, and HBL-1 cells were treated with the Syk kinase inhibitor R406, and HCK transcription levels were then assessed. Differences in HCK expression were observed between MYD88 mutated WM and ABC DLBCL cells following R406, supporting a contributing role for BCR signaling in ABC DLBCL but not WM cells to HCK expression. Our data provide critical new insights into HCK regulation, and a framework for targeting pro-survival HCK signaling in WM and ABC DLBCL cells dependent on activating MYD88 mutations. DisclosuresCastillo:Biogen: Consultancy; Otsuka: Consultancy; Millennium: Research Funding; Janssen: Honoraria; Abbvie: Research Funding; Pharmacyclics: Honoraria. Treon:Janssen: Consultancy; Pharmacyclics: Consultancy, Research Funding.