Waldenstrom Macroglobulinemia Cell Research Articles

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

Pirtobrutinib (LOXO-305) Is Active and Overcomes ERK Related Pro-Survival Signaling in Ibrutinib Resistant, BTK Cys481 Mutant Expressing WM and ABC DLBCL Lymphoma Cells Driven By Activating MYD88 Mutations

MYD88 mutations are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of Diffuse Large B-cell Lymphoma (ABC DLBCL). Mutated MYD88 activates BTK, and triggers downstream pro-survival signaling that includes NF-kB and ERK (Yang et al, Blood 2013; Blood 2016). ERK related signaling triggers inflammatory cytokine production including IL-6 and IL-10 (Chen et al, Blood 2016). Ibrutinib covalently binds to BTK Cys481 and inactivates BTK and downstream NF-kB and ERK signaling. Ibrutinib is approved for the treatment of WM and is associated with high overall response rates (>90%) and long term progression free survival in WM though intolerance to therapy, as well as resistance related to acquired BTK Cys481 mutations frequently leads to treatment discontinuation. We therefore investigated a novel, non-covalent BTK-inhibitor, pirtobrutinib that binds to BTK at non-Cys481 amino acids (G473-K483). Pirtobrutinib showed highly selective anti-proliferative activity against MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8 and HBL-1) versus MYD88 wild-type (OCI-Ly7, OCI-Ly19, Ramos, and RPMI-8226) cells, with marked apoptotic effect exhibited against primary MYD88 mutated WM cells at pharmacologically achievable levels (100-500 nM). Importantly, pirtobrutinib blocked BTK activity and overcame ibrutinib resistance in BCWM.1 WM and TMD-8 ABC DLBCL cells transduced to express both wild-type and mutated BTK (BTK Cys481Ser) with similar efficacy. The downstream signaling consequences of pirtobrutinib in vector only, wild-type and mutant BTK Cys481 expressing BCWM.1, MWCL-1, TMD-8 and HBL-1 cells was also examined. Treatment of vector only and wild-type BTK Cys481 expressing WM and ABC DLBCL cells with ibrutinib or pirtobrutinib abrogated both p-BTK and p-ERK signaling. In contrast, only pirtobrutinib blocked p-BTK and p-ERK signaling in mutant BTK Cys481 expressing WM and ABC DLBCL cells. In previous studies, we showed that inflammatory cytokine production that included IL-6 and IL-10 driven by ERK triggered ibrutinib resistance in wild-type BTK Cys481 MYD88 mutated lymphoma cells co-cultured with their mutated BTK expressing counterparts (Chen et al, BLOOD 2018). ERK-driven cytokine resistance to ibrutinib was postulated to explain how disease progression occurs in patients with modest variant expression of mutated BTK Cys481 (Woyach et al, JCO 2017; Xu et al, Blood 2017). Co-culture of BTK Cys481 mutated expressing TMD-8 cells with wild-type BTK expressing TMD-8 cells triggered resistance of the latter to ibrutinib. Treatment with pirtobrutinib blocked IL-6 and IL-10 production and overcame the protective effects conferred by BTK Cys481 mutated TMD-8 cells in these experiments. Lastly, oral administration of pirtobrutinib blocked p-BTK and p-ERK in BTK Cys481 mutated TMD-8 tumors xenografted in mice. Our findings therefore show that pirtobrutinib inhibits growth of MYD88 mutated lymphoma cells in a highly selective manner and can trigger apoptosis of primary WM patient BM lymphoplasmacytic cells at levels comparable to ibrutinib. Moreover, pirtobrutinib effectively blocked mutated BTK Cys481 driven BTK and ERK1/2 activation and produced similar cellular efficacy in both BTK wild-type and BTK Cys481 mutated cells. Pirtobrutinib also blocked the protective effect conferred to BTK wild-type cells through paracrine cytokines released by BTK Cys481Ser expressing cells. Lastly, pirtobrutinib blocked BTK and ERK1/2 activation in TMD8-BTK Cys481Ser xenografted mice. The findings support the development of pirtobrutinib in MYD88 driven lymphomas, including those resistant to ibrutinib on the bases of BTK Cys481 mutations. DisclosuresBranagan: Adaptive Biotechnologies: Consultancy; BeiGene: Consultancy; CSL Behring: Consultancy; Karyopharm: Consultancy; Pharmacyclics: Consultancy; Sanofi Genzyme: Consultancy. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.

The Oral Proteasome Inhibitor Ixazomib, Alone and in Combination with Ibrutinib, Induces Lethality in Waldenstrom Macroglobulinemia Cells That Are Resistant to Ibrutinib

Introduction: Dysregulated B-cell receptor (BCR) signaling drives proliferation of Waldenstrom macroglobulinemia (WM) cells, which characteristically secrete excessive IgM. To maintain IgM production and preserve cellular homeostasis, WM cells rely on optimally functioning proteasomes to degrade and recycle proteins. This biological dependence has been clinically exploited using the proteasome inhibitor (PI), bortezomib (Btz), which is capable of inducing remission in 85% of WM patients (Treon et al Clin Canc Res, 2007) as a single agent. Despite, its activity, Btz use is associated with a high incidence of sensory and autonomic neuropathies as well as its cumbersome dosing schedule and non-oral route of administration. Thus, new agents that 1.) build on the activity of Btz, 2.) have a more favorable toxicity profile and 3.) can be combined with standard of care agents such as, ibrutinib (Ibr), are highly attractive and should be prioritized for investigation. Here we describe the preclinical activity of ixazomib (Ixa), a PI that is administered orally and is associated with significantly less peripheral neuropathy; in combination with Ibr, in WM cells.Methods: WM cell lines (BCWM.1 and RPCI.WM1) and their drug-resistant subclones were used. Genomic data including whole exome sequencing (WES) and copy number variations (CNV) was used in creation of digital simulation avatars of RPCI-WM1 and RPCI-WM1/IR (Ibr-resistant, Ibr-R variant), where the salient and prominently dysregulated cellular pathways were identified. Ixa +/- Ibr was tested in digital avatars and dose-dependent impact on simulated disease growth was assessed (Fig 1A). The predictive results were experimentally validated. Cell viability was measured using trypan blue or CellTiter Glo. Apoptosis was examined by annexin-V/PI staining of drug-treated cells followed by flow cytometry analysis. Mitochondrial membrane permeability (MOMP) was determined by TMRM staining of drug-treated cells followed by flow cytometry.Results: We first profiled the growth inhibitory action of Ixa in WM cell lines (n=9); all of which were sensitive to Ixa irrespective of acquired resistance to Btz, Ibr or carfilzomib with a median IC50 of 95.59nM. We then tested whether Ixa could cooperate with Ibr to enhance WM cell kill. Indeed, the Cellworks modeling tool, predicted a significant loss of viability and induction of apoptosis in both wildtype and Ibr-R cells treated with Ibr + Ixa, as compared to either single agent (Fig 1B-D). We confirmed this in vitro, where viability of wildtype WM cells treated with Ixa (20nM), Ibr (5uM) or Ibr + Ixa for 6h significantly decreased; most notable with Ixa + Ibr (25.5% viable cells). And this effect was similar in Ibr-R cell lines (30.3% viable cells) after combination treatment. Further confirming in silico predictive modeling results, we noted a significant increase in annexin-V/PI positivity in Ixa treated wildtype (24.5%) and Ibr-R WM cell lines (33.1%). Whereas Ibr alone exhibited minimal apoptosis in WM cell lines (~11 - 15%), the combination of with Ixa + Ibr enhanced programmed cell death in wildtype (40.2%) and Ibr-R cells (50.5%). As intrinsic apoptosis is triggered through mitochondrial disruption, we also examined mitochondrial transmembrane permeability (MOMP) in cells treated with Ibr, Ixa or Ixa + Ibr. In wildtype cells, mean MOMP induced by single agent Ixa was 23.5% and in Ibr-R cells 33%; increasing to 39% and 50.4%, respectively, after treatment with Ibr + Ixa. Mechanistic studies revealed changes in proteins regulated by the ubiquitin proteasome system and those associated with BTK signaling. While Ixa alone downregulated Bcl-2 and Mcl-1 levels, this effect was more marked in cells (both wildtype and Ibr-resistant) treated with Ixa + Ibr combination. Similar effects were noted in pPLCg2, albeit with some variability between the cell lines.Conclusion: Using a combination of in silico modeling and experimental approaches, we provide first preclinical evidence on the antitumor effects of Ibr combined with the oral PI, Ixa, in WM cells, including those resistant to Ibr. Whereas further investigation on the precise molecular mechanisms of how Ibr + Ixa cooperate to induce apoptosis in WM cell is ongoing, our studies provide the basis for clinical testing of the Ibr + Ixa regimen; with a phase II clinical study in development. [Display omitted] DisclosuresSingh:Cellworks Research India Pvt. Ltd: Employment. Kumar:Cellworks: Employment. Basu:Cellworks: Employment. Trivedi:Cellworks: Employment. Majumdar:Cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Martin:Janssen: Consultancy, Honoraria, Other: travel expenses; Genentech: Consultancy; Celgene: Consultancy; Teva: Research Funding; Novartis: Consultancy; Gilead: Consultancy, Other: travel expenses. Ansell:Bristol-Myers Squibb: Research Funding; Merck: Research Funding; Celldex: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding. Ailawadhi:Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pharmacyclics: Research Funding; Takeda: Consultancy, Honoraria.

Targeting CD38 with Daratumumab Suppresses B-Cell Receptor Signaling and Enhances the Activity of Ibrutinib in Waldenstrom Macroglobulinemia Cells

Introduction: Waldenstrom macroglobulinemia (WM) is characterized by malignant B-lymphoplasmacytic cells that rely on B-cell receptor (BCR) signaling, which is clinically exploited with ibrutinib (Ibr). Although effective, Ibr tx. mostly results in partial remission of disease. This signifies the need to examine combination tx. strategies that can lead to superior disease eradication and counteract onset of Ibr resistance. WM cells express several surface antigens, including CD38 and Daratumumab (Dara) is a first-in-class monoclonal antibody that binds CD38; inducing an immune effector-mediated cytolytic reaction. CD38 also functions as a molecular hub for integration of signals transmitted through the B-cell receptor complex (BCR) to promote cell proliferation. We hypothesized that cotargeting BTK (with Ibr) and CD38 (with Dara) would induce both an immune-mediated and cell intrinsic mode of death to result in enhanced WM cell kill.Methods: Wildtype WM cell lines (BCWM.1, RPCI.WM1) and ibrutinib-resistant (IR) subclones were used in experiments. CD38 MFI and surface antibody binding (sAbc) was determined by flow cytometry. Ibr (1uM), Dara (0.1ug/mL) or Ibr + Dara-mediated death through antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) was measured using the Calcein AM assay at an effector to target (E:T) ratio of 50:1. Antibody-dependent cellular phagocytosis (ADCP) was assessed using human macrophages at an E:T ratio of 2:1. An IgG1 isotype Ab was used as a control in experiments.Results: CD38 sAbc in BCWM.1 and RPCI-WM1 cells was 4864 and 35892, respectively; consistent with previously noted MFI's (Paulus et al PLOS One, 2016). Specific lysis induced by single agent Dara in wildtype WM cells, showed a mean ADCC of 24.46%, CDC: 20.29% and ADCP: 20.12%. Dara can also induce apoptosis and we noted an average of 33.35% annexin-V/PI+ WM cells after 24h Dara tx. We surmised the anti-WM activity of Dara could be enhanced by Ibr as 1.) the BTK inhibitor has been shown to improve anti-tumor T-cell responses and 2.) the combined effects of each drug could synergistically attenuate BCR-associated signaling. As anticipated, mean ADCC, CDC, ADCP and apoptosis increased to 52.7%, 38.41%, 30.19% and 57.53%, respectively in Ibr + Dara-treated wildtype WM cells. While potential mechanisms of the enhanced immune-mediated specific lysis from the combination tx. are being investigated, we observed cell signaling changes that may account for the increased cell death. As expected, Dara decreased pLyn, pSyk, pBTK, pPLCG2 and pERK as well as pAKT, pmTOR and pS6K levels in WM cells. And these proteins decreased further in Ibr + Dara treated cells. Next, we asked the question whether Dara +/- Ibr would be active in IR WM cells, which had lower CD38 sAbc (mean 8400, range 1024 - 15776). Mean ADCC by Dara in IR WM cells was 19.52%, CDC: 31.85%, ADCP: 27.5% and apoptosis at 31.3%. While ADCC was expectedly lower in IR vs WT cells, CDC and ADCP were significantly higher. We then tested the combination of Ibr + Dara in IR WM cells and noted greater ADCC (63.97%), CDC (60.34%) and apoptosis (60.23%), whereas ADCP did not increase (29.45%). We also analyzed BCR-associated protein expression and noted single agent Dara decreased pBTK, pPLCG2, pERK and pS6K, whereas the effects of Ibr + Dara were more cell line specific. As IR WM subclones are less reliant on BTK/BCR-mediated signaling, cell death from Dara (+/- Ibr) may be due to other mechanisms possibly related to alterations in the mitochondria (intrinsic to IR cells, Paulus et al BCJ 2017).Conclusion: We demonstrate in vitro activity of Dara and Ibr in WM cells, showing that 1.) Dara induces cell death through various mechanisms (ADCC, CDC, ADCP and apoptosis); 2.) while higher CD38 sAbc appears to correlate with greater ADCC, this was not the case for CDC, ADCP or apoptosis; 3.) Dara significantly modulates BCR-associated signaling; an effect preserved in IR WM cells and documented for the first time and 4.) the combination tx. exerts significantly more WM cell death in nearly all the aforementioned assays than either single agent alone and in some cases more so in IR WM cells. With further preclinical results supporting these data, to be shared at the conference, our investigations provide rationale for clinical testing of Ibr + Dara in WM. DisclosuresSher:LAM Therapeutics, Inc: Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Merck: Research Funding; Celldex: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding. Malavasi:Jaansen Pharmaceuticals: Research Funding. Ailawadhi:Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pharmacyclics: Research Funding; Takeda: Consultancy, Honoraria.

Soluble PD-1 Ligands Regulate T-Cell Function in Waldenstrom's Macroglobulinemia

While immune checkpoint molecules contribute to the malignant development of several cancers, including certain types of lymphomas, their potential significance in malignant progression of Waldenstrom's Macroglobulinemia (WM), a low-grade B-cell lymphoma, has not fully been described. The main characteristic of WM is infiltration of lymphoplasmacytic cells in the bone marrow (BM) and increased monoclonal IgM production by malignant cells within BM microenvironment. While next generation sequencing analysis has identified mutations in the MYD88 gene as the driver of WM development, studies by our group have also highlighted a role for the BM microenvironment in pathophysiology of WM and shown that elevated levels of certain cytokines in the BM, such as IL-6 and IL-21, promote both cell proliferation and IgM secretion in WM.In this study, we investigated the impact of cytokines on the immune checkpoint receptor Programmed Death-1 (PD-1) and its ligands PD-L1 and PD-L2 and evaluated how these molecules contribute to malignant progression in WM.Using RT-PCR analysis we have shown that treatment of WM cell lines with IL-21 and IL-6 significantly induce PD-L1 and PD-L2 gene expression, implying that elevated levels of these cytokines within the BM microenvironment could be associated with increased PD-L1 and PD-L2 expression by malignant cells in WM patients. We therefore performed RT-PCR analysis on cells isolated from patients' BM and confirmed increased PD-L1 and PD-L2 expression by both WM and BM cells when compared normal BM cells. In contrast, flow cytometry analysis showed minimal expression of these ligands on the surface of the WM cells, with no significant difference between WM and normal BM cells, indicating that there might be a regulatory mechanism controlling surface expression of these ligands. Further analysis using ELISA and Western Blotting showed that PD-L1 and PD-L2 are found as secreted forms in both BM plasma and peripheral blood serum of the WM patients and their level is significantly higher in WM as compared to normal subjects. Interestingly, in vitro treatment of the WM cell lines by IL-21 and IL-6 increased secreted PD-L1 and PD-L2 in the culture media of the cells, providing an additional line of evidence for elevated PD-L1 and PD-L2 levels in WM patients. Subsequent analysis showed that the secreted form of the PD-L1 and PD-L2 binds to T-cells and induces signals that reduce T-cell proliferation and cell cycle progression, the effects that are associated with a reduction in p-ERK and cyclin A levels.In summary, our data identifies that secreted form of PD-1 ligands is elevated in WM and play an important role in regulating T-cell function. Given the capability of secreted form to be bioactive at distant sites, soluble PD-1 ligands may have potential to promote disease progression. DisclosuresAnsell:Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Affimed: Research Funding; Merck: Research Funding; Celldex: Research Funding.

Characterization of Chromosome 6q Deletions in MYD88 Mutant and Wild-Type Waldenström's Macroglobulinemia

Introduction: MYD88 L265P mutations have been described in more than 90% of Waldenström's Macroglobulinemia (WM) patients. MYD88 wild-type (wt) WM represent less than 10% of WM, and associate with shorter overall survival versus MYD88 mutated (mut) WM, as well as with lack of major responses and inferior progression-free survival to ibrutinib. Mutated MYD88 triggers WM cells growth and survival through NF-kB activation via IRAK1/4, BTK and HCK transactivation. CXCR4 mutations promote pro-survival AKT and ERK1/2 signaling and are associated with delayed response to ibrutinib. Deletions involving the long arm of chromosome 6 (Del6q) are highly recurrent in various B-cell malignancies, particularly those that are driven by mutated MYD88, and have been observed in up to half of WM patients. Chr6q genomic loss in WM affect important modulators of NFkB (TNFAIP3, HIVEP2), BCL2 protein family (BCLAF1), apoptosis and IGF1/PI3K/AKT signaling (FOXO3), and BTK (IBTK). The frequency and impact of Del6q on the different patient genotype remains to be clarified.Patients and Methods: Based on location and known regulation of key pathways in WM, copy number alterations (CNA) and gene expression level were assessed for IBTK, FOXO3, BCLAF1, TNFAIP3 and HIVEP2 genes, along with CXCR4 transcriptional levels. DNA and RNA from CD19+ sorted BM lymphoplasmacytic cells from 32 untreated WM and one IgM and IgG-secreting lymphoplasmacytic lymphoma patients were analyzed. Paired CD19-depleted peripheral-blood mononuclear cells (PBMCs) were used as germline controls. Paired CD19+ and CD19- PBMCs from 6 healthy donors were analyzed to rule out possible B-cell specific findings. CNA were measured in quadruplicate and gene expression in triplicate using TaqMan real-time polymerase chain reaction (RT-PCR) assays. Deletions affecting less than 20% of WM cells were considered to be beneath the RT-PCR detection threshold. The cohort included 33 patients (21 males, 12 females), with a median age of 62 (range 35-91) years, BM involvement of 60% (range 2.5-90%), serum IgM levels of 3010 (range 257-6910) mg/dl, and hemoglobin of 10.9 (range 8.4-14.4) g/dl. MYD88 L265P mutations were detectable in 25 (76%) patients, 11 of whom (44%) also carried CXCR4WHIM mutations. Eight patients (24%) were MYD88 and CXCR4 wt.Results: Somatic Del6q were observed in 4/8 (50%) and in 20/25 (80%) patients in the MYD88 wt and mut population, respectively (p=NS). In contrast, no CNA were present in the HDs CD19+/CD19- compartments. IBTK remained intact in all 8 MYD88 wt WM, whereas resulted deleted in 13/25 (52%) MYD88 mut patients (p=0.01). BCLAF1 deletion frequency was significantly different among the two subgroups, with only 2/8 deleted MYD88wt (25%) and 19/25 (76%) deleted MYD88 mut patients (p=0.02; Figure 1). Eight MYD88 mut WM (32%) showed contiguous fully clonal Del6q, that spanned all studied genes in 7/8 (87.5%) cases; in contrast, contiguous subclonal deletions occurred only in 3/12 (25%) cases (p=0.02). All 8 fully clonal Del6q patients were CXCR4 wt; in contrast, CXCR4 mut were detected in 9/12 (75%) patients with subclonal Del6q (p=0.001; Figure 2). Unlike the MYD88 mut population, all 4 MYD88 wt patients presented with non-contiguous subclonal deletions (p=NS). Fully clonal deletions of IBTK, BCLAF1 and HIVEP2 significantly reduced the respective gene transcriptional levels (p= 0.03, 0.01 and 0.01, respectively), while CNA in TNFAIP3 and FOXO3 did not affect their expression. The MYD88 wt patients showed significant decreases in the transcriptional levels of BCLAF1 related to gene subclonal deletions, and reduced CXCR4 levels compared to MYD88 mut WM (p=0.02 and p=0.02, respectively).Conclusions: Our findings demonstrate that gene loss of IBTK, FOXO3, BCLAF1, TNFAIP3 and HIVEP2 occurs in most WM patients, but at a lower frequency in MYD88 wt. Unlike the MYD88 mut patients, those MYD88 wt showed no contiguous fully clonal deletions. The latter correlate with CXCR4 wt and reduce only IBTK, BCLAF1 and HIVEP2 levels, suggesting that regulatory mechanisms may compensate for FOXO3 and TNFAIP3 gene loss. Significant variation in IBTK and BCLAF1 deletion rate suggest that BCR and BCL2 signaling may play a different role in MYD88 mut and wt WM. The findings provide valuable insights into MYD88 wt WM pathogenesis, and may be relevant to understanding therapeutic outcome with agents that target MYD88, CXCR4, BTK and BCL2. [Display omitted] DisclosuresCastillo:Millennium: Research Funding; Abbvie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Arcaini:Pfizer, Celgene, Bayer, Roche: Membership on an entity's Board of Directors or advisory committees; Celgene, Roche, Sandoz: Consultancy; Gilead: Research Funding. Varettoni:Jannsen: Consultancy, Other: travel expenses. Treon:Pharmacyclics: Consultancy, Research Funding.

Mutated MYD88 Activates the BCR Component SYK and Provides a Rationale Therapeutic Target in Waldenstrom's Macroglobulinemia

Background: Mutations in the Toll receptor (TLR) pathway are highly prevalent in Waldenstrom's Macroglobulinemia (WM) and ABC DLBCL, wherein mutated MYD88 triggers NF-kB pro-survival signaling through BTK/IRAK. Activation of B-cell receptor (BCR) signaling can also be triggered by activating mutations in CD79A/B in ABC DLBCL, though are rare in WM (5-8%). Despite these findings, there is evidence for BCR activation in WM (Argyropoulos et al, Leukemia 2016). We hypothesized that crosstalk between TLR and BCR might account for aberrant BCR signaling in WM. We therefore performed PhosFLow studies on MYD88 and BCR signaling components and observed the hyperactivation of SYK (pY525-pY526) in MYD88 mutated WM (BCWM.1, MWCL-1) cells compared to wild-type MYD88 expressing control cells. We next treated MYD88 mutated WM cells with a peptide inhibitor of MYD88 homodimerization, and observed that the phosphorylation of SYK at tyrosine 525 and 526 (pY525-pY526) together with phosphorylation of BTK at tyrosine 223 (pY223) was markedly reduced. In contrast, the MYD88 inhibitor had no impact on either SYK or BTK activation in MYD88 wild-type Ramos cells. Knockdown of MYD88 by lentiviral transduction in MYD88 mutated BCWM.1 cells confirmed the reduction of SYK (pY525-pY526) by two shRNAs. Cell viability analysis by CellTiter-Glo® assay showed that the SYK inhibitor R406 was cytotoxic to MYD88 mutated WM cells at pharmacologically attainable drug levels (EC50 0.247 uM for BCWM.1; 0.315 uM for MWCL-1), versus >1 uM for MYD88 wild-type control cells. Furthermore, the combination of R406 with ibrutinib produced synergistic cytotoxicity in MYD88 mutated WM cells. Conclusion: Our findings demonstrate that in addition to TLR path activation, mutated MYD88 activates the BCR component SYK tyrosine kinase. These findings provide the rationale for combined therapeutic targeting of BCR and TLR pathways in MYD88 mutated WM. DisclosuresCastillo:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

Mast Cell Density and Its Clinical Relevance in Waldenstrom's Macroglobulinemia

Background. Waldenström's macroglobulinemia (WM) is characterized by a lymphoplasmacytic infiltration of the bone marrow (BM) along with a serum monoclonal IgM and includes MYD88 L265P and CXCR4 mutations in around 90% and 25% respectively (Treon, NEJM 2012; Hunter, Blood 2014). CXCR4WHIMmutations confer worse outcome and are associated with larger BM involvement and less adenopathies (Treon, Blood 2014). One hallmark and histological criteria for WM diagnosis is the presence of numerous mast cells (MC) segregating with tumor cells in the BM (Waldenström, Acta Haematologica 1958). MC have been shown to support lymphoplasmacytic cell growth, through CD154/CD40 signaling (Tournilhac, Ann Oncol 2006), but there is so far no clear demonstration of the prognostic impact of BM MC density in WM.Aim. We proposed to investigate BM MC density by using sensitive and specific digital quantification, allowing the analysis of large tumor cell infiltrated BM area, in order to assess its clinical relevance in WM.Methods. A total of 65 WM patients were investigated, including 54 pts at diagnosis and 11 at relapse. Mutational status could be determined in 35 pts: allele specific PCR found MYD88 L265P mutation in 32/35 pts (91%) and deep next-generation analysis found CXCR4WHIMmutation in 7/35 (20%) pts. Diagnostic and treatment criteria for WM fulfilled recommendations of the 8th international WM workshop (Castillo, BJH 2016 ; Leblond, Blood 2016). MC density was explored using a digital tool previously used for quantifying immune cells infiltrates on tumor tissues sections (Galon, Science 2006). Tryptase and CD20 immunohistochemisty stainings were done on contiguous sections of deparaffinized BM trephine biopsies. After numerization of each section, BM surface area was manually marked out, excluding bone framework and adipocytes, in order to limit subsequent analyses to hematopoietic tissue. We then defined optimal thresholds for horseradish peroxidase signal detection. MC were counted up with “Immunoscore module” (Definiens Developer XT), on the hematopoietic tissue of the whole BM section : MC density was automately recorded as the number of tryptase positive cells per unit of selected bone marrow tissue surface area.Results. MC density was found to be heterogeneous over BM, with a mean of 106 MC.mm-2 of hematopoietic tissue (Figure 1). Higher MC density (> 100 MC.mm-2) was associated with larger BM involvement by WM cells (BM deep infiltration (p=0.039) and diffuse tumor pattern (p=0.003)) and with less frequent spleen involvement (p=0.011) and a trend towards less lymph node (p=0.072) and liver (p=0.054) involvement. Higher MC density was also associated with features of advanced disease such as anemia (<115g.L-1 ; p = 0.041), thrombocytopenia (<100G.L-1 ; p=0.01) and lower albumin rate (<35g.L-1 ; p=0.03)). Furthermore MC density was 194 MC.mm-2 in patients with high IPSS, 82.4 MC.mm-2 in those with intermediate IPSS and 67.9 MC.mm-2 in those with low IPSS (p=0.0003). Regarding outcome, patients with higher MC density had a shorter median OS (56.5 months vs non reached in patients with low MC density, p = 0.0139)) (Figure 2). Among the pts scheduled to receive a 1st line treatment for an active disase (n=43) and molecularly characterized (n=20), those with CXCR4WHIM mutations had a significantly higher MC density (240.7 MC.mm-2vs 90.9 MC.mm-2 for CXCR4 WT pts ; p=0.0221).Conclusion. By using specific digital tool on well-outlined hematopoietic tissue surface, MC density can be accurately measured in WM patients. High MC density is associated with aggressive features, poor clinical outcome and possibly with CXCR4WHIM mutation. [Display omitted] DisclosuresYsebaert:Janssen: Consultancy, Research Funding, Speakers Bureau. Leblond:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria; Novartis: Honoraria; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hermine:Hybrigenics: Research Funding; INatherys: Equity Ownership, Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; Novartis: Research Funding; Celgene: Research Funding. Guieze:GILEAD: Other: Educational Presentation; ABBVIE: Other: Educational Presentation; JANSSEN: Other: Educational Presentation. Tournilhac:Janssen: Honoraria, Other: travel funding; AMGEN: Other: Travel funding, Research Funding; ROCHE: Honoraria, Other: Travel funding, Research Funding; GILEAD: Honoraria, Other: Travel Funding, Research Funding; Abbvie: Honoraria, Other: Travel funding.

MYD88 Mutation Status Impacts Overall Survival and Risk of Histological Transformation in Waldenstrom's Macroglobulinemia

Activating mutations in MYD88 and CXCR4 are present in 93-95% and 30-40% of patients with Waldenstrom's Macroglobulinemia (WM), respectively. Mutations in MYD88 trigger NF-KB dependent growth and survival of WM cells through BTK/IRAK, and AKT/ERK through HCK (Yang et al, Blood 2013; 2016). Patients with MYD88 mutations show high levels of response activity to ibrutinib, that targets BTK and HCK, while responses are poor in MYD88WT patients denoting biological differences between these two subgroups (Treon et al, NEJM 2015). The mutational landscape of MYD88 wild-type (WT) patients is under investigation, though is likely to involve alternative signaling pathways based on transcriptome studies (Hunter et al, Blood 2016). In a previous study, we observed that patients with MYD88WT were at higher risk of death, while CXCR4 mutation status had no impact on overall survival (Treon et al, Blood 2014). Given the uncommon nature of MYD88WT WM disease, we sought in this study to investigate the impact of MYD88WT status in a larger population of WM patients. WHO and WM Consensus guidelines were utilized to establish WM diagnosis, and thorough pathological and laboratory testing was undertaken to exclude non-WM IgM secreting B-cell entities. We utilized sorted CD19+ lymphoplasmacytic cells (LPC) obtained from the bone marrow of WM patients, and determined MYD88 mutation status by highly sensitive and specific AS-PCR for MYD88L265P as before (Xu et al, Blood 2013). Patients with wild-type MYD88 by AS-PCR underwent Sanger sequencing to exclude non-L265P MYD88 mutations as before (Treon et al, NEJM 2015). CXCR4 mutation status was determined in sorted LPC by AS-PCR and Sanger sequencing as before (Xu et al, BJH 2016). We identified 46 MYD88WT WM patients by these efforts, and compared their findings at time of diagnosis, and survival outcome to 262 patients with MYD88 mutated (MYD88MUT) disease who were diagnosed over the same time-period. The median follow-up for all patients was 74.7 (0.5-324.9 months), and was similar for MYD88WT and MYD88MUT WM patients (64.1 vs. 73.7 months, respectively; p=0.71). Patient characteristics are shown in Table 1.Among 262 MYD88MUT patients, 261 had L265P and 1 had the S243N MYD88 mutations; 101 (38.5%) were also CXCR4 mutated. Four (8.6%) MYD88WT patients were also CXCR4 mutated. During the follow-up period, there were 11 (23.9%) and 15 (5.7%) deaths among the MYD88WT and MYD88MUT patients (p=0.003). Figure 1A shows overall survival based on MYD88 and CXCR4 mutation status from time of diagnosis for all patients. The estimated 10-year survival was 73% (95% CI 52-86%) and 90% (95% CI 82-95%) for MYD88WT and MYD88MUT patients, respectively (Log-rank p<0.001), and did not differ for MYD88MUT patients by CXCR4 mutation status (Log-rank p=0.69). Multivariate analysis that included age, gender, serum IgM, hemoglobin, BM disease involvement, serum B2M, MYD88 and CXCR4 mutation status only showed MYD88 mutation status (p<0.001) as a significant determinant for overall survival. Transformation to diffuse large B-cell lymphoma occurred in 7 (15.2%) and 2 (0.76%) of MYD88WT and MYD88MUT patients, respectively (p<0.0001), with odds ratio of transformation for MYD88WT versus MYD88MUT of 23.3 (95% CI 4.2-233.8; p<0.001). Among MYD88WT patients who transformed, overall survival was shorter (Figure 1B; Log-rank p=0.08).Our findings affirm the prognostic significance for MYD88 mutation status as an important determinant of overall survival in WM, and identify a high risk of disease transformation in patients with MYD88WT disease. [Display omitted] DisclosuresTreon:Pharmacyclics: Consultancy, Research Funding. Castillo:Pharmacyclics: Consultancy, Research Funding; Abbvie: Research Funding; Millennium: Research Funding; Janssen: Consultancy, Research Funding.

Halting the FGF/FGFR axis leads to antitumor activity in Waldenström macroglobulinemia by silencing MYD88

The human fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) axis deregulation is largely involved in supporting the pathogenesis of hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb because of disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGFR system blockade in WM by using a pan-FGF trap molecule (NSC12). Wide-transcriptome profiling confirmed inhibition of FGFR signaling in NSC12-treated WM cells; unveiling a significant inhibition of MYD88 was also confirmed at the protein level. Importantly, the NSC12-dependent silencing of MYD88 was functionally active, as it led to inhibition of MYD88-driven pathways, such as BTK and SYK, as well as the MYD88-downstream target HCK. Of note, both canonical and noncanonical NF-κB cascades were downregulated in WM cells upon NSC12 treatment. Functional sequelae exerted by NSC12 in WM cells were studied, demonstrating significant inhibition of WM cell growth, induction of WM cell apoptosis, halting MAPK, JAK/STAT3, and PI3K-Akt pathways. Importantly, NSC12 exerted an anti-WM effect even in the presence of bone marrow microenvironment, both in vitro and in vivo. Our studies provide the evidence for using NSC12 as a specific FGF/FGFR system inhibitor, thus representing a novel therapeutic strategy in WM.

Progression from Monoclonal gammopathy of undetermined significance of the immunoglobulin M class (IgM-MGUS) to Waldenstrom Macroglobulinemia is associated with an alteration in lipid metabolism

The molecular events that modulate the progression of monoclonal gammopathy of undetermined significance of the immunoglobulin M class (IgM-MGUS) to Waldenstrom Macroglobulinemia (WM) are mostly unknown. We implemented comparative proteomics and metabolomics analyses on patient serum samples to identify differentially expressed molecules crucial to the progression from IgM-MGUS to WM. Our data identified altered lipid metabolism as a discriminating factor between MGUS, WM, and matched normal controls. Levels of many fatty acids, including polyunsaturated fatty acids and dicarboxylic acids, were significantly downregulated in WM sera when compared to MGUS. These reductions were associated with diminished 15-LOX and PPAR protein expression and increased 5-LOX and GPX4 expression in WM versus MGUS patients’ samples. Furthermore, WM serum samples showed increased lipid peroxidation compared to MGUS. Treatment with IL-6 or TNFα, upstream regulators of differentially expressed proteins between MGUS and WM, increased lipid absorption and lipid peroxidation in WM cell lines. Knock-down of 15-LOX expression increased WM cell survival, an effect accompanied by increased 5-LOX and GPX4 expression. In summary, our data show that reduced fatty acid and lipid metabolite levels in the serum of the WM patients are associated with increased lipid peroxidation and that downregulation of 15-LOX increases the survival of WM cells. These data are highly significant in identifying the biomarkers of disease progression and designing targeted therapeutic intervention.

Targeting of CXCR4 by the Naturally Occurring CXCR4 Antagonist EPI-X4 in Waldenström's Macroglobulinemia.

CXCR4 expression and downstream signaling have been identified as key factors in malignant hematopoiesis. Thus, up to 40% of all patients with Waldenström's macroglobulinemia (WM) carry an activating mutation of CXCR4 that leads to a more aggressive clinical course and inferior outcome upon treatment with the Bruton's tyrosine kinase inhibitor ibrutinib. Nevertheless, little is known about physiological mechanisms counteracting CXCR4 signaling in hematopoietic neoplasms. Recently, the endogenous human peptide EPI-X4 was identified as a natural CXCR4 antagonist that effectively blocks CXCL12-mediated receptor internalization and suppresses the migration and invasion of cancer cells towards a CXCL12 gradient. Here, we demonstrate that EPI-X4 efficiently binds to CXCR4 of WM cells and decreases their migration towards CXCL12. The CXCR4 inhibitory activity of EPI-X4 is accompanied by reduced expression of genes involved in MAPK signaling and energy metabolism. Notably, the anti-WM activity of EPI-X4 could be further augmented by the rational design of EPI-X4 derivatives showing higher binding affinity to CXCR4. In summary, these data demonstrate that a naturally occurring anti-CXCR4 peptide is able to interfere with WM cell behaviour, and that optimized derivatives of EPI-X4 may represent a promising approach in suppressing growth promoting CXCR4 signaling in WM.

Epigenetic Targeting of Waldenström Macroglobulinemia Cells with Bet Inhibitors Synergizes with BCl2 or Histone Deacetylase Inhibition

Aim: Waldenström macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by overproduction of monoclonal IgM. To date, there are no therapies that provide a cure for WM patients, and therefore, it is important to explore new therapies. Little is known about the efficiency of epigenetic targeting in WM. Materials & methods: WM cells were treated with BET inhibitors (JQ1 and I-BET-762) and venetoclax, panobinostat or ibrutinib. Results: BET inhibition reduces growth of WM cells, with little effect on survival. This finding was enhanced by combination therapy, with panobinostat (LBH589) showing the highest synergy. Conclusion: Our studies identify BET inhibitors as effective therapy for WM, and these inhibitors can be enhanced in combination with BCL2 or histone deacetylase inhibition.

FGF/FGFR Axis-Blockade Leads to Anti-Tumor Activity in Waldenstrom's Macroglobulinemia By Silencing MYD88

The human fibroblast growth factor/fibroblast growth factor-receptor (FGF/FGFR) axis deregulation is largely involved in supporting the pathogenesis of hematologic malignancies, including Waldenstrom's Macroglobulinemia (WM). Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We investigated the role of FGF/FGFR system blockade in WM by using a pan-FGF trap molecule, NSC12, a small molecule identified using pharmacophore modeling of the interaction of a minimal PTX3-derived FGF-binding pentapeptide with FGF2. By interrogating the transcriptome signature of patients' BM-derived CD19-positive cells (GEO9656, GEO6691), we found a significant enrichment of FGF/FGFR-driven signaling cascades, including PI3K-AKT, MAPK and STAT3 pathways (FDR&amp;lt;0.25; P&amp;lt;0.05); coupled with higher expression of FGFs (FGF2, FGF7, FGF12, FGF18; P&amp;lt;0.05) in WM cells as compared to their normal cellular counterpart. FGFRs are also shown to be overexpressed in WM. We performed transcriptome profiling of NSC12-treated WM cells, confirming the blockade of the FGFR-signaling blockade; and, importantly, we discovered the efficacious silencing of MYD88 and MYD88-dopwnstream target HCK in WM cells. These findings were confirmed at protein level, showing inhibition of MYD88-driven pathways, such as BTK-, and SYK-phosphorylation. As a further demonstration of the functional impact of NSC12-dependent targeting of MYD88, we could confirm the inhibition of both canonical and non-canonical NF-kB in NSC12-treated WM cells, as assessed at nuclear protein level by WB and by using the NFkB activity assay. In addition, the NSC12-dependent inhibition of MYD88 resulted in silencing of the MAPK-ERK signaling cascade, thus leading to NSC12-induced Myc-silencing in WM cells. We next confirmed the efficacy of NSC12 in silencing bone marrow stromal cell (BMSC)-induced FGFR3 phosphorylation; paralleled by inhibition of of pro-survival pathways, including pAKT, the AKT-downstream pGSK3β; p-ERK; and p-STAT3. Functional sequelae of the FGF/FGFR blockade in WM cells were studied, demonstrating inhibition of WM cell growth, induction of apoptosis, enhanced ER stress and initiation of UPR. Of note, anti-WM activity of NSC12 was also documented using primary bone marrow-derived CD19+ cells isolated from patients with WM. In contrast, NSC12 did not show cytotoxicity on PBMC-derived CD19+ cells isolated from healthy donors. The anti-WM activity exerted by NSC12 was confirmed also within the context of the supportive bone marrow milieu, as shown both in vitro and in vivo. BCWM.1, MWCL1 cells were cultured with NSC12 in the presence or absence of primary WM BMSCs: adherence of WM cells to BMSCs triggered a significant increase in the proliferation, which was inhibited by NSC12in a dose-dependent manner (P&amp;lt;0.05). Using a disseminated humanized WM model, BCWM.1 M-Cherry-Luc tumor-bearing mice were treated with either NSC12 or vehicle control. NSC12-treated mice presented with a significant inhibition of WM tumor growth as demonstrated by bioluminescence imaging analysis. NSC12-dependent reduction of WM tumor cell BM infiltration was confirmed by IHC performed on harvested femurs, in comparison to vehicle-treated mice. Importantly, we assessed MYD88-mRNA expression using harvested bone marrow femurs; and found a significant reduction of MYD88- and MYD88-downstream target HCK in NSC12-treated mice was confirmed ex vivo (P&amp;lt;0.05). Overall, our studies are reporting on the use of NSC12, as a novel potential therapeutic strategy to specifically halt the FGF/FGFR axis in WM; and demonstrate how the observed anti-WM activity exerted by NSC12 may be driven, at least in part, by inhibition of MYD88. Disclosures Motta: Roche: Honoraria; Janssen: Honoraria. Rossi:Alexion: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Advisory board; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria; Amgen: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Roccaro:Amgen: Other; AstraZeneca: Research Funding; Celgene: Other; Janssen: Other; Italian Association for Cancer Research (AIRC): Research Funding; Transcan2-ERANET: Research Funding; European Hematology Association: Research Funding.

Discovery of a Selective, Covalent IRAK1 Inhibitor with Antiproliferative Activity in MYD88 Mutated B-Cell Lymphoma.

Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating the innate immune response against foreign pathogens. Additionally, dysregulation of IRAK1 signaling plays a role in neoplastic disorders. For example, IRAK1 was shown to be important for survival and proliferation in many B-cell lymphomas, including Waldenström's macroglobulinemia (WM) and ABC subtype Diffused Large B-cell Lymphoma (DLBCL) cells. Here, we report the discovery of a highly potent and selective covalent inhibitor of IRAK1, JH-X-119-01. Intact protein MS labeling studies confirmed that JH-X-119-01 irreversibly labels IRAK1 at C302. This compound exhibited cytotoxic activity at single digit micromolar concentrations in a panel of WM, DLBCL, and lymphoma cell lines expressing MYD88. Cotreatment of JH-X-119-01 with the BTK inhibitor ibrutinib resulted in synergistic killing effects in these systems. Taken together, JH-X-119-01 represents a highly selective probe of IRAK1 for further development.

Increased glutathione utilization augments tumor cell proliferation in Waldenstrom Macroglobulinemia

Metabolic reprogramming is a hallmark of cancer cells. In Waldenstrom Macroglobulinemia (WM), the infiltration of IgM-secreting lymphoplamacytic cells into the bone marrow (BM) could shift the homeostasis of proteins and metabolites towards a permissive niche for tumor growth. Here, we investigated whether alerted metabolic pathways contribute to the pathobiology of WM and whether the cytokine composition of the BM promotes such changes.Metabolomics analysis on WM patients and normal donors’ serum samples revealed a total of 75 metabolites that were significantly altered between two groups. While these metabolites belonged to amino acids, glucose, glutathione and lipid metabolism pathways, the highest number of the differentially expressed metabolites belonged to glutathione metabolism. Proteomics analysis and immunohistochemical staining both confirmed the increased protein levels mediating glutathione metabolism, including GCLC, MT1X, QPCT and GPX3. Moreover, treatment with IL-6 and IL-21, cytokines that induce WM cell proliferation and IgM secretion, increased gene expression of the amino acid transporters mediating glutathione metabolism, including ASCT2, SLC7A11 and 4F2HC, indicating that cytokines in the WM BM could modulate glutathione metabolism. Glutathione synthesis inhibition using Buthionine sulphoximine (BSO) significantly reduced WM cells proliferation in vitro, accompanied with decreased NFκB-p65 and MAPK-p38 phosphorylation. Moreover, BSO treatment significantly reduced the tumor growth rate in a WM xenograft model, further highlighting the role of glutathione metabolism in promoting tumor growth and proliferation.In summary, our data highlight a central role for glutathione metabolism in WM pathobiology and indicate that intervening with the metabolic processes could be a potential therapy for WM patients.

The BLIMP1-EZH2 nexus in a non-Hodgkin lymphoma.

Waldenström's macroglobulinemia (WM) is a non-Hodgkin lymphoma, resulting in antibody-secreting lymphoplasmacytic cells in the bone marrow and pathologies resulting from high levels of monoclonal immunoglobulin M (IgM) in the blood. Despite the key role for BLIMP1 in plasma cell maturation and antibody secretion, its potential effect on WM cell biology has not yet been explored. Here we provide evidence of a crucial role for BLIMP1 in the survival of cells from WM cell line models and further demonstrate that BLIMP1 is necessary for the expression of the histone methyltransferase EZH2 in both WM and multiple myeloma cell lines. The effect of BLIMP1 on EZH2 levels is post-translational, at least partially through the regulation of proteasomal targeting of EZH2. Chromatin immunoprecipitation analysis and transcriptome profiling suggest that the two factors co-operate in regulating genes involved in cancer cell immune evasion. Co-cultures of natural killer cells and cells from a WM cell line further suggest that both factors participate in immune evasion by promoting escape from natural killer cell-mediated cytotoxicity. Together, the interplay of BLIMP1 and EZH2 plays a vital role in promoting the survival of WM cell lines, suggesting a role for the two factors in Waldenström's macroglobulinaemia.

CXCR4 mutational status does not impact outcomes in patients with Waldenström macroglobulinemia treated with proteasome inhibitors.

CXCR4 mutational status does not impact outcomes in patients with Waldenström macroglobulinemia treated with proteasome inhibitors.

Overcoming the Supportive Stroma-Induced Proliferation in Waldenstrom's Macroglobulinemia By Selective Inhibition of the FGF/FGF-Receptor Axis

The human fibroblast growth factor receptor (FGF-R) family plays an essential role in a wide range of cellular processes, such as cell growth, proliferation, differentiation, migration and survival. It has been reported that FGF-Rs are expressed in hematopoietic cells; and FGF/FGFR signaling deregulation is largely involved in hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb due to disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGF-R system in FGF-dependent WM cell lines by using an anti-pan FGF trap molecule (NSC12), responsible for FGF/FGF-R blocking. We first interrogated the GSE9656 dataset in order to confirm the expression of FGFs and FGF-Rs in WM cells, demonstrating an enrichment of several FGF- and FGF-R-isoforms in primary WM patients' derived tumor cells compared to the normal cellular counterpart (P&lt;0.05); and demonstrated the ability of NSC12 to inhibit FGF-secretion within the conditioned media of NCS12-treated WM cells, as shown by ELISA. Wide-transcriptome profiling of NSC12-treated WM cells (BCWM.1; MWCL1) revealed a significant inhibition of Myc-target related genes, coupled with silencing of genes involved in cell cycle progression, cell proliferation, PI3K-AKT-mTOR signaling, oxidative phosphorylation (Hallmark; FDR&lt;0.25; P&lt;0.05). This prompted us to evaluate the anti-tumor functional sequelae exerted by NSC12 in WM cells: NSC12 induced significant inhibition of WM cell growth (BCWM1 and WMCL1) in a dose-dependent fashion (0.1-10μM; IC50 ~3μM), even in the presence of bone marrow microenvironment. In addition, a significant effect was also observed in primary tumor cells from WM patients; while no effect was observed on healthy donor-derived peripheral blood mononuclear cells. The growth inhibitory effect was associated with induction of apoptotic cell death, caspase activation and PARP cleavage, as demonstrated by flow cytometry and western blot, respectively. Moreover, we also observed a NSC12 dose-dependent increase of mitochondrial reactive oxigen species (ROS), at protein level. Cell cycle analysis revealed a reduction of the S-phase and increase of G0/G1 phase. Mechanistically, NSC12 targeted WM cells by inhibiting MAPK, JAK/STAT3 and PI3K-Akt pathways known to be FGFRs-activated signaling cascades. Importantly, the same effect was maintained in WM cells even in the presence of the supporting BM microenvironment. Functional studies demonstrated the ability of NSC12 to impair the adhesion of both cell lines to the supportive primary bone marrow stromal cells, in vitro. NCS12-dependnet anti-WM activity was also tested in combination with bortezomib, carfilzomib, everolimus and ibrutinib: the combinatory treatment (48h) resulted in a more significant dose-dependent inhibition of WM cell survival and proliferation (P&lt;0.05), thus suggesting the rational for combining FGF-blockade with proteasome-, mTOR-, or BTK-inhibitors. In vivo studies are being performed, in order to further corroborate the anti-WM activity of NSC12 using WM animal models. Disclosures Ronca: Associazione Italiana per la Ricerca sul Canctro (AIRC): Research Funding. Rossi:Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Roccaro:AstraZeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; European Hematology Association: Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding.

MLL1 Modulates IgM and Inflammatory Cytokines in Waldenstrom's Macroglobulinemia

Waldenström macroglobulinemia (WM) is a low-grade B-cell lymphoma associated with the accumulation of lymphoplasmacytic cells in the bone marrow (BM) and a high level of monoclonal IgM protein in the serum. Genome-wide studies revealed genomic alternations in WM, with the most prevalent being a point mutation in the myeloid differentiation primary response gene 88 (MyD88) resulting in an amino acid substitution (L265P) that is present in over 90% of WM patients. MyD88 is an effector of Toll-like receptor (TLR) signaling pathway that mediates inflammatory cytokine expression and secretion upon activation. Here, we found that activation of the TLR-MyD88 pathway in WM cells carrying the MyD88 L265P somatic mutation increases the expression of IL-6 and CCL2, two cytokines known to participate in the pathobiology of several neoplasms including B cell malignancies. Furthermore, IL-6 has been shown to modulate IgM secretion and malignant B cell growth in WM. We demonstrate that an active ERK1/2 pathway, which is elevated in WM patient samples, is required to maintain IL-6 and CCL2 expression in BCWM.1 (p=0.0339) and RPCI-WM1 (p=0.0005) cells. Further characterization identified a change in the chromatin landscape in response to TLR-MyD88 activation in WM cells. We found increased levels of trimethylation of histone 3 lysine 4 (H3K4me3) at the promoters of the inflammatory cytokines IL-6 and CCL2 by chromatin immunoprecipitation (ChIP) assay followed by qPCR (ChIP-qPCR). The H3K4me3 modification is catalyzed by six related homologs of the yeast histone methyltransferase (HMT) family. Further analysis identified the Mixed-lineage leukemia 1 (MLL1) as the enzyme bound to these promoters in response to TLR-MyD88 stimulation of WM cells. Analysis of WM cell lines and primary WM patient cells showed that MLL1 and its binding partner, menin, are expressed at significantly higher levels in CD19+CD138+cells from WM patients compared to CD19+B cells from peripheral blood of healthy donors (p&lt;0.001). We also found an increase in H3K4me3 deposition on IL-6 and CCL2 promoters during early (1-3 hr) and late (12-24 hr) kinetics following TLR-MyD88 stimulation. This also coincided with increased deposition of MLL1 on these cytokine promoters by ChIP-qPCR. Disruption of menin-MLL1 using the MI-2 or MI-503 menin-MLL1 inhibitors significantly reduced IL-6 and CCL2 expression in WM cell lines (BCWM.1 p=0.039; RPCI-WM1 p=0.0041). This also results in significantly reduced IgM expression (p&lt;0.0001) and secretion at inhibitor dose (5 μM) that has no effect on cell proliferation. Finally, MLL1 knockdown using RNAi significantly reduces IgM expression in BCWM.1 (p&lt;0.0001), MWCL (p=0.0016) and RPCI-WM1 (p&lt;0.0001) and IgM secretion in BCWM.1 (p&lt;0.0001) and MWCL-1 (p&lt;0.0001). Taken together, these results identify a novel role for menin-MLL1 in regulating inflammatory cytokines and IgM expression and secretion in WM and provide the rationale for targeting these molecules in WM patients. Disclosures Ansell: Mayo Clinic Rochester: Employment; Mayo Clinic Rochester: Employment; Regeneron: Research Funding; Seattle Genetics: Research Funding; Trillium: Research Funding; Seattle Genetics: Research Funding; Regeneron: Research Funding; Trillium: Research Funding; Bristol-Myers Squibb: Research Funding; Mayo Clinic Rochester: Employment; Trillium: Research Funding; Affimed: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Trillium: Research Funding; Mayo Clinic Rochester: Employment; Bristol-Myers Squibb: Research Funding; Mayo Clinic Rochester: Employment; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; LAM Therapeutics: Research Funding; LAM Therapeutics: Research Funding.