WM Cells Research Articles

Comparative Analysis on the Anti-Tumor Activity of Venetoclax and Obinutuzumab (VO) Versus Venetoclax and Rituximab (VR) in Primary CLL Cells, Ex Vivo

INTRODUCTION: Treatment of chronic lymphocytic leukemia (CLL) has expanded significantly with the approval of multiple small molecule inhibitors. This is of great significance for patients with adverse cytogenetic features who tend to respond poorly to standard chemo-immunotherapy (CIT). While single agent ibrutinib and venetoclax (V) have shown high rates of overall response, complete remission and minimal residual disease (MRD) eradication rates remain low. This argues for further testing and development of various combination strategies. The MURANO trial compared venetoclax and rituximab (VR) versus bendamustine and rituximab (BR) in patients with relapsed/refractory CLL reporting clear superiority of VR over BR. MRD rates in the bone marrow were reported to be 27.3% for VR versus 1.5% for BR. Given much higher rates of MRD eradication with combination of small molecule inhibitors and monoclonal antibodies (mAb) compared to standard CIT, we performed a comparative investigation into the direct and immune-mediated cytolytic effects of VR versus V + Obinutuzumab (O, type II anti-CD20 mAb) in primary CLL cells and B-lymphoid cell lines.METHODS: CD19+ B-cells were isolated from PBMCs of CLL patients (N=3). For all experiments using primary CLL cells, concentration of VR and VO was 3nM (V) and 10ug /ml (R, O), respectively. For cell lines, VR and VO was used at 5uM (V) and 10ug /ml (R, O), respectively. Apoptosis was determined by annexin-V/PI staining followed by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by VR and VO was assessed in Calcein AM labeled CLL cells or cell lines co-cultured with healthy donor PBMCs (E:T ratio, 40:1); complement-dependent cytotoxicity (CDC) was measured using 10% serum from a healthy human donor.RESULTS: We assessed the ability of V+/-O or V+/-R to induce apoptotic cell death in the CD20+ BCWM.1 cell line (Waldenström's macroglobulinemia [WM] phenotype) and the MEC-1 cell line (B-PLL phenotype); with CD20- RPCI-WM1 (WM cells, negative control). Notably, Bcl-2 protein is expressed in all the aforementioned cell lines. We observed that single agent V, O and R induced ~30%, 61% and 13.64% annexin V/PI positivity in BCWM.1 cells, respectively. However, a significant degree of cell death was noted in VO-treated cells (~74%) compared to VR-treated cells (~40%) (p<0.01). Next, we examined for apoptosis in MEC1 cells and noted a similar trend; where the VO combination induced markedly more cell death (~71%) than VR (~57%). Contrastingly, in RPCI-WM1 cells neither single agent O or R could elicit >12% annexin V/PI positivity and where the addition of V increased apoptosis by only 3 - 4%. We also examined the apoptotic potential of VO or VR in tumor cells from low, intermediate and high-risk CLL patients. In low and intermediate-risk CLL cells from low and intermediate-risk patients, V alone induced ~30% cell death, which increased significantly with the addition of O (VO) to between 48 - 52%. Contrastingly, the combination of VR did not induce more than 29 - 32% apoptosis. In CLL cells from high-risk patient, we noted that exposure to single agent V induced ~ 28% cell death and in VO-treated cells, this number increased to 47%. We also examined for ADCC and CDC in the same cell lines and primary CLL cells. Despite considerable variability, single agent O and VO treatment of tumor cells resulted in greater ADCC than VR treatment. By contrast, in single agent R or VR-treated cells, more CDC was observed.CONCLUSION: Our preliminary investigation in VR- and VO-treated cell lines and primary CLL cells suggests the VO combination may be superior to VR in induction of direct tumor cell death. Mechanistic experiments underway will provide further insight and can aid in design of future VO-based clinical studies in CLL. DisclosuresAilawadhi:Pharmacyclics: Research Funding; Takeda: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy.

BTKCys481Ser Mutation Drives Ibrutinib Resistance through ERK1/2 Hyperactivation, and Can Confer a Protective Effect on Bystander Waldenstrom's Macroglobulinemia and ABC DLBCL Cells through Paracrine Mediated Pro-Survival Signaling

▪Activating mutations in MYD88 promote NF-kB survival signaling in WM and ABC DLBCL cells through BTK and HCK, both targets of ibrutinib (Yang et al, Blood 2013; 2016). These findings likely explain the high rates of response observed in MYD88 mutated WM and ABC DLBCL patients (Treon et al, NEJM 2015; Wilson et al, Nature Med 2015). Resistance to ibrutinib is increasingly being recognized with prolonged therapy in patients with various B-cell malignancies. Mutations at the BTKCys481 site are observed in half of WM patients with disease progression following initial response (Xu et al, Blood 2017), and are also associated with clinical resistance in patients with CLL and MCL. However, in many WM and CLL cases, BTKCys481 mutations are subclonal and their relevance to clinical disease progression remains unclear. Moreover, the signaling mechanisms that promote ibrutinib resistance remain to be clarified. We therefore performed lentiviral transduction studies in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1) cells with vector alone, wild-type BTK (BTKWT), and the BTK variant most frequently observed in ibrutinib resistant WM and CLL cases (BTKCys481Ser). BTKCys481Ser but not BTKWT or vector only transduced WM and ABC DLBCL cells uniformly demonstrated persistent BTK and PLCγ2 activation in the presence of ibrutinib (100, 500 nM), along with a 1-3 log fold increase in EC50 to ibrutinib. Western blot analysis showed persistent BTK signaling in BTKCys481Ser expressing cells that was accompanied by ERK1/2 hyperactivation. Use of highly selective and potent ERK1/2 inhibitors (ulixertinib, GDC-0994) induced apoptosis of BTKCys481Ser expressing WM and ABC DLBCL cells, and showed synergistic tumor cell killing with ibrutinib by CellTiter-Glo® Luminescent Cell Viability Assays using Calcasyn, as well as enhanced apoptosis by Annexin V staining. Using a multiplex cytokine assay and confirmed by TaqMan® quantitative RT-PCR assay for cytokine mRNA levels, we identified that ERK1/2 activation in ibrutinib treated BTKCys481Ser WM and ABC DLBCL cells was accompanied by persistent release of pro-inflammatory and growth enhancing cytokines including CXCL13, IL-2R, IL-6, IL-10, IL-12p40, and TNF-a that was blocked by ulixertinib. In contrast, ibrutinib blocked their release in BTKWT WM and ABC DLBCL cells. We next sought to clarify if cytokine release by ibrutinib treated BTKCys481Ser WM and ABC DLBCL could impact survival of bystander tumor cells. We performed experiments using a Transwell system in which BTKCys481Ser or BTKWT transduced WM or ABC DLBCL cells were co-cultured with their native counterpart cells in the presence or absence of ibrutinib (Figures 1A, B). These studies showed that native WM or ABC DLBCL cells were rescued in the presence of ibrutinib when co-cultured with their BTKCys481Ser but not BTKWT transduced counterparts. Finally, use of IL-6 and IL-10 blocking antibodies abolished the protective effect on native tumor cells conferred by co-culture with BTKCys481Ser expressing cells in the presence of ibrutinib (Figures 1C, D).Our findings show that BTKCys481Ser mutation drives ibrutinib resistance through hyperactivation of ERK1/2, and that paracrine mediated ERK1/2 dependent pro-survival signaling by BTKCys481Ser expressing cells can confer a protective effect on bystander Waldenstrom's Macroglobulinemia and ABC DLBCL cells in the presence of ibrutinib. [Display omitted] DisclosuresCastillo:Janssen: Consultancy, Research Funding; Abbvie: Research Funding; Pharmacyclics: Consultancy, Research Funding; Millennium: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

A Novel, Highly Selective IRAK1 Inhibitor Jh-X-119-01 Shows Synergistic Tumor Cell Killing with Ibrutinib in MYD88 Mutated B-Cell Lymphoma Cells

▪Activating mutations in MYD88 are present in many B-cell lymphoproliferative disorders including WM (95%), ABC DLBCL (39%), Primary CNS Lymphoma (80-90%), Marginal Zone Lymphoma (6-10%) and Chronic Lymphocytic Leukemia (4-8%). In MYD88 mutated WM and ABC DLBCL cells, MYD88 triggers NF-kB pro-growth and survival signaling through divergent pathways driven by BTK and IRAK4/IRAK1 (Ngo et al, Nature 2011; Yang et al, Blood 2013). Ibrutinib targets BTK, and has shown high levels of activity in MYD88 mutated WM, ABC DLBCL and PCNSL. Responses however are partial, and complete remissions are lacking. We therefore sought to clarify if persistent IRAK4/IRAK1 signaling was responsible for survival of malignant lymphoplasmacytic cells (LPC) in WM patients on ibrutinib. We observed by flow cytometric analysis that while BTK activation was suppressed in WM patients on ibrutinib for >6 months, both IRAK4 and IRAK 1 remained hyperactivated. Treatment of LPC from patients on ibrutinib with a toolbox IRAK4/IRAK1 inhibitor resulted in marked reduction of NF-kB and induction of apoptosis. Subsequently, we performed lentiviral transduction studies in MYD88 mutated BCWM.1 cells, and observed in an inducible model system that knockdown of IRAK1 produced more robust apoptotic effects versus IRAK4 (Figure 1A). Given these findings, we have pursued a medical chemistry campaign to develop a potent and highly selective inhibitor of IRAK1 kinase activity, JH-X-119-01. JH-X-119-01 inhibited IRAK1 biochemically with an IC50 of 9.3 nM while exhibiting no inhibition of IRAK4 at concentrations up to 10 µM, and showed exceptional Kinome selectivity with off-target inhibition of only two kinases, YSK4 and MEK3 (Figure 1B). Mass spec labelling studies were used to confirm that JH-X-119-01 irreversibly labelled IRAK1 at cysteine 302. JH-X-119-01 showed antiproliferative effects on MYD88 mutated WM and ABC DLBCL cells. Importantly, the combination of JH-X-119-01 with Ibrutinib led to synergistic tumor cell killing in MYD88 mutated WM and ABC-DLBCL cells, and suppression of NF-ĸB activation (Figure 1C). In vivo PK studies revealed a favorable profile for JH-X-119-01 with a moderate half-life of 1.61 hours, a Cmax of 9.95 µM, and a low clearance of 18.84 mL/min/kg when dosed IV. Our findings evidence the development of a novel, highly selective IRAK1 kinase inhibitor, JH-X-119-01, that shows specific inhibition of IRAK1 kinase activity and a reduction of tumor cell survival in MYD88 mutated WM. Importantly, JH-X-119-01 shows synergistic tumor cell killing with ibrutinib in MYD88 mutated WM and ABC-DLBCL cells. This study provides a framework for the development of highly selective IRAK1 inhibitors for use alone, and in combination with ibrutinib in MYD88 mutated B-cell lymphomas. [Display omitted] DisclosuresCastillo:Janssen: Consultancy, Research Funding; Abbvie: Research Funding; Millennium: Research Funding; Pharmacyclics: Consultancy, Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

Abstract 4823: Characterization of newly established WM-1-SNU cell line harboring negative for MYD88 L265P derived from Waldenstrom macroglobulinemia patient's peripheral blood using xenograft model

Abstract Waldenstrom macroglobulinemia (WM) is a rare hematologic malignancy that is a lymphoplasmacytic lymphoma a type of B-cell disease. The common characteristics of WM cells are expression of B cell markers, CD19 and CD20 and secretion of monoclonal immunoglobulin (Ig) M. The other critical feature of WM is MYD88 L265P that is found almost 90% patients. MYD88 gene is related with immune response and inflammatory signaling. Many researches are underway about MYD88 L265P role in WM. However, there is a lack of functional studies in the negative for MYD88 L265P in WM due to the absence of the MYD88 L265P negative cell line. Each cell lines represent the heterogeneity and molecular diversity by genetic features, it is important to establish the MYD88 L265P negative WM cell line for elucidating the biological characteristics of WM. Mononuclear cells obtained from patient’s peripheral blood by ficoll separation and were injected to NRG (JAX, USA) mouse via tail vein. After 24 days, mouse sacrificed and extracted bone marrow (BM) cells from mouse tibia by syringe flushing. Mouse BM cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 20% heat-inactivated FBS, Penicillin-Streptomycin (10,000 U/mL) (Gibco, USA). G-banding was used to confirm the human chromosomes. Sanger sequencing conducted for detecting the MYD88 L265P mutation. Flow cytometry was used for screening cell surface markers and ELISA was used for the detection of immunoglobulin level. For confirm the tumorigenicity of the cell line, we re-injected the cell line to NSG (JAX, USA) mouse by subcutaneously. After 3 weeks, tumor mass and metastatic tissues were cultured in conditioned medium. Tumor cells have had adherent phenotype and human chromosomes were identified by G-banding. MYD88 L265P mutation that genetic signature of WM was not detected both patient’s peripheral blood and established cell line. CD19 and CD20 expressions were not detected, but CD138 highly expression was confirmed about 90%. Lambda and Kappa secretion were not detected in cell culture supernatant. We confirmed the tumorigenicity through re-injection the cell line by subcutaneously. Tumor mass was formed with a 100% probability and metastasis to lung and bone marrow occurred in about 66%. Tumor mass and metastatic tissues were cultured in in vitro and confirmed the reproducibility. In this study, we describe the newly established human WM cell line derived from peripheral blood of the WM patient who negative for MYD88 L265P using xenograft model having unique biological characteristics comparing to MYD88 L265P positive cells that high tumor formation ability and high expression of CD138 and does not secrete IgM. Citation Format: Hyejoo Park, Youngil Koh, Kwang-Sung Ahn, Hyo Jung Kim, Sung-Soo Yoon. Characterization of newly established WM-1-SNU cell line harboring negative for MYD88 L265P derived from Waldenstrom macroglobulinemia patient's peripheral blood using xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4823. doi:10.1158/1538-7445.AM2017-4823

Drug Resistance Alters CD38 Expression and in Vitro Response to Daratumumab in Waldenstrom Macroglobulinemia Cells

Introduction: With approval of Daratumumab (Dara), targeting CD38 has become an attractive therapeutic option for various B cell cancers including WM. Using preclinical models of WM we investigated CD38 expression patterns, the impact of drug-resistance and the anti-WM effects of Dara, in vitro. An important aspect of our evaluation was the identification of optimal CD38 expression that correlates with maximal cytotoxic effects of Dara; such an approach has not been attempted previously in WM.Methods: WM cell lines (BCWM.1, MWCL.1 and RPCI.WM1) with isogenic proteasome inhibitor-resistant (CR, resistant to carfilzomib, bortezomib and ixazomib) and ibrutinib-resistant subclones (IR) were used. CD38 expression was measured by flow cytometry and a grading system was developed based on increasing CD38 MFI values with 1+= <50,000, 2+ = >50,000 and 3+ = >100,000. Daratumumab-induced specific lysis due to antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) was measured using the Calcein AM assay at an effector to target ratio (E:T) of 100:1. Peripheral blood mononuclear cells (PBMCs) and complement-containing serum were obtained from healthy human donors. Daratumumab, carfilzomib and ibrutinib were obtained from commercial sources.Results: Expression of CD38 (MFI) was highest in RPCI-WM1 (91295.6) > BCWM.1 (13865.3) > MWCL-1 (9804.19) (Figure 1A.). Intriguingly we observed an interesting change in CD38 expression with induction of drug resistance; all CR clones showed a decrease in MFI (RPCI-WM1/CR; øMFI 55960.5 > MWCL-1/CR; øMFI 3890.0 > BCWM.1/CR; øMFI 1381), while decreased expression was noted in all IR clones except one (MWCL-1/IR; øMFI 6272.96, BCWM.1/IR; øMFI 3076 vs. RPCI-WM1/IR; _MFI 461784.6) (Figure 1B). When we applied our CD38 expression grading system, RPCI-WM1/IR cells classified as Grade 3+, RPCI-WM1 and its CR clones as Grade 2+, while all other WM cells were Grade 1+. We noted that utilization of this grading system was helpful in correlating the extent of cytotoxicity to the level of CD38 expression (Table 1). We next treated WM cells with Dara and observed direct lysis in only RPCI-WM1/CR (43%) > BCWM.1/CR (19%) and RPCI-WM1 (11%) cells. However, when Dara-treated WM cells were cocultured with PBMCs, significantly increased ADCC was noted in RPCI-WM1 (90.3%), RPCI-WM1/IR (88.3%) and RPCI-WM1/CR (68.0%) cells (Figure 1C). Although CDC has been reported as a mechanism of mAb-mediated cell death in WM our evaluation of CDC as a potential effector mechanism in daratumumab-treated WM was negative as expected, highlighting ADCC rather than CDC as the primary cytotoxic mechanism.Conclusion: Our analysis of CD38 in WM preclinical models highlights several important findings: 1.) Expression of CD38 changes as WM tumor clones evolve with induction of drug-resistance; an effect that may also be cell-type specific as evidenced by increased CD38 expression in RPCI-WM1/IR cells, 2.) Daratumumab can exert a direct lethal effect in WM cells but this effect is variable and cell line dependent, 3.) Expression of CD38 is an important factor directing cytotoxicity of Dara however antigen density appears to be an important factor and a grading system can be beneficial for future preclinical and/or clinical studies, 4.) ADCC and not CDC appear to be the prominent mechanism of action and 5.) ADCC-mediated cytolytic activity of Dara is contingent on CD38 expression as evidenced by the most specific lysis being noted in CD38 Grade 2+ and 3+ expressing WM cells (Table 1.). These observations can help direct optimal design of clinical studies with Dara in WM where drug resistance and CD38 expression should be further explored as factors contributing to the clinical efficacy of this new agent. [Display omitted] [Display omitted] DisclosuresAilawadhi:Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding.

Waldenstrom Macroglobulinemia Cells Modulate Mitochondrial Bioenergetics and Induce a Respiratory Hyper-Drive State upon Acquisition of Ibrutinib-Resistance

Background: Mitochondrial activity is critical to maintain a high cellular proliferation especially in drug-resistant clones. Ibrutinib-resistance (IR) in CLL is associated with highly aggressive clinical behavior, carrying a median survival of ~3 months. While these outcomes are not yet reported in WM, resistance to ibrutinib is foreseeable considering no WM patients achieve a complete remission with BTK inhibitors (reported series). The transition to a highly proliferative drive in the IR clone remains a biological dilemma and understanding this will allow rationale development of therapeutic strategies. It is established that increased cell proliferation is highly dependent on the functional capabilities of the mitochondria. Yet the respiratory proficiencies and adaptation of mitochondrial machinery in the IR state remains unknown. Here we interrogated mitochondrial function and its relevance in IR cells using our WM models.Materials: WM cell lines (BCWM.1, non-IR) and its isogenic IR clone (BCWM.1/IR; 20-fold resistant, without BTK or CXCR4WHIM-like mutations) were used. Mitochondrial respirometry was measured by Seahorse XF96 flux analyzer (Fig. 1a). Drug sensitivity and cytotoxicity were determined by Cell-Titer Glo cell viability assay. Whole exome sequencing (WES) was performed on an Illumina HiSeq2000.Results: IR WM cells were noted to be in G1 arrest with altered morphologic features and growth kinetics. Despite growth arrest, IR cells showed increased basal oxygen consumption rate (OCR) (>30% increase) vs. parental (non-IR) cells. Consistent with this we observed enhanced mitochondrial ATP production suggesting increased mitochondrial utilization by the IR clones. We then measured maximal respiratory potential of WM cells with FCCP pre-treatment, which evaluates cellular potential to respond to increased ATP demand. We observed upregulation of mitochondrial spare respiratory capacity in BCWM.1/IR cells vs. parental BCWM.1 cell. In addition, a >50% increase in non-mitochondrial oxygen consumption/respiration was also noted (Fig.1b, c). To identify potential genomic changes that could explain mitochondrial overdrive, WES of the non-IR and IR cells was performed. Interestingly, this analysis did not uncover any mutational alterations in genes directing cellular oxidative phosphorylation (NDUF, COX1, COQ7, SURF1, etc.), glycolysis (PDK1, HK2, PKM2, ALDOA, etc.) or ATP production (ATP5, ND1, UCP1 and others). We then asked if mitochondrial function impacts therapeutic potential of a specific drug (or a class of drug), focusing specifically on proteasome disruption. First, we observed that proteasome inhibition- independent of the site (20S vs. 19S), retained high cytotoxic potential, wherein IR cells showed exquisite sensitivity to proteasome disruption. Interestingly, VLX1570 (targets 19S proteasome) had a 2-fold lower IC50 in IR clones than its non-IR counterpart. Cell respirometry demonstrated that compared to parental cells, VLX1570 (500nM) treatment significantly (p<0.002) and rapidly (2hr) attenuated basal respiration in IR clones at concentrations half of those needed to achieve similar effects in BCWM.1 cells. A concomitant decrease in ATP production as well as spare respiratory capacity was observed at these time points/drug concentrations (significantly more evident than in BCWM.1 cells). While our focus was mitochondrial bioenergetics and its therapeutic relevance in drug-resistant WM, unexpectedly we observed for the first time, evidence that VLX1570 induces changes in mitochondrial bioenergetics as early as 2hrs leading to increased mitochondrial membrane permeability and cell apoptosis in 6-12hrs. Comparable observations were made with bortezomib (targeting 20S).Conclusions: This is the first report identifying the role of mitochondrial hyper-drive in WM cells with resistance to ibrutinib. Of note, enhancedmitochondrial bioenergetics in the IR state increased sensitivity to proteasome inhibition and this was independent of the site of disruption in the proteasome pathway. Although, WES findings alone were not sufficient in explaining mitochondrial overdrive noted in our system, ongoing analysis of various mitochondrial functions (Fig. 1d) at the gene expression, copy number and epigenetic level may elucidate the underlying biomechanics of mitochondrial disposition in IR WM cells. [Display omitted] DisclosuresAnsell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Linder:Vivolux, Ab: Other: Shareholder. Ailawadhi:Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy.

Whole Genome and Transcriptome Analysis of Waldenstrom's Cell Lines Show Preservation of Many Important Genomic Features of Primary Disease, and Identify a Non-L265P Mutation in MYD88

Background: WM cell lines represent a vital tool for discovery of mechanisms responsible for WM pathogenesis, cell signaling, development of targeted therapeutics, and evaluation of mechanisms responsible for drug resistance. To date, there has been no comprehensive genomic and transcriptomic profiling of WM cell lines using next generation sequencing.Methods: We performed whole genome (WGS) and transcriptome sequencing (WTS) on three established (BCWM.1, MWCL-1, RPCI-WM1), and one novel (BCWM.2) cell line established from continuous culture of CD19-selected bone marrow cells from a WM patient with an immunophenotype consistent with primary WM disease (CD5-, CD10-, CD19+, CD20+, sIgM+, λ+, CD11c-, CD38+, and CD138+). Samples from primary tumor and germline from the founder of BCWM.2, along with tumor samples from 57 WM patients, as well as memory (CD19+ CD27+) and non-memory (CD19+ CD27-) B-cells from 9 healthy donors were also sequenced. WGS was performed using Complete Genomics platform, and WTS using Illumina HiSeq. Reads were aligned to KnownGene HG19/GRCh37 reference using STAR. Read counts per gene were obtained using featureCounts from Rsubread, and analyzed using voom from the edgeR/limma Bioconductor packages in R. Differential isoform expression was assessed using the Cufflinks software suite.Results: WGS revealed no cytogenetic level amplifications or deletions in BCWM.1, whereas MWCL-1 harbored monoallelic deletions in 17p and 17q, and amplifications in 11q. RPCI-WM1 showed deletions in 3p, 6q, 9p, 13q, 18q, 19q and X, with amplifications in 5p, 6p, 7q, 11q, 14q, 18q, 21q, and acquired uniparental disomies on 17p, 18q, and X. BCWM.2 matched its parent tumor profile with trisomy 3 and 12, as well as deletion of the entire arm of 6q and corresponding amplification of 6p. All 4 cell lines were wild type for CXCR4 and all but BCWM.2 harbored the MYD88 L265P mutation. Like its founding clone, BCWM.2 carried an activating heterozygous MYD88 G/A S243N mutation (Nature 470(7332):115-9) previously described in ABC DLBCL. In addition, a novel I318N mutation in LYN that was predicted to be activating was found in BCWM.2, but not in the founder primary tumor or germline samples. While WTS analysis of RPCI-WM1 is pending, principal component analysis showed that BCWM.1, BCWM.2 and MWCL-1 cells clustered with primary WM tumor cells, and were distinct from memory and non-memory B-cell samples from healthy donors. Likewise, multidimensional scaling of pairwise similarity clustered BCWM.2 directly with its corresponding primary sample (Figure 1). BCWM.1, BCWM.2, and MWCL-1 cells showed a high degree of recapitulation with primary MYD88mutated CXCR4wild-type WM cells including IL17RB, CABLES1, WNT5A, GPER, or WNK2 over-expression. As a group, WM cell lines were found to over-express many cancer associated genes, including MUC13, MMP7, LMO3, IL4I1, and several SRC family kinases.Conclusions: This study provides the first ever comprehensive genomic and transcriptomic profiling of WM cell lines by next generation sequencing. The findings show preservation of many important genomic features of primary WM disease, and identified the existence of a non-L265P activating MYD88 mutation, as well as dysregulation of many cancer associated genes in WM. The findings provide an important new resource for the study of WM disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Aurora Kinase Is a Therapeutic Target in Ibrutinib-Resistant Waldenstrom Macroglobulinemia: In-Silico Target Identification and in-Vitro Validation

Background: Current tumor profiling analytics provide some insight into the various molecular abnormalities and their individual consequences on oncogenic signaling. However, these analyses are limited by their lack of integration where the combined effect of individual mutations, gene copy number variations and chromosomal aberrations are not consolidated to create the global molecular architecture that supports neoplastic growth, particularly in the context of drug resistance. Consequentially, identities of the preferential oncogenic pathway(s) tumor cells employ to oppose the effects of targeted therapies remain cryptic and unactionable. Here we present a simulation-based method, which not only replicates the molecular architecture of ibrutinib-resistant Waldenstroms Macroglobulinemia (WM, for which ibrutinib is the only FDA-approved agent) in silico, but also predicts cell sensitivity towards existing drugs, which we validated experimentally for potential clinical translation.Materials: We used the newly established human WM cell line, RPCI-WM1/IR, as a surrogate model of ibrutinib-refractory WM. Genomic data including whole exome sequencing (WES) and copy number analysis (CNA) was utilized for the creation of an avatar of RPCI-WM1/IR, which through simulation identified the salient and prominently dysregulated cellular pathways. Importantly, illustrating these pathways highlights common convergence points on increased proliferation and viability. These convergence points were then directly and indirectly targeted by simulated testing of a library of FDA approved drugs and those impacting these dysregulated pathways were nominated. Importantly, this simulation avatar approach not only looks for agents acting on the specific gene mutation, but also predicts the convergence points to be attacked. The personalized simulation avatar technology is a comprehensive functional proteomics representation of the WM physiology network. A standardized library of equations models all the biological reactions such as enzymatic reactions, allosteric binding and protein modulation by phosphorylation, de-phosphorylation, ubiquitination, acetylation, prenylation and others.Results: Several genomic aberrations were used to create the RPCI-WM1/IR simulation avatar. Functional activity (based on mutation or copy number alteration) of several ibrutinib targets or transcription factors associated with BTK activity such as FYN, SP1, BMX and FRK were predicted to be lost. Increased expression of CAV1, which also inhibits BTK mediated signaling, was increased. An increase in CSNK2B, which activates PU.1- a transcriptional target of BTK, was also observed. Of note, no CXCR4 mutations, which have been shown to impact ibrutinib response, were observed. Next, the cytotoxic potential of over 150 FDA approved drug (and some in experimental stages) were simulated individually and in combination on the RPCI-WM1/IR avatar. In silico modeling predicted aberrant activity of aurora kinase A (AURKA) and its associated signaling partners, which could be disrupted with the (AURKA) inhibitor, tozasertib. AURKA activation was predicted as upregulated due to alterations in several genes: RASA1 loss and SOS1 increase --> increased ERK --> increased ETS1 --> increased AURKA. High beta-catenin signaling (high CTNNB1 and FZD1/4 and low AXIN1 and GSK3B) was also shown to increase AURKA. The simulation predictions were experimentally validated in vitro where AURKA inhibition with tozasertib significantly inhibited proliferation of RPCI-WM1/IR cells (IC50~14nM) as well as inducing apoptosis (48hr, 20nM treatment) and cell-cycle arrest.Conclusions: Our data demonstrates the potential of in silico modeling in predicting novel drug targets, allowing guidance in 1.) Delineating operational oncogenic circuits in an ibrutinib-resistant state by reanimation of the molecular architecture in silico, 2.) Calculating the impact of individual genomic abnormalities and their collective influence on maintaining tumor survival and 3.) Performing a rapid in-silico drug-sensitivity screen directed by the pathway analyses, which can be validated experimentally using standard assays. This novel approach holds tremendous potential in creating highly personalized therapies for ibrutinib-refractory WM patients based on unique genetic signatures. DisclosuresVali:Cellworks Group, Inc.: Employment, Equity Ownership. Kumar:Cellworks Group, Inc.: Employment. Singh:Cellworks Group, Inc.: Employment. Abbasi:Cellworks Group, Inc.: Employment, Equity Ownership.

A Systemic Xenograft Model of Waldenström’s Macroglobulinemia Demonstrates the Potent Anti-Tumor Effect of Second Generation CD19 Directed Chimeric Antigen Receptor Modified T Cells in This Disease

Chimeric antigen receptor (CAR) modified T-cell therapy consists of ex-vivo genetic manipulation of autologous lymphocytes in order to establish robust T-cell mediated anti-tumor immunity. Our group was the first to design and evaluate a CAR targeted toward the B cell antigen CD19 in mice. Currently we utilize a second generation CAR comprised of a single-chain variable fragment (scFv) derived from an antibody against CD19 fused to the CD3 ζ chain and the CD28 intracellular signaling domain (19-28z) to provide the necessary signal 1 and signal 2 for enhanced T-cell activation and persistence. We have gone on to test the safety and efficacy of 19-28z CAR T-cells in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We observed rapid complete molecular remissions in the first 14/16 patients treated with relapsed/refractory ALL. We hypothesize that contributing to the enhanced efficacy seen in ALL, when compared to solid tumors or extra-medullary CLL, is the fact that ALL is a bone marrow predominant disease, which may provide a microenvironment more amenable to T-cell therapy.Waldenström’s Macroglobulinemia (WM) is an ideal disease to test 19-28z CAR-modified T-cell therapy, as it is often bone marrow predominant, and WM cells from patient samples typically uniformly express high levels of CD19. Furthermore, despite recent progress made with novel BCR-directed therapy, complete eradication of the WM clone from the bone marrow niche remains elusive, therefore providing an ideal clinical scenario for treatment consolidation via alternative cytotoxic methods such as cellular immunotherapy.Using the human WM cell line, BCWM.1, we evaluated the in vitro efficacy of 19-28z CAR-modified T-cells when compared to mock transduced T cells or T cells transduced with an irrelevant second generation CAR directed towards the ovarian antigen MUC16. In a 4 hour co-culture assay, we observed significant cytotoxicity, even at low effector:target ratios (52% lysis at 1:1; 92% lysis at 10:1; p<0.01). This corresponded to increased secretion of INFγ and IL-2, markers of T-cell activation (p<0.01).We then conducted in vivo studies using sublethally irradiated SCID/beige mice to generate a systemic model of WM via tail vein injection of 1x106 luciferase transduced BCWM.1 cells. This model is characterized by tumor growth in the bone marrow followed by rapid spread to the liver, lungs, kidney, and CNS. Mice were monitored by weekly bioluminescent imaging (BLI) and ultimately were sacrificed when they developed hind leg paralysis. 19-28z CAR modified T-cells administered at day 7, after tumor establishment, when compared to non-treated and irrelevant CAR-modified T cell controls, delayed the progression of disease and doubled the median survival time of the mice after treatment (p=0.001).Taken together, the pre-clinical efficacy demonstrated in this abstract and the clinical features of WM, listed above, provide the rational for testing 19-28z CAR modified T cells clinically for WM. We have now opened a clinical trial for patients with relapsed or refractory WM, in which chemotherapy preconditioning is followed by a single dose of 19-28z CAR modified autologous T-cells (NCT00466531). DisclosuresBrentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

A Novel and Personalized Method Using Simulation for Predicting Effective Therapeutics for Waldenströms Macroglobulinemia

Background: The concept of treatment personalization in cancer often means associating a drug with an actionable mutation. One limitation of this approach is that most tumors have multiple aberrations, which are not actionable. Here we present a predictive simulation based approach which models the molecular architecture and resulting physiology of advanced stage Waldenströms Macroglobulinemia (WM, a B-lymphoplasmacytic lymphoma) as well as the design of novel personalized treatments using existing drug agents for rapid clinical translation with validations.Methods: We used the human WM cell line, RPCI-WM1, as a surrogate model of refractory, advanced patient sub-groups. Publically available as well as proprietary genomic and cytogenetic data was utilized for the creation of an avatar of RPCI-WM1, which through simulation identified the salient and prominently dysregulated cellular pathways. Importantly, illustrating these pathways highlights common convergence points on increased proliferation and viability. These convergence points were then directly and indirectly targeted by simulated testing of a library of FDA approved drugs and those impacting these dysregulated pathways were shortlisted. Importantly, this simulation avatar approach not only looks for agents acting on the specific gene mutation, but also predicts the convergence points to be attacked. The personalized simulation avatar technology is a comprehensive functional proteomics representation of WM physiology network. A standardized library of equations models all the biological reactions such as enzymatic reactions, allosteric binding and protein modulation by phosphorylation, de-phosphorylation, ubiquitination, acetylation, prenylation and others. A library of over 150 digital functional library of FDA approved drug agents and those in clinical study has been developed and was simulated individually and in combination on the RPCI-WM1 (advanced stage WM patient) avatar.Results: Overall, there were 272 gene aberrations used to create the RPCI-WM1 simulation avatar. Importantly, presence of MYD88L265P mutation, absence of CXCR4 mutations and additional chromosomal aberrations (derivatives, translocations, deletions and amplifications of chromosomes 3, 6, 9, 13, 18 and 19) were taken into account.The RPCI-WM1 patient avatar predicted increased IRAK1/4 engagement due to MYD88 mutation and high copy number (CN) of IL18, to increased NFkB via TAK1, and increased ERK signaling through IRAK1/4 mediated down regulation of DUSP1. The RPCI-WM1 model also indicated high AKT due to indirect convergence of multiple aberrations. Another key characteristic of the genomic aberrations driving proliferative phenotype was low CN of RB1, FOXO3, CDKN2A, CDKN2B, and CEBPa.Modeling predicted sensitivity to the aurora kinase (AURKA) inhibitor, tozasertib and resistance to cell cycle cyclin D-CDK4/6 pathway inhibitors., Although there was no CN variation in AURKA, as per simulation its expression and activity was upregulated due to high CN of ETS1 and PAK1 and increased activation of NFkB, HIF1A and STAT5 in the disease network. Despite high proliferation, LEE011 (representative CDK4/6 inhibitor) was predicted to exert no cytotoxic effect, due to the presence of an RB1 deletion. Through phosphorylation of RB1, the CDK4-CyclinD1 complex aids in release of E2F1 from the RB1 sequestered complex, to drive proliferation. However, predictive modeling suggested that with an RB1 deletion present, this regulation becomes irrelevant and therefore the inhibition of the CDK4/6-CyclinD1 complex would be ineffective.The simulation predictions were experimentally validated. As predicted, AURKA inhibition with tozasertib significantly inhibited viability and proliferation of RPCI-WM1 cells (IC50 ~7nM) whereas inhibition of CDK4/6 with LEE011 had no effect on tumor cell survival.Conclusions: This study demonstrates the utility of a novel technology for rapid translation of a (WM) genetic signature towards a personalized therapeutic strategy. This simulation avatar based approach holistically integrates all genomic aberration information to design personalized therapies architected from FDA approved and/or clinical drug agents with therapeutic potential for WM patients based on unique genetic signatures. DisclosuresVali:CellWorks: Employment. Kumar:CellWorks: Employment. Singh:CellWorks: Employment. Kapoor:Cellworks Research India Limited: Employment. Abbasi:CellWorks: Employment, Equity Ownership.

C1013G/CXCR4 acts as a driver mutation of tumor progression and modulator of drug resistance in lymphoplasmacytic lymphoma

AbstractThe C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in modulating cell trafficking in hematopoietic stem cells and clonal B cells. We screened 418 patients with B-cell lymphoproliferative disorders and described the presence of the C1013G/CXCR4 warts, hypogammaglobulinemia, infections, and myelokathexis–associated mutation in 28.2% (37/131) of patients with lymphoplasmacytic lymphoma (Waldenström macroglobulinemia [WM]), being either absent or present in only 7% of other B-cell lymphomas. In vivo functional characterization demonstrates its activating role in WM cells, as demonstrated by significant tumor proliferation and dissemination to extramedullary organs, leading to disease progression and decreased survival. The use of a monoclonal antibody anti-CXCR4 led to significant tumor reduction in a C1013G/CXCR4 WM model, whereas drug resistance was observed in mutated WM cells exposed to Bruton’s tyrosine kinase, mammalian target of rapamycin, and phosphatidylinositol 3-kinase inhibitors, but not proteasome inhibitors. These findings demonstrate that C1013G/CXCR4 is an activating mutation in WM and support its role as a critical regulator of WM molecular pathogenesis and as an important therapeutic target.

Tumor Hypoxia Promotes Dissemination and Tumor Colonization In Waldenström Macroglobulinemia

IntroductionWaldenström Macroglobulinemia (WM) is a rare, low-grade B-cell lymphoma characterized by lymphoplasmacytic cells spread widely in the bone marrow (BM) and overproduction of monoclonal immunoglobulins M (IgM). Previous studies showed that tumor hypoxia develops in the BM of other hematologic malignancies and promotes dissemination. In this study, we tested the effect of hypoxia on cell proliferation, cell cycle and apoptosis; on egress and homing of WM cells from and into the BM; and on recovery and tumor colonization in the new BM niche. MethodsWe characterized the effect of tumor progression on generation of hypoxic conditions in the BM in vivo, by injecting BCWM1-mCherry cells to SCID mice, letting them grow for two weeks, analyzing the hypoxic state of the WM cells in the BM using pimonidazole, and testing the number of circulating cells. Moreover, we tested the effect of hypoxia on the homing of WM cells to the BM by injecting normoxic and hypoxic cells to mice and monitoring the number of the circulating WM cells in the blood at different time points by flow cytometry. Cancer cell colonization was assessed 1 and 3 days post IV injection of normoxic and hypoxic cells to mice; mononuclear cells were isolated from the BM, fixed, permeabilized and stained with antibodies for p-Rb and cyclin-E. The percentile of WM cells in the BM and the expression of cell cycle proteins were analyzed by flow cytometry.BCWM1 cells were exposed to normoxia (21% O2) or hypoxia (1% O2) in vitro for 24hrs, and n some cases reoxygenated for 24hrs. The expression of E-cadherin, VLA-4 and CXCR4 was analyzed by western blot or flow cytometry. We tested the effect of hypoxia on adhesion of WM cells to BM stroma and fibronectin. We further tested the effect of hypoxia on chemotactic properties of WM cells towards SDF-1 using a transwell migration chamber. In addition, we tested the effect of hypoxia on WM cell survival (by MTT assay), apoptosis and cell cycle (by using AnnexinV-PI and PI, respectively), and signaling pathways associated with survival, apoptosis and cell cycle (by western blotting). ResultsTumor progression was shown to increase hypoxic conditions in the BM in vivo. We found a direct correlation between the percent of WM cells in the BM to the level of hypoxia. The level of hypoxia was in a direct correlation with the number of circulating WM cells in vivo. Then we mimicked the hypoxic conditions in vitro and found that cell progression (MTT) and cell cycle (PI staining) were decreased, but apoptosis of WM cells was not affected (AnnexinV-PI staining). These results were confirmed by decreased activation of the PI3K signaling pathway (p-PI3K, p-AKT, p-GSK) and decreased expression of cell cycle proteins (p-Rb, CDK2, CDK4, cyclin-D1 and p-cyclin-E); however, no change was observed in apoptosis-related proteins (PARP, cleaved caspase-3, -8 and -9). Moreover, hypoxia decreased the expression of E-cadherin which contributed to reduction of adhesion of WM cells to the BM stromal cells. At the same time, hypoxic WM cells exhibited increased CXCR4 surface expression and augmented migratory abilities in the presence of SDF-1. Neither the expression of integrins (VLA-4) nor the adhesion of WM cells to fibronectin was affected by hypoxia. This data indicates the conservation of the homing machinery of the WM to the BM despite the hypoxic conditions accompanied by increased chemotactic ability. When hypoxic and normoxic cells were injected to naïve mice, hypoxic cells showed enhanced homing to the BM and tumor colonization. Similarly, hypoxic cells which were reoxygenated in vitro showed more proliferation, cell cycle and activation of proliferative signaling pathways compared to normoxic cells. ConclusionsWe report that WM tumor growth in the BM increases hypoxia, and that hypoxia induces cell cycle arrest, and less proliferation of cells with no apoptosis. At the same time, hypoxia induces egress of WM cells from the BM through reduction of E-cadherin expression and decreased adhesion. When in the circulation, previously hypoxic cells home more efficiently to the BM through increased expression of CXCR4 and chemotaxis, and through maintaining expression of integrins and adhesion to fibronectin. When in the new oxygenated BM niche, hypoxic WM cells recover and colonize the new niche better than normoxic cells, and reoxygenated hypoxic cells have faster cell cycle and proliferation rate. Disclosures:No relevant conflicts of interest to declare.

Interference Of The Tumor Supportive Effects Of BCL2 and MCL1 Sensitize Malignant Plasma Cells To The Lethal Effects Of Lenalidomide and Dexamethasone Regimen: An Important Clinical Path For BCL2 Targeting Drugs

BackgroundMultiple myeloma (MM) is the second most common hematologic malignancy in the U.S. and is characterized by increased immunoglobulin production and infiltration of the bone marrow by malignant plasma cells. Myeloma cell growth is supported by both the elements present in the bone marrow microenvironment as well as deregulation of internal cellular systems associated with proliferation and apoptosis. Defective programmed cell death by BCL2 or MCL1 upregulation is observed in >80% of myeloma cases and is associated with an aggressive clinical course. Remarkably, there is no approved drug with the ability to target BCL2 or MCL1. Various strategies to mitigate the effects of increased BCL2 functionality, via small molecule inhibitors or BCL2-specific antisense oligonucleotides have been previously examined in MM. These studies revealed that 1) apart from BCL2, additional anti-apoptotic members belonging to the BCL2 family (i.e. MCL1, BCL-xL) play integral roles in maintenance of the myeloma cell phenotype, 2) drugs, which target only BCL2 offer limited therapeutic advantage and 3) given that survival factors engaged in MM are multifactorial, perhaps isolated targeting of the BCL2 pathways may not be sufficient. We therefore hypothesized that the clinical development of drugs that target various antiapoptotic BCL2 family members will require a partnership with established anti-MM regimens whose activity can potentially be further enhanced with correction of the apoptotic response system. AimSince lenalidomide and dexamethasone (LD) is an established therapy for plasma cell cancers, we investigated if the pan-BCL2 inhibitor AT-101 (BH3 mimetic), which has high binding specificity for BCL2, MCL1 and BCL-xL, can be an effective therapeutic partner to enhance anti-MM effects of LD. MethodsHuman MM (KMS11, U266, OPM2) and WM (BCWM.1) cell lines along with their corresponding bortezomib resistant (BR) clones, (KMS11/BR, U266/BR, OPM2/BR and BCWM.1/BR) which were developed in our laboratory were used in this study. For gene expression profiling, the Illumina HumanHT-12 v3 whole-genome gene expression array and Nanostring nCounter mRNA quantification assays (NanoString, Seattle, WA) were utilized. Statistical analysis was conducted using R-based packages and the MeV software (TIGR). Apoptosis was measured by annexin-v/PI staining, and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiles were ascertained by western blot. ResultsGene expression and immunoblot analysis of six plasma cell cancer models showed upregulation of various BCL2 family members, notably MCL1 and BCL-xL. In a dose-dependent manner, AT-101 was able to downregulate BCL2 and MCL1 and induced apoptotic cell death in MM and WM cells in a dose dependent manner. Tumor cell death was associated with caspase and PARP-1 cleavage accompanied by an increase in MOMP. This cytotoxic effect and BCL2 downregulation were further potentiated when AT-101 was combined with lenalidomide/dexamethasone (LDA). Nanostring nCounter mRNA quantification and IPA analysis revealed differential changes in the CCNA, FRZB, FYN, IRF1, PTPN11 genes in LDA treated cells. IPA canonical pathway analysis demonstrated the p53 signaling and the cyclins and cell cycle regulation pathways to be the most significantly activated by LDA therapy. SummaryIn summary, we describe for the first time the cellular and molecular events associated with the use of AT-101 in combination with lenalidomide/dexamethasone in preclinical models of plasma cell malignancy. This study lays the rationale for engaging and handicapping the intrinsic apoptotic system through the pan-BCL2 targeting capabilities of AT-101; thus enhancing the anti-MM effects of lenalidomide/dexamethasone. Disclosures:Foran:Celgene: Research Funding.

Inhibition Of The Deubiquitinating Enzymes UCHL5 and USP14 Is Lethal To Waldenströms Macroglobulinemia Cells

BackgroundWaldenströms macroglobulinemia (WM) is a lymphoplasmacytic subtype of Non-Hodgkins lymphoma in which specific molecular changes lead to unrestricted cell proliferation and overproduction of immunoglobulin-M. These processes require exquisite coordination between growth promoting transcription factors (NFkB) and cell homeostatic systems. The bioavailability of various transcription factors and cell cycle proteins is primarily regulated by the ubiquitin proteasome system (UPS). Moreover, the proteolytic machinery ensures proper elimination of unwanted or misfolded proteins, which when disrupted leads to protein accumulation and apoptotic cell death. The 26S proteasome is a barrel shaped structure with a 20S catalytic core that is flanked by 19S caps on either side. 26S proteasomes are critical for WM cell survival, which is clinically evident, as up to 80% of WM patients treated with the proteasome inhibitor (PI) bortezomib have derived significant benefit. Traditional PI such as bortezomib and carfilzomib have been designed to target the B5 catalytic core within the 26S proteasome. However, proteasomes are capable of altering conformation of their proteolytic subunits in response to inflammation and stress, which can lower the effectiveness of these PI. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14 results in cell death in a variety of tumor types but more so in cancer cells that rapidly divide or have a high protein turnover rate. AimTo define the anti-WM activity of the novel DUB inhibitor, b-AP15 in preclinical models of WM. MethodsThe WM cell lines BCWM.1, RPCI-WM1 and MWCL-1 were used for experiments in this analysis. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Apoptosis was determined by annexin-V/PI staining and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiling was done via immunoblot analysis. ResultsAs reported by D'Arcy et al in their characterization of the b-AP15 molecule, inhibition of UCHL5 and USP14 by b-AP15 does not disrupt proteasomal activity conferred by the b-catalytic subunits. We confirmed these findings by treating WM cells with single agent bortezomib (10nM), carfilzomib (10nM), b-AP15 (100nM) or all three agents in combination to assess chymotrypsin-like, caspase-like and trypsin-like proteasomal activity using fluorogenic peptide substrates. Further, when used in combination with the other PI, PI-mediated chymotrypsin-like activity remained inhibited in the presence of b-AP15. Next, we sought to determine if b-AP15 treatment could induce WM cell death. Annexin-v staining showed that b-AP15 (500uM) induced apoptosis in all WM cell lines (28 – 60% cell death) with variable responses noted among the 3 models. Treatment with b-AP15 increased MOMP while also inducing the cleavage of caspases-9 and 3 and PARP-1 suggesting that the anti-WM activity of b-AP15 is mediated through the intrinsic apoptotic pathway. As components of the NFkB signaling cascade lie downstream to the UPS, we examined the effects of b-AP15 on this pathway. When treated with the DUB-inhibitor, nuclear translocation of NFkB was reduced in a time dependent manner. Interestingly, treatment with b-AP15 increased the phosphorylation of the mitogen activated protein kinase-p38 (p38) in all the WM cell lines tested. Treatment of WM cells with b-AP15 in presence of the p38 inhibitor, SB203580, resulted in a synergistic induction of cell death.Conclusionb-AP15 is a novel DUB inhibitor that disrupts two regulatory enzymes (UCHL5 and USP14) present in the 19S proteasomal cap. Results presented here demonstrate that b-AP15 induces apoptosis in in vitro models of WM and with in vivo analysis underway, is a potential therapeutic in WM.We would like to acknowledge the Waterfall Waldenström Macroglobulinemia Research Fund, the Leukemia and Lymphoma Society and the International Waldenström Macroglobulinemia Foundation for their continued support. Disclosures:No relevant conflicts of interest to declare.

Somatic Activating Mutations In CXCR4 Are Common In Patients With Waldenstrom's Macroglobulinemia, and Their Expression In WM Cells Promotes Resistance To Ibrutinib

Background Waldenstrom's macroglobulinemia (WM) is an indolent non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells in the bone marrow. CXCR4 is a chemokine receptor that promotes the survival, migration, and adhesion to the bone marrow stroma of WM lymphoplasmacytic cells (LPC) through interactions with its ligand CXCL12. Through whole genome sequencing, we identified somatic mutations in CXCR4 that affected 1/3 of WM patients. These mutations were identical or functionally similar to those associated with Warts, Hypogammaglobulinemia, Infection, and Myelokathexis (WHIM) syndrome (Hunter et al, ASCO 2012), a rare autosomal dominant genetic disorder that is caused by frame shift or nonsense mutations in the carboxyl-terminal cytoplasmic tail of CXCR4. In WHIM syndrome, loss of the c-terminal tail of CXCR4 impairs receptor internalization, thereby prolonging G-protein and β-arrestin signaling (Lugane et al, Blood 2008). Ibrutinib induces WM cell death, and is highly active in WM (Treon et al, ICML-12, 2013). Since the target of ibrutinib (BTK) is a known downstream target of CXCR4, we sought to clarify if ibrutinib activity in WM LPCs was modulated by WHIM-like mutations in CXCR4. Methods We first sought to confirm the frequency of WHIM-like mutations in 87 untreated WM patients by Sanger sequencing. The most common CXCR4 somatic mutation identified (S338X) in these studies was then cloned by PCR from CD19+ LPCs from a WM patient with this somatic mutation. Wild type (WT) and S338X CXCR4 cDNAs were subcloned into plenti-IRES-GFP vector, and transduced using an optimized lentiviral based strategy into BCWM.1 WM cells. Five days after transduction, GFP positive cells were sorted and used for functional studies. Surface expression of CXCR4 was determined by flow cytometeric analysis using a PE-conjugated anti-CXCR4 monoclonal antibody. The expression of phosphorylated BTK, AKT, and ERK1/2 was determined by western blot analysis. Cell proliferation was measured with alamar blue. Results Sanger sequencing identified nonsense or frame shift mutations (WHIM-like) in the c-terminal tail of CXCR4 in 28 of 87 (32%) patients, the most common of which was a non-sense mutation (S338X) that was present in 12 patients. BCWM.1 cells were then transduced with control vector, CXCR4 wild type or CXCR4 S338X mutant expressing vectors. Expression was confirmed by cDNA Sanger sequencing. Stably transduced cells exposed to ibrutinib (0.5uM or 1uM) showed significantly reduced cell proliferation (p&lt;0.005). Ibrutinib treated control vector and CXCR4 wild-type transduced cells showed suppressed tumor cell growth even in the presence of the CXCR4 ligand CXCL12 (20 nM), whereas cells transduced with CXCR4 S338X WHIM-like mutation demonstrated resistance to ibrutinib growth effect (p&lt;0.005). In turn, this rescue could be blocked by treatment with 30uM of the CXCR4 specific inhibitor AMD3100 confirming that this effect was mediated through CXCR4 (p&lt;0.005) (Figure 1). Phosphorylated BTK, ERK1/2 and AKT signaling increased following CXCL12 stimulation in all transduced cells, while ibrutinib inhibited their activation in control vector and CXCR4 wild-type, but not CXCR4 S338X mutant cells. CXCR4 triggered signaling by CXCL12 in these experiments was confirmed by pre-treatment with AMD3100. Conclusions By Sanger sequencing, WHIM-like CXCR4 somatic mutations are observed in 1/3 of untreated WM patients. WHIM-like CXCR4 mutations are associated with resistance to ibrutinib mediated ERK1/2 and AKT signaling, as well as growth suppression in the presence of the CXCR4 ligand, CXCL12, in WM cells. These studies may have important implications for CXCR4 modulation in the treatment of WM, as well as potential use of CXCR4 mutations in predicting outcome for patients undergoing ibrutinib therapy. Disclosures: No relevant conflicts of interest to declare.

Small Molecule PRIMA-1 met Sensitizes Waldenstrom Macroglobulinemia Cells To Apoptosis and Displays Synergistic Cytotoxicity With Bortezomib

BackgroundWaldenstrom macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by heterogeneous infiltration of bone marrow and IgM monoclonal gammopathy. WM constitutes 1-2% of haematological malignancies and patients exhibit diverse outcome. Given the current therapies WM remains incurable with high rate of relapse, therefore, the quest for finding a more effective drug continues. PRIMA-1met is a small molecule with the ability to restore wild type conformation to mutant P53 and to activate wild type P53. PRIMA-1met has shown significant anti-tumour activity both in vitro and in vivo by inducing apoptosis in several human cancers including haematological malignancies and is currently under phase I/II clinical trials. To date, the effects of PRIMA-1met have not been explored in WM. Here we examined the antitumor activity of PRIMA-1met alone and in combination with dexamethasone or bortezomib in WM. DesignWM cell lines, BCWM-1(Wild type P53) and MWCL-1 (mutant P53), were used for these studies. Cellular proliferation, viability, migration and percentage of cells undergoing apoptosis in WM cells treated with PRIMA-1met or DMSO control were evaluated using trypan blue, MTT assay, Boyden chamber assay and Annexin-V staining methods respectively. The status of key role players in apoptosis and cell cycle regulators were examined following treatment with PRIMA-1metusing qRT-PCR and Western blot analyses. Cell viability was also investigated by combining PRIMA-1metwith either dexamethasone or bortezomib in WM cells by MTT assay. ResultBoth BCWM-1 and MWCL-1 cells showed significant decrease in cell viability after exposure to escalating doses of PRIMA-1met with observed IC50 of 30 μM in BCWM-1 cells and 28 μM in MWCL-1 cells after 48 hours. Two primary WM patients' samples exposed to increasing concentration of PRIMA-1metdemonstrated significant decrease in cell viability with IC50of 30 and 10 μM after 48 hours. Importantly, peripheral and bone marrow mononuclear cells of 3 healthy donors were subjected to PRIMA-1metand no significant cytotoxicity was observed. Both cell lines showed an increase in apoptosis after PRIMA-1mettreatment in a dose-dependent manner compared to control DMSO treatment. In BCWM-1, cell proliferation, measured by counting viable cells after 7 days, was significantly inhibited by PRIMA-1metcompared to DMSO control (∼54% fewer cells when treated with 25 μM PRIMA-1met, p<0.05). Decrease in cell migration by as much as 28% at 25 μM compared to DMSO control (p<0.001) was also observed after 8 hours of treatment with PRIMA-1met. Expressions of P53 and its downstream target p21 were elevated in PRIMA-1mettreated cells compared to DMSO control. In addition, qRT-PCR studies of 50 genes associated with apoptosis also demonstrated enhanced expression of pro-apoptotic markers such as PUMA, Noxa, caspase 8 and 9, BIM and BAD, which were further confirmed for their protein expressions by Western blot analysis. Finally, PRIMA-1met(25 μM) was found to exert synergistic cytotoxic effect in combination with dexamethasone (2 μM) in MWCL-1 cells (Combination index (CI)=0.5). PRIMA-1met(25 μM) also displayed synergism with bortezomib (3 μM) in its cytotoxicity toward BCWM-1 cells (CI=0.8). ConclusionsOur results demonstrate that PRIMA-1met induces apoptosis irrespective of P53 status in WM cells. It also provides the preclinical framework for evaluation of PRIMA-1met either alone or in combination with bortezomib or dexamethasone as a novel therapeutic approach for the treatment of WM patients. Disclosures:Chen:Roche: Honoraria; Johnson & Johnson: Consultancy, Research Funding; Lundbeck: Consultancy; Celgene: Consultancy, Research Funding; GlaxoSmithKline: Research Funding.

Development Of a Human IgM Secreting Model Of Rpci-WM1 Through Xenografting In SCID Mice For In Vivo Drug Evaluation

BackgroundWaldenstršms macroglobulinemia (WM) is a rare and incurable indolent Non-Hodgkin's lymphoplasmacytic lymphoma. Rarity and limited availability of authentic preclinical models has been a major constraint in drug development for WM. We recently reported the establishment and characterization of a novel human WM cell line, RPCI-WM1. Thereafter we evaluated the tumorigenic potential of this unique WM cell line in a xenograft mouse model along with its ability to produce human IgM in vivo. Moreover, we assessed its potential as a possible model for enhanced understanding of WM cell biology in vivo and a mechanism to conduct functional studies and preclinical anti-WM drug evaluation. AimThe primary goal of this experiment was to develop and validate an in vivo WM model that can be used to investigate WM cell biology as well as drug evaluation. MethodsThe RPCI-WM1 cell line (1x106 cells) was injected both subcutaneously and via tail vein into female SCID mice. Engraftment was validated via detection of human IgM (ELISA) and tumor growth (as well as response to therapy) was monitored by weekly assessment of IgM levels and external tumor formation measurements. ResultsAfter subcutaneous injection (flank) into mice, the RPCI-WM1 cells developed localized tumors that secreted human IgM detectable in serum as early as 14 days. Progressive increase in IgM was measured in the serum and this corresponded with tumor growth in a time-dependent fashion. On average, tumor tissues grew to a median of 3 cm and IgM levels reached a median of 823 ng/mL by Day 30 post-implantation. We also assessed the practical utility of this xenograft mouse model for evaluation of therapeutics, using a compound X (propriety drug with demonstrable in vitro anti-WM effects). Eleven days post-implantation, the mice were randomized into 3 groups receiving either DMSO (n=6), 100 mg of compound X (n=7) or 200 mg/kg of compound X (n=7) every alternate day via intraperitoneal injection for 26 days. On Day 37, mice were sacrificed and tumor volume was measured. We observed a 48% reduction in tumor volume in the 100mg/kg cohort, with no additional anti-WM benefit noted in the 200mg/kg cohort. Human IgM secreted by the xenografted tumors was measured by ELISA and was significantly lower in mice treated with compound X versus the control group (Figure 1). We also conducted immunohistochemistry (IHC) analysis on tumor tissues obtained from 9 mice treated with compound X (both dose cohorts) or DMSO alone. IHC staining demonstrated changes in the known target proteins of compound X (such as BCL2, p53, Ki-67) between the treated and untreated mice. [Display omitted] SummaryThus we demonstrate that the RPCI-WM1 cell line can be dependably used to induce tumor growth through xenotransplantation in SCID mice and this mimics the IgM secreting clinical disease model. This can then be used to serve as a platform for preclinical testing of novel anti-WM therapeutics and allow for subsequent correlative studies for pharmacodynamics purpose. Comprehensive data regarding this model its utility in evaluation of various biological questions such as those relevant to the tumor microenvironment as well therapeutic modeling will be presented.We are grateful to the Leukemia and Lymphoma Society and the International Waldenstršm Macroglobulinemia Foundation for their continued support. Disclosures:No relevant conflicts of interest to declare.

Clonotypic Analysis Of Immunoglobulin Heavy Chain (IgH) Sequences In Patients With Waldenström's Macroglobulinemia. Correlation With MYD88 L265P Somatic Mutation Status, Clinical Features and Outcome

Background and aimWaldenström's Macroglobulinemia (WM) cells express a unique clonotypic rearrangement of heavy chain immunoglobulin (IgH) genes in each individual patient. Recent studies demonstrate high frequency of the MYD88 L265P somatic mutation in patients with WM, implying a new pathway for the pathogenesis of this disease. In this study we characterized clonal IgH genes rearrangements, IGHV gene use, somatic hypemutations (SHM) and presence of the ΜΥD88 L265P mutation in a cohort of WM patients and we investigated any eventual correlation with patients' clinical features. Patients and methods36 patients with WM were studied (median age 64y, 19 males and 17 females, symptomatic 72%, asymptomatic 28%) of whom 45%, 39% and 16% were stage IPSSWM 1, 2 and 3, respectively. Median time to treatment initiation was 13 months and median overall survival was 61 months. DNA was extracted from patients' bone marrow/blood samples and clonal IgH VDJ rearrangements were amplified by PCR using the Biomed-2 Concerted Action protocol. Monoclonal VDJ rearranged PCR fragments were sequenced and analyzed using the IMGT database (www.imgt.org). Thirty-one patients' DNA samples were also analyzed for the presence of the ΜΥD88 L265P mutation by using a mutation specific PCR assay. ResultsThe most frequent clonal rearrangements observed in our patients were in the IGVH3 family (73% of cases); the IGVH3-23 gene and the IGVH3-74 gene were used in 24.4% and 18,9% of cases, respectively. Somatic hypermutation (>2%) was seen in all but three cases (92%). Mean percentage of mutations in all cases, IGVH3 family, IGVH3-23 and IGVH3-74 genes was 7,5%, 8%, 9,4% and 7,5%, respectively. Sixty-two percent of the patients were positive for the ΜΥD88 L265P mutation and notably all three unmutated WM cases were negative for this MYD88 point mutation. No correlation between VH or DH gene usage, CDR3 length, and mutational status with clinical parameters, time to treatment initiation, or survival was found. ConclusionsWM IgH genes repertoire, as expected, differs from that observed in normal B-cells and other B-cell diseases such as MZL, MCL and B-CLL/SLL. In addition to the known hyper-representation of the IGVH3-23 gene, another member of the VH3 family, the IGVH3-74 gene is also over-represented in WM. The high IGVH mutation rate supports a derivation of WM cells from postgerminal center memory B cells in the majority (92%) of WM patients. However, the identification of a minority of WM patients (3 of 36) with unmutated IGHV gene segments, negative for the ΜΥD88 L265P mutation, supports the hypothesis that they represent a subgroup of WM not arising from post-germinal B cells with a different disease pathogenesis. Disclosures:No relevant conflicts of interest to declare.

Candidate genes of Waldenström's macroglobulinemia: current evidence and research.

Waldenström’s macroglobulinemia (WM) is a relatively uncommon, indolent malignancy of immunoglobulin M-producing B cells. The World Health Organization classifies it as a lymphoplasmacytic lymphoma and patients typically present with anemia, hepatosplenomegaly and diffuse lymphadenopathies. Historically, the genetic characterization of the disease has been hampered by the relatively low proliferative rate of WM cells, thus making karyotyping challenging. The use of novel technologies such as fluorescence in situ hybridization, gene array, and whole genome sequencing has contributed greatly to establishing candidate genes in the pathophysiology of WM and to identifying potential treatment targets, such as L265P MYD88. The discovery of microRNAs and the recognition of epigenetics as a major modulatory mechanism of oncogene expression and/or oncosuppressor silencing have aided in further understanding the pathogenesis of WM. Once thought to closely resemble multiple myeloma, a cancer of terminally differentiated, immunoglobulin-secreting plasma cells, WM appears to genetically cluster with other indolent B-cell lymphomas such as chronic lymphocytic leukemia/small cell lymphoma. The relative high incidence of familial cases of WM and other B-cell malignancies has been helpful in identifying high-risk gene candidates. In this review, we focus on the established genes involved in the pathogenesis of WM, with special emphasis on the key role of derangement of the nuclear factor kappa B signaling pathway and epigenetic mechanisms.

MYD88 L265P Promotes Survival of Waldenstrom's Macroglobulinemia Cells by Activation of Bruton's Tyrosine Kinase

Abstract 897 Background:MYD88 is an adapter molecule which promotes Toll-Like Receptor and IL-1 Receptor signaling through the IRAK 1/4/TRAF6/NF-κB pathway. MYD88 L265P is a recently described somatic mutation that is widely expressed (>90%) in patients with Waldenstrom’s Macroglobulinemia (Treon et al, NEJM 2012). The signaling pathway by which MYD88 L265P promotes WM growth and survival remains to be fully clarified. We therefore sought to identify pathway(s) responsible for MYD88 L265P pro-survival signaling. Patients and Methods:Primary WM-LPCs were isolated from bone marrow biopsy specimens from WM patients. Normal CD19+ B-cells were isolated from the peripheral blood of healthy donors. Western blot analyses was performed using total and phospho-specific antibodies in the MYD88 L265P expressing WM cell lines BCWM.1 and MCWL-1 following MYD88 knockdown by lentiviral transduction, and/or use of MYD88 or IRAK signal inhibitors. WM cells were also treated with the BTK inhibitor PCI-32765, in the presence or absence of an IRAK1/4 inhibitor. Cell survival was evaluated by Annexin V/PI staining and/or AlamarBlue® or ATPLite® assays after treatments with inhibitors. Synergistic interactions were assessed with CalcuSyn software. Results:Lentiviral knockdown of MYD88 and/or use of an inhibitor of MYD88 homo-dimerization led to decreased survival of MYD88 L265P expressing BCWM.1 and MWCL-1 cells, as well as primary WM LPCs expressing MYD88 L265P. In contrast minimal killing was observed in MYD88 Wild Type (WT) expressing Ramos, MM.1S, or healthy donor CD19+ selected B-cells. Phospho-specific western blot analysis showed decreased IRAK1, NF-kB and STAT3 signaling following inhibition of MYD88 signaling in L265P expressing WM cells. Conversely, use of an IRAK1/4 kinase inhibitor showed reduced tumor cell killing, NF-kB and STAT3 signaling. We therefore performed co-immunopreciptation experiments to identify other MYD88 binding partners in L265P expressing WM cells. These studies identified Bruton's Tyrosine Kinase (BTK) in complex with MYD88, with MYD88 showing more robust binding to phospho-BTK in L265P expressing WM cells. Lentiviral knockdown of MYD88 and/or use of an inhibitor of MYD88 homodimerization led to decreased BTK phosphorylation. Moreover, overexpression by lentiviral transduction of MYD88 L265P showed more robust BTK phosphorylation versus WM cells transduced with MYD88 WT. Lastly, the use of a BTK-inhibitor (PCI-32765) blocked NF-kB and STAT3 signaling, as well as induced WM tumor cell killing that was synergistically enhanced by the presence of an IRAK1/4 kinase inhibitor. Conclusion:MYD88 L265P promotes survival of WM cells by activation of BTK. These studies provide the framework for the investigation of BTK inhibitors in WM, as single agents and in combination with other MYD88 pathway inhibitors. Disclosures:Treon:Onyx: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Cephalon: Consultancy; Avila: Consultancy.