Paulus, Aneel

Abstract Background: The observation that B-cell receptor (BCR) signaling play a critical role in the survival of malignant Waldenstrom macroglobulinemia (WM) cells is highlighted by clinical efficacy of the BTK-inhibitor, ibrutinib in WM patients. BCR signaling leads to downstream activation of several intermediary components whose activity results in increased cell proliferation. Although disruption of this axis at the proximal end (BCR) has proved successful in WM, the exact role and impact of targeting the distal intermediaries remains unknown, particularly in aggressive forms of the disease. This is important as induction of resistance to ibrutinib is clearly documented which can either be mediated through mutational changes in BTK or other members within the BCR signalosome. Using a novel simulation-based approach, we reverse-engineered the WM cell molecular architecture to uncover the role of oncogenic intermediaries distal to the BCR complex in advanced-stage WM and conducted a virtual drug-screen targeting these pathways with in vitro validation. Methods: The human WM cell line, RPCI-WM1, was used in simulation and validation experiments. RPCI-WM1 was established from a highly drug-refractory advanced disease stage patient refractory to both rituximab and bortezomib. Publically available as well as proprietary genomic and cytogenetic data was utilized for the creation of the RPCI-WM1 patient avatar, 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. 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 FDA approved drug agents (n~150) 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: MYD88L265P mutation, absence of CXCR4 mutations and additional chromosomal aberrations (derivatives, translocations, deletions and amplifications of chromosomes 3, 6, 9, 13, 18 and 19), which are notable features of RPCI-WM1 cells were configured and modeled in silico. The RPCI-WM1 patient avatar was predicted to have increased IRAK1/4 engagement due to MYD88 mutation and high copy number (CN) of IL18. Downregulation of DUSP1 through MYD88-mediated signaling was noted to result in high ERK activity. Increase gene copy number of both FOS and ETS1, which are downstream of ERK, were noted. Notably, FOS is a key regulator of the AP1 complex, whose activity is regulated upstream by ERK. The transcription factor, ETS1 was also predicted as over expressed and ERK-mediated phosphorylation of ETS1 regulates the activity of ETS1. The RPCI-WM1 patient avatar model also indicated high AKT due to indirect convergence of multiple aberrations. Drug screening revealed sensitivity to the MEK inhibitor, binimetinib. Although no copy number variation in MEK-ERK pathway genes were detected, per simulation, MYD88 mutation through inactivation of DUSP1 activated ERK and downstream associated survival pathways. The simulation predictions were experimentally validated. As predicted, binimetinib significantly inhibited proliferation and viability of RPCI-WM1 cells (IC50 <100nM). Conclusions: Our study demonstrates the functional role and impact of MEK1/2 - an oncogenic component distal to the BCR, and whose activity can be targeted with binimetinib to elicit a lethal effect in advanced-stage WM cells. We also show the utility of a novel technology, which is capable of integrating genome-wide molecular data points to emulate the salient genomic drivers of a given tumor cell and test its sensitivity to numerous drugs in a high-throughput manner for truly personalized therapy. Disclosures Vali: Cellworks Group, Inc.: Employment, Equity Ownership. Kumar:Cellworks Group, Inc.: Employment. Singh:Cellworks Group, Inc.: Employment. Usmani:3Cellworks Research India Limited: Employment. Grover:3Cellworks Research India Limited: Employment. Abbasi:Cellworks Group, Inc.: Employment, Equity Ownership.

Abstract Background: Outcomes in multiple myeloma (MM) have improved significantly over the years but disparities in outcomes exist among MM patients (pts) belonging to different racial subgroups. Previous studies have explored healthcare access and utilization disparities for pts of different races as potential causes, but genomic differences, which could alter disease biology and clinical behavior, have not been studied. We utilized the MMRF CoMMpass database, which prospectively captures clinical and molecular characteristics of MM patients to explore race-based differences at the genomic level. Methods: The publically available CoMMpass trial database (data release version IA8) was utilized. Pts with race category of White (total n=698) and non-White (total n=223, comprising 129 Blacks, 19 Asians and 45 others) were analyzed for genomics including cytogenetics (fluorescent in situ hybridization; FISH), tumor diploidy (conventional karyotyping), gene expression profiling (GEP), gene copy number variations (CNV), gene single nucleotide variations (SNV) and genomic translocations analysis focused on genes from the FoundationOne Heme panel¨ (402 genes). GEP was considered significantly different between categories for more than or equal to a 2-fold change for a given gene (p<0.05). Categorical variables for FISH probes and diploidy status were compared by Chi-square or Fischer exact test, wherever applicable. Results: GEP clustering analysis uncovered 67 genes differentially expressed between Whites vs. non-Whites (Fig 1A). Baseline cytogenetics aberrations between both race cohorts showed statistically insignificant differences. Multiple cytogenetic abnormalities were seen most commonly in both race groups (~31%) with the distribution of others (in descending order) as such: Del13 > No abnormalities > t(11;14) > 1q amplification > Del17p or p53 > t(4;14) > Del1p > t(14;16) > t(14;20). Aneuploidy status showed insignificant differences between race groups with ~34% of patients from both groups having no aneuploidy. SNV data was present for 90% of Whites and 57% of non-Whites. Overall, 84% of genes had>1 SNV (84%), 2% translocations only and 14% had neither SNVs or translocations. SNVs were classified as Non-specific Non-Synonymous coding (NNSCs), Splice-site (SS) or Tier 1 (T1, codon deletion, insertion, frame-shift, etc.). A total of 880 NNSCs SNVs were observed: 670 in Whites across 227 genes, 210 in non-Whites across 120 genes. In whites, 57% of NNSC SNVs consisted of transition-type mutations and 43% as transversions. In non-Whites, transition mutations comprised 64% of all NNSC SNVs and transversions in the remaining 36%. A total of 38 SS SNVs were observed, 30 in Whites and 8 in non-Whites observed in 25 and 6 genes, respectively. In Whites, 57% of SS SNVs were transition type and remaining 43% were transversion. In non-Whites, an equal % of SS transition and transversion mutations were noted. A total of 240 T1 SNVs were noted; 182 in Whites seen in 82 genes; 58 in non-Whites seen 6 genes. In both Whites and non-Whites, frameshifts (41% in Whites, 47% in non-Whites) and stop-gained (46% in Whites, 45% in non-Whites) SNVs were most common. A total of 17 genes with>10 distinct SNVs were identified in Whites; 5 genes in non-Whites and 4 genes commonly seen in both cohorts (Fig 1B). In Whites, 17 genes were found to have translocations (intrachromosomal TRAF3 and PIK3R2 most common); in non-Whites, 5 genes had translocations (intrachromosomal CIC and EMSY most common). CNV analysis demonstrated 21 genes that were significantly altered with either copy number gain or loss between Whites vs. non-Whites (Fig 1C). Conclusions: We present the first race-based comprehensive genomic analysis utilizing the public interface of the CoMMpass trial. We discovered genomic differences between Whites and non-Whites at several levels including GEP, SNV and CNV, but not at the level of cytogenetics, which are in fact the most commonly performed clinical genomic analysis. Correlation of these variations with MM pt outcomes would be invaluable in understanding disease biology and hopefully mitigating some of the outcome disparities by pt race. As the database grows and becomes more enriched, separate analyses for Blacks, Asians and Hispanics would be attempted. Acknowledgments: We would like to thank the MMRF, Ms. Mary Derome and Dr. Jonathan Keats for providing scientific overview and technical support. Disclosures Fonseca: Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; AMGEN: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Celgene: Consultancy; BMS: Consultancy; Bayer: Consultancy; Novartis: Consultancy; Sanofi: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Millennium, a Takeda Company: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Bayer: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Novartis: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Sanofi: Consultancy; Janssen: Consultancy; Millennium, a Takeda Company: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms. Ailawadhi:Takeda Oncology: Consultancy; Amgen Inc: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy.

Abstract Introduction. In comparison toCLL, single agent clinical efficacy of venetoclax (a Bcl-2 inhibitor; Bcl-2i) in multiple myeloma (MM) has been modest (ORR of ~12%; Kumar et al ASCO 2016). Clinical experience to date unequivocally directs towards a combination strategy in MM for venetoclax. Our prior work has demonstrated that interaction between tumor and stromal cells upregulates phospho-Bcl-2 (pBcl-2, more stable than Bcl-2) which undermines activity of Bcl-2i. We also noted that this can be overcome using agents such as lenalidomide, which can impair the protective effect of stromal cells. Our goal in this investigation was to determine if the antitumor effect of lenalidomide/dexamethasone (Ld) regimen can be further optimized through leveraging the Bcl-2 targeting properties of venetoclax thereby sensitizing MM cells to enhanced lethality. Given the known effects of Ld on activating host immune effector cells, we hypothesized that immune mediated cytotoxicity (engaged by Ld) can be further enhanced through concurrent treatment with venetoclax. Data presented here provides preclinical rationale and proof-of-principle for clinical development of Bcl-2i in combination with Ld. Methodology. Malignant plasma cell cancer models (MM1.S, BCWM.1, KMS11) and stromal cell line (HS5) were used. Bcl-2 and pBcl-2 were determined by flow cytometry. Drug cytotoxicity was determined by MTS assay and/orAnnexin-V/PI staining. SCID mice were used for xenograft studies. Bcl-2 inhibitors (venetoclax; Bcl-2 specific inhibitor, AT-101; pan Bcl-2 inhibitor) as well asLd were purchased from commercial sources. Results. Tumor cell lines were treated with Bcl-2i for 24hrs and showed significant loss of viability (51 - 68%) and induction of apoptosis (~40%) in vitro. To replicate microenvironment conditions, we co-cultured tumor and stromal cells for 48hrs followed by treatment with Bcl-2i (1uM). Under these conditions, we noted a significant reduction in apoptosis (~50%) in Bcl-2i-treated cells indicating the protective effect of the stroma (Fig.1a). Under similar settings, we examined Bcl-2 species in tumor cells +/- stromal cells. The ratio of pBcl-2:Bcl-2 in tumor cells without stromal contact was 0.7, whereas, the ratio increased to 1.8 when tumor and stromal cells were cocultured suggesting increased pBcl-2 as a protective mechanism (Fig.1b,c). We then investigated the potential of Ld (1uM) +/- Bcl-2i to downregulate pBcl-2:Bcl-2 and whether this correlated with increased tumor lethality. While solo Bcl-2i treatment of tumor + stromal cells downregulated pBcl-2 and Bcl-2 (ratio 1.5), a marked reduction was noted in Ld + Bcl-2i treated tumor cells (ratio of 0.3). And this correlated with enhanced tumor cell apoptosis; analogous to that observed in tumor cells without stroma (Fig.1a and c). As these experiments indicated the triple combination to overcome protective effects of the stroma through decreasing bioavailability of pBcl-2 in vitro, we next validated these observations in a xenograft model. MM1.S cells were subcutaneously implanted in SCID mice with local tumor growth monitored by caliper measurement and systemic dissemination measured by Human-Ig lambda levels. DMSO-treated mice demonstrated tumors averaging ~410mm3 by Day 24 post-tumor implantation, whereas mice treated with Bcl-2i (35mg/kg QD, oral gavage) or Ld alone (Len 10mg/kg QD; Dex 0.5mg/kg QD, IP injection) showed tumors ~330mm3. In Ld + Bcl-2i- treated mice, tumor size was ~250mm3- significantly lower than DMSO (p<0.001) or Bcl-2i orLd group mice (p<0.054) (Fig.1d). A similar trend was seen when systemic Human-Ig-lambda was measured (Fig.1e). Notably, mice tolerated the triple combination treatment well; with no significant weight loss or other toxicities noted. Conclusions. Regulation of the Bcl-2 pathway is an important approach for controlling tumor cell proliferation; most recently validated with the approval of venetoclax for treatment of CLL. Herein we demonstrateLd as an attractive partner regimen to enhance the effects of not only Bcl-2i but also that ofLd in MM. Ongoing work is investigating the combination ofLd + venetoclax and preliminary in vitro results in venetoclax-insensitive MM cells (IC50 >5uM) show significant apoptosis induction with combination therapy (Fig.1f). Altogether our studies provide the rationale for clinical evaluation of this combination platform (Len/Dex + Bcl-2i). Disclosures Ailawadhi: Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy.

Abstract Waldenströms Macroglobulinemia (WM) is a unique form of lymphoplasmacytic lymphoma, in which the tumor compartment is comprised of B-lymphocytes, plasmacytoid lymphocytes and plasma cells. The majority of WM cases are immunophenotypically characterized by expression of surface antigens that are present on both B-cells (CD19/20) and plasma cells (CD38/138). However, in advanced stage disease, which has been treated with various chemoimmunotherapeutics the molecular profile of WM cells can drastically shift resulting in a majority of the WM tumor compartment exhibiting loss of CD20 and concurrent upregulation of atypical surface antigens (Barakat et al, Am J Clin Pathol 2011; Morice et al Mod Pathol 2009). These external immunophenotypic changes are associated with underlying epigenetic and genomic alterations, whose integrated study may provide further insight into the biology of advanced stage WM. Moreover, such an analysis holds clinical potential by uncovering of therapeutic vulnerabilities, which can direct the selection of appropriate therapeutics for patients with CD20- WM. With this in mind, we conducted comprehensive molecular testing coupled with functional studies to understand if underlying genomic/epigenetic changes correlated with drug sensitivity using appropriate preclinical models of CD20+ WM (BCWM.1 cell line) and CD20- WM (RPCI-WM1 cell line). Importantly, the index patient from whom the CD20- cells were developed had received a total of 6 lines of treatment, two of which contained rituximab and was documented as being refractory to rituximab. Immunophenotyping of CD20- cells showed differential downregulation of 19 and upregulation of 7 CD antigens relative to CD20+ WM cells (cutoff at 1.5 fold; >20% gated expression). Upregulated antigens were associated with a phenotype reminiscent of a memory B-cell with plasmacytoid features. Next, we conducted RNA-Seq analysis (Illumina HiSeq), which revealed downregulation of 6,673 and upregulation of 4,594 genes (>1.5 fold cutoff) in CD20- WM cells compared with CD20+ cells. Gene pathway enrichment analysis (NextBio, Illumina Inc.) of mRNA differentially expressed in CD20- cells showed significant enrichment of upregulated genes corresponding to Protein Transport (p<1.8E-98) and Response to Unfolded Protein Gene Ontology groups (p<5.9E-20). Interestingly, pathway analysis of downregulated genes in CD20- WM cells showed enrichment within the B-cell receptor-signaling (BCR) pathway (Broad MSigDB) (p<6.1E-14) relative to CD20+ WM cells. BCR signaling has been linked to dysregulated expression of several micro-RNAs (miRs). Consistent with pathway analysis results, we observed decreased levels of numerous miRs (Exiqon qPCR) in CD20- vs. CD20+ WM cells, which target and regulate components involved in BCR activation. To examine further epigenetic changes that may account for decreased BCR signaling, we examined the global methylation profile (Infinium HumanMethylation 450K array) of CD20- and CD20+ WM cells and noted hypermethylation of the promoter sites (200 base pairs upstream) for a number of transcription factors involved in the BCR pathway. We therefore examined sensitivity of CD20- WM cells to the BTK inhibitor ibrutinib, which abrogates BCR signaling. As anticipated, CD20- WM cells retained >90% viability and demonstrated significantly less apoptosis in presence of ibrutinib (4uM) compared to CD20+ cells, suggesting decreased reliance on canonical BCR signaling for survival. In parallel and guided by pathway analysis results, we exposed both CD20- and CD20+ WM cells to a novel 19S proteasome inhibitor (VLX1570), which caused profound proteotoxicity and ER stress at concentrations ranging between 250 – 500nM. We noted CD20- cells were significantly more sensitive (p<0.01) to 19S proteasome inhibition than CD20+ WM cells indicating the formers critical dependence on tightly regulated protein homeostasis. In summary we describe for the first time, a comprehensive molecular and immunophenotypic investigation of CD20- WM cells compared to CD20+ WM cells, using validated preclinical models of the two disease phenotypes. Overall our data suggests significant architectural differences between CD20+ and CD20- WM, which can potentially impact the behavior of the cells and their sensitivity towards novel therapeutics being developed for treatment of advanced stage WM. Disclosures Martin: Janssen: Honoraria.

Abstract 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. Disclosures Vali: CellWorks: Employment. Kumar:CellWorks: Employment. Singh:CellWorks: Employment. Kapoor:Cellworks Research India Limited: Employment. Abbasi:CellWorks: Employment, Equity Ownership.

Abstract Background Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells in the peripheral blood, bone marrow, lymph nodes, and spleen. This is due to a combined effect of deferred apoptosis with slow, but persistent proliferation of malignant cells. In CLL, tumor-sustaining homeostasis is critically maintained by the ubiquitin proteasome system. The proteasome mediates degradation of various transcription factors such as TP53 as well as upholding a balance between the anti and pro apoptotic proteins of the BCL2 family. Previous studies have demonstrated that the clinical course of the disease is negatively associated with malfunctioning apoptotic pathways that result in increased levels of BCL2. Thus, identification and correction of defects that affect programmed cell death offer therapeutic vantage to reset and engage cell death pathways in CLL. Aim Examination of the anti-CLL properties of the investigational agent MLN2238 (Millennium Pharmaceuticals, Inc., Cambridge, MA) and its ability to inhibit the proteasomal machinery; induce CLL cell death and downregulate BCL2. MLN2238 activity was also investigated in conjunction with anti-CLL therapies such as fludarabine and dexamethasone along with the BH3 mimetic BCL2 inhibitor, AT-101 (Ascenta Pharmaceuticals, Malvern, PA). Methods CLL cells with >90% CD19+ tumor population were obtained from 28 patients with a confirmed diagnosis of CLL. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. 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. Results MLN2238 inhibited the chymotrypsin-like proteasomal activity by more than 90% (p<0.005) in all patient samples without altering PSMB5 protein levels. Moderate to minimal inhibitory effect on caspase-like and trypsin-like proteasomal activities, respectively, was also noted. CLL cells showed a concentration dependent decrease in viability in response to treatment with MLN2238 at an IC50 of 50 nM. MLN2238 treated cells underwent apoptosis in a dose dependent manner with a median dose effect (cell death) observed in 42% of cells at 25 nM (range 10% - 54%) and 60% of cells at a 50 nM concentration (range, 25% - 73%). PARP-1 and caspase-3 cleavage along with an increase in MOMP was also noted after CLL cells were treated with MLN2238; however, apoptosis was only partially blocked by the pan-caspase inhibitor z-VAD.fmk. BCL2 downregulation was dose-dependent and was observed as early as 12 hours. We sought to determine whether directly disrupting BCL2 function with AT-101 could enhance the anti-CLL effects of MLN2238. When used at sub-IC50 concentrations, AT1-10 synergized with MLN2238 to induce CLL cell death. Synergy was also observed when MLN2238 was paired with the cytotoxic agent fludarabine, whereas the combination of MLN2238 and dexamethasone resulted in additive anti-CLL activity. Conclusion While PI have made an important impact in various B cell cancers, their role in CLL has not been well established. We investigated preclinically, a novel PI and noted that targeting the proteasome with MLN2238 resulted in lethal events in CLL cells, which were further enhanced by disruption of the BCL2 prosurvival pathway. Moreover, proteasome disruption sensitized CLL cells to the cytotoxic effect of fludarabine, an important therapeutic in CLL. These data provides the mechanistic basis for evaluation of MLN2238 in CLL through rationale design of drug combination strategies based on CLL biology. We would like to acknowledge the Leukemia and Lymphoma Society (A.C.-K. is a Leukemia and Lymphoma Scholar in Clinical Research) for their ongoing support. We are also grateful to Mary Ella Mahoney Davidson (Millennium Pharmaceuticals) for providing logistical support. Disclosures: Foran: Celgene: Research Funding.

Abstract Background: Patient (Pt) outcomes have improved significantly in multiple myeloma (MM) due to widespread use of novel therapeutic agents and stem cell transplant (SCT) butoutcome disparities among racial/ethnic groups exist. Differences in access to and utilization of novel therapeutics are likely contributing factors. We performed an analysis of the SEER-Medicare database to explore such differences. Methods: Pts with a confirmed MM diagnosis between 2007-2009 and continuous Medicare coverage from 1 year prior to diagnosis through end of 2012 were included. Medicare Part D data (oral medications) is not available prior to 2007 so utilization analysis of lenalidomide (LEN), thalidomide (THAL) and bortezomib (BORT) was performed from 2007-2009. Demographic/survival data (PEDSAF files) and drug/SCT utilization (NCH, OUTSAF, PDESAF files) were obtained (HCPCS/NCH codes). Categorical and continuous variables were compared using /Fisher tests and Wilcoxon tests, respectively. Gender, age, and year-adjusted p-values were calculated using logistic regression. A proportional odds model was used to detect time-dependent trends in drug usage within 12 months of MM diagnosis across year of diagnosis by race. Separate multivariate models were fit for each drug adjusting for gender, age, drug use and race. Associations between drug use and overall survival (OS) were examined using a time dependent proportional hazards model. Results: A total of 5358 MM pts were included (any Medicare claims) with 2733 (51%) males. Mutually exclusive race categories were White (W; 67%), African-American (AA; 18%), Hispanic (H; 10%) and Asian (A; 5%). Median follow up was 2.4 yrs. Baseline pt characteristics were significantly different for age with AA being the youngest at diagnosis (p<0.001) and for gender (p<0.001) with W having the largest (53.4%) and AA the smallest (45.2%) proportion of males. Medicare claims capture only pts without any supplemental insurance and so drug utilization in this database was much lower than expected for all subgroups. Year-wise comparison showed that LEN use within the first year of MM diagnosis increased from 2007 to 2009 (16.5% to 27.7%; p<0.001) and there was a significant decrease in median days to first dose (p=0.002). Over the same period, use of THAL decreased (26.6% to 14.6%; p<0.001) while use of BORT (7.8% to 15.4%; p<0.001), SCT (3.3% to 6.2%; p<0.001) and more than 1 novel agent (2.5% to 5.7%; p<0.001) increased significantly. Utilization patterns within first year of MM diagnosis were significantly different among racial subgroups for LEN, THAL, BORT, SCT and >1 novel agent use. (Figure 1) No significant difference was seen for days to first dose of LEN/THAL for any race but for BORT, H got first dose in median 117 days after diagnosis as compared to median 46-51 days for other races (p=0.025). During the first year after diagnosis, W and AA had higher BORT only usage while H and A had a higher LEN/THAL only usage (p<0.001). For drug use by year and race, a significant increase in LEN use was seen from 2007 to 2009 in W (p<0.001), AA (p=0.001) and A (p<0.001) but not H (p=0.26) while THAL use decreased significantly for all races. BORT use increased significantly for all except A (p=0.602) and SCT use increased significantly for all except AA (p=0.068). Inferior OS was significantly associated with older age (p<0.001) and males (p<0.001). LEN/THAL use within the first year of diagnosis did not affect OS while BORT use within the first year of diagnosis increased mortality risk (HR 1.18, p=0.021). SCT use improved OS (HR 0.52, p<0.001). Conclusions: In this comprehensive analysis of MM therapeutics utilization, we noted significant variability among different racial groups. The use of LEN, BORT and SCT has increased while THAL has decreased over time but disparities exist in utilization among different races, with lower use of LEN in AA, lower use of BORT in A and significantly delayed use of BORT in H, which are all notable since racial/ethnic minorities are fast growing in the US population mix and seem to have inequitable utilization and/or access. Inferior survival with early BORT use may represent selection bias of pts with more advanced/aggressive disease. These trends need to be examined in larger sample sizes especially from commercial payers to eliminate drug access and utilization disparities and achieve equitable benefit of therapeutic advances across all racial subgroups. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Ailawadhi: Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy.

Abstract Background: The proteasome is an established and druggable target for the treatment of plasma cell-related malignancies including Waldenstrom macroglobulinemia (WM). WM cells as a consequence of high immunoglobulin production and increased B-cell receptor (BCR) mediated proliferation upregulate proteasome activity. Additionally, microenvironmental influence mediated through BCR signaling directly influences Bcl-2 and its BH3 family members, supporting tumor cell survival. Indeed, WM patients derive significant clinical benefit from proteasome-inhibitor (PI) based therapy with agents such as bortezomib and carfilzomib. However, resistance to PI develops over time and for these patients the optimal choice and sequence of therapy has yet to be determined. Using our WM models of PI-resistance we interrogated molecular events within the BCR and Bcl-2 pathways to determine therapeutic potential of targeting these crucial pathway in PI-resistance. Materials & Methods: WM cell lines (BCWM.1 and MWCL-1) and carfilzomib-resistant (CR) subclones (BCWM.1/CR and MWCL-1/CR) were used in experiments. Gene-expression and long-noncoding (LNC) RNA analysis was performed (Arraystar) and validated by real-time PCR. Bortezomib, carfilzomib, ABT199 and ibrutinib were purchased from Sellekchem. Results: To determine the functional impact of BCR and Bcl-2 signaling in PI-resistance, as well as therapeutic sensitivity of PI-resistant cells to their inhibitors (ibrutinib, ABT-199, respectively), we established and characterized WM cell lines resistant to carfilzomib. BCWM.1/CR cells showed approximately 20-fold resistance to carfilzomib (IC50 = 92.75nM) and MWCL-1/CR cells approximately 10-fold resistance (Fig. 1A). Both CR clones also displayed some cross-resistance to bortezomib. Gene expression and LNC-RNA profiling demonstrated several changes between carfilzomib-resistant vs. sensitive WM cells. Analysis of proteasome-related mRNA revealed downregulation of PSMB5, PSMB1, PSMB2 and PSMB8. Similarly, profiling of BCR-associated genes demonstrated decreased expression of several components, including BTK and SPI1. This observation functionally manifested as reduced sensitivity to the BTK-inhibitor ibrutinib, wherein CR cells displayed 1.5 - 2 fold growth resistance to ibrutinib on 72hr MTS assay. Next we examined the expression of Bcl-2 family members in CR cells. Intriguingly, we observed that Bcl-2 and Mcl-1 were significantly downregulated but XIAP (inhibitor of apoptosis) was significantly increased in CR cells vs. wildtype WM cells- both at transcriptional and translational levels. This suggested that upon acquisition of CR, a transcriptional shift towards XIAP occurs to accommodate sustained therapeutic stress from carfilzomib and maintain steady antiapoptotic composure. To test if the PI-resistant cells have moved away from their survival dependence on Bcl-2, we treated CR cells to increasing concentrations of the Bcl-2-specific inhibitor, ABT199, and as anticipated, CR cells displayed reduced apoptosis in presence of ABT199 compared to wildtype WM cells (32% annexin-V staining vs. 50%, respectively) (Fig. 1B). Conclusions: Our study sheds insight into the differential drivers of PI-resistance particularly towards carfilzomib, in WM cells. We demonstrate that acquisition of CR is associated with downregulation of Bcl-2 and Mcl-1, which is countered by upregulation of XIAP- an event that renders CR cells resistant to ABT199 (as it targets only Bcl-2). Likewise, downregulation of BCR-related components in CR cells was associated with reduced sensitivity towards ibrutinib. These observations suggest that acquisition of resistance to PI such as carfilzomib can impact future treatment with agents such as ibrutinib or ABT199. Our preclinical models provide rationale or early sequencing of ibrutinib or ABT199 in therapeutic planning of WM patients prior to induction of PI resistance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract Background: While B-cell receptor (BCR) signaling is essential for the development, of normal B cells, its aberrant hyper-activation results in neoplastic transformation of B-lymphocytes. Recent investigations using small molecule inhibitors validate the BCR pathway as a valuable target. Bruton’s tyrosine kinase (BTK) is one of the components of a signaling hub that transduces signals from the BCR into the cell for its activation and has been shown to be a therapeutic target. Ibrutinib (PCI-32765), an irreversible BTK inhibitor has shown clinical efficacy in CLL, mantle cell lymphoma (MCL) and Waldenströms macroglobulinemia (WM). Ibrutinib binds to cysteine-481 of the BTK protein and blocks its phosphorylation, resulting in termination of BCR-mediated activation of cells with a concomitant induction of death. Despite the clinical success of ibrutinib, a high percentage of patients achieve only partial response and eventually acquire resistance to the drug, resulting in aggressive relapse of the disease. A mutation of Cys481-Ser in BTK (ibrutinib-BTK binding site) has been reported to be one of the reasons for the development of ibrutinib resistance (IR). To understand the mechanisms resulting in acquisition of IR, we developed preclinical models of IR in WM and MCL. Materials: Ibrutinib was obtained from Pharmacyclics, CA. Validated human WM models (BCWM.1, RPCI-WM1 and MWCL.1 cell lines) and human MCL models (Jeko-1 and Maver cell lines) were used for the study. Results: BTK was constitutively phosphorylated at Y223 and Y551 in all the cell lines tested and this was inhibited by ibrutinib in a dose dependent manner. Phosphorylation of other kinases in the cascade such as SYK (Y323 and Y525/526) and PLCg2 (Y759 and Y1217) were also inhibited while AKT phosphorylation at both Ser473 and Thr308 was consistently increased in presence of ibrutinib. Treatment with ibrutinib induced cell cycle arrest in the G1 phase by 24h followed by apoptosis. Cell growth assays (MTS assay) showed that BCWM.1 was the most sensitive cell line followed by MWCL-1, RPCI-WM1, Maver and Jeko-1. Exposure of WM and MCL cells for prolonged periods of time with progressively increasing concentrations of ibrutinib resulted in outgrowth of clones (IR WM and MCL cell lines) that were resistant to apoptosis with a slow growth rate as compared to wild type parental cells. IR cells attained 2 – 20 fold resistance to ibrutinib as compared to the respective parental lines as determined by MTS assay. Sequence analysis of the BTK gene in all the cell lines revealed no mutation in IR cells at Cys481 suggesting that in an acquired IR state, resistance to ibrutinib can be developed independent of BTK Cys481 mutation. Interestingly, we found p-BTK levels to be markedly reduced in IR cells. Ibrutinib reversal experiments suggested that while a continuous presence of ibrutinib is needed for inhibition of BTK phosphorylation, a stable IR state could be maintained (for >1 month) in the absence of ibrutinib. This suggested the cells reliance on a parallel survival pathway, independent of BTK phosphorylation. Focused mRNA (Nanostring nCounter assay) and immunoblot analysis revealed significant changes in the expression profiles of several cellular elements. These included transcription factors such as PU.1, IRF4, BLIMP1, BCL-6 b-catenin as well as the phosphorylated ERK1/2, STAT1 and 3 suggesting a reprogramming of critical cellular networks, which IR tumor cells might be utilizing to overcome ibrutinib-induced cytotoxicity. Importantly, we observed that IR cells retained high levels of p-AKT and showed an increase in expression of BCL2 family members, as well as BCL-2 itself. Treatment of IR cells with ibrutinib +/- MK2206 (AKT inhibitor), or ABT-199 (BCL-2 inhibitor), synergistically induced cytotoxicity in IR cells, suggesting the importance of these parallel survival pathways (AKT/BCL2) in maintaining an IR state. Conclusion: Here we demonstrate that in the absence of BTK Cys481 mutation, an IR state is associated with reprogramming of transcriptional networks countering ibrutinib-induced toxicity by activation of AKT and BCL-2. Our current data exposes multiple vulnerabilities within IR cells, which can be therapeutically exploited to potentially delay onset of IR, by targeting alternative oncogenic mechanisms that are activated in presence of sustained BTK inhibition. Disclosures No relevant conflicts of interest to declare.

Abstract Background:The pathological behavior of Waldenströms macroglobulinemia (WM) cells relies on aberrant B-cell receptor (BCR), and Toll-like receptor (TLR) signaling and is intricately supported by malfunctions in initiation of apoptosis. In this dynamic, a complex shift between functionally active pro-apoptotic and anti-apoptotic proteins from the Bcl-2 family occurs and bestows the malignant cells with a significant survival advantage. This defective apoptotic signaling is associated with aggressive clinical behavior, chemoresistance, and a poor prognosis in many B-cell cancers, including WM. Thus, BCR/TLR induced proliferation and abnormal apoptotic signaling due to dysregulation of Bcl-2 signaling, act in concert for the assurance of the malignant WM cells survival. Clinically, mitigation of BCR signaling with the first in class BTK inhibitor, ibrutinib, has shown promising results in WM and other B-cell cancers. However, clinical responses to the BTK inhibitor alone are less than optimal and development of ibrutinib-resistant disease is being recognized as an important and impending dilemma. We have previously shown the utility of disrupting pro-apoptotic Bcl-2 protein interactions in WM, chronic lymphocytic leukemia and multiple myeloma and that this functional interference results in re-sensitization (or heightened sensitivity) of tumor cells towards chemoimmunotherapy. As such, we examined the effects of ABT-199, a Bcl-2 specific small molecule inhibitor, in preclinical models of WM. Importantly; we conducted this investigation in proprietarily developed WM cells that have acquired resistance mechanisms towards either ibrutinib (IR WM subclones) or bortezomib (BR WM subclones). Materials:WM cell lines (BCWM.1, MWCL.1 and RPCI-WM1; WT cells) including their bortezomib resistant (BR) or ibrutinib resistant (IR) subclones were used in this study. Results: An initial comparison of Bcl-2 family members, Bcl-2, Mcl-1, BCL-xL, A1 (anti-apoptotic members), Noxa, Puma, Bim (pro-apoptotic initiators) and Bak and Bax (pro-apoptotic effectors), revealed that BR WM cells express increased levels of pro-apoptotic Bcl-2 family members, particularly Bcl-2, relative to WT cells. Similarly, A1 and Bcl-2 levels were up regulated in IR cell lines suggesting that the Bcl-2 mediated survival pathway is involved in resistance to both bortezomib and ibrutinib. Cell growth (MTS) assays using ABT-199 indicated that both BR and IR cells were equally sensitivity to Bcl-2 inhibition, with an IC50 ranging between 2 – 4mM. ABT-199 induced a dose dependent increase in MOMP with concomitant induction of apoptotic cell death, the latter confirmed by observation of PARP-1 cleavage. ABT-199 induced an intrinsic apoptotic program with the activation of caspase 9 and caspase 3 in all the cells tested. This data indicated the Bcl-2 pathway as being relevant and essential in WM cell lines even under drug resistant conditions. To evaluate the priming effect of ABT-199 on bortezomib and ibrutinib induced cell death, MTS assays were performed with these drugs in presence of IC25 concentrations of ABT-199. The addition of ABT-199 significantly reduced the IC50of both bortezomib and ibrutinib in WT, BR and IR cells. This suggested that re-sensitization of BR and IR cells towards either bortezomib or ibrutinib, respectively, was possible in presence of ABT-199. Further, combined addition of ABT-199 with bortezomib or ibrutinib showed synergistic cell death (combination index range: 0.004 – 0.6, CalcuSyn, Biosoft UK) in BR or IR WM cells, respectively. Conclusion: Dysregulation of Bcl-2 signaling is associated with the pathogenesis and promotion of drug resistance in B-cell cancers, including WM. Our report is first to highlight in WM preclinically, the antiproliferative effect of disrupting Bcl-2, through use of ABT-199. Altogether, our data suggests that therapeutic interference of Bcl-2 function with ABT-199 complements the anti-WM activity of ibrutinib or bortezomib in a synergistic manner and can in fact restore sensitivity of either drug in WM cells that are resistant to proteasome or BTK inhibitors. Focused mRNA and protein profiling analyses are currently underway to delineate the mechanisms accounting for synergy observed between concomitant Bcl-2/BTK inhibition as well Bcl-2/proteasome inhibition. Disclosures Ansell: Biothera: Research Funding. Martin:Janssen: Honoraria. Coleman:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Speakers Bureau.