Galardy, Paul J.

Abstract Gene expression profiling has identified two major subclasses of diffuse large B-cell lymphoma. Cases resembling germinal center B-cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-germinal center cells (ABC-DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here we show that UCH-L1 reflects germinal center lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in germinal center B-cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biologic diversity in this subset of disease. We also find that UCH-L1 synergizes with BCL6 in a mouse model of germinal center B-cell lymphoma, but not with the development of multiple myeloma derived from post-germinal center cells. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in germinal center B-cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL. Disclosures Galardy: Mission Therapeutics: Research Funding.

Abstract Abstract 3691 The mTOR kinase is the core component a master regulatory network that controls nutrient-sensitive mRNA translation and the activity of the Akt growth and survival pathway. The importance of this kinase in cancer has made it the target of several new agents for a variety of cancers. Recently, prospective trials have shown strong responses in patients with refractory B-cell non-Hodgkin's lymphomas treated with mTOR inhibitors. We recently demonstrated that the de-ubiquitiinase UCH-L1 is a potent oncogene that drives the development of B-cell lymphomas in mice and is frequently over-expressed in human B-cell lymphomas of a variety of histologies. UCH-L1 promotes signaling through the Akt pathway, though the molecular basis for this is unknown. Here we describe a novel mechanism by which UCH-L1 regulates the mTOR/Akt signaling pathway and provide data suggesting that UCH-L1 expression predicts a poor response to mTOR inhibition in patients with B-cell lymphoma. Depletion of UCH-L1 in malignant B-cells leads to a dramatic decline in Akt phosphorylation that is not due to changes in the levels of the kinases PDK1, mTOR, or any mTOR complex component. We observe, however, a striking UCH-L1 driven increase in the association of mTOR with the mTORC2 component rictor, and a reciprocal decline in the mTORC1 subunit raptor, suggesting that UCH-L1 promotes mTORC2 assembly. This is verified when we introduce UCH-L1 into HeLa cells and observe an increase in the co-precipitation of rictor with mTOR, with a corresponding decrease in the recovery of raptor. Consistent with these results, we find that mTORC1 activity is greatly reduced in cells with high levels of UCH-L1, and conversely is enhanced in tissues from Uchl1 null mice. This effect is entirely dependent on DUB activity, as expression of a catalytic mutant UCH-L1 construct has no effect on complex assembly. We find that UCH-L1 forms a novel association with the ubiquitin-ligase DDB1 and that the formation of this complex leads to the displacement of DDB1 from raptor, which then leads to the destabilization of mTORC1. As expected, depletion of DDB1 itself produces a phenocopy of UCH-L1 over-expression. We hypothesize that the suppressive effect of UCH-L1 effect on mTORC1 signaling would render malignant cells resistant to mTOR inhibition, as they likely have adapted to low mTORC1 activity. To test this hypothesis, we analyzed data from a recent prospective trial in which patients with refractory mantle cell lymphoma received the mTOR inhibitor temsirolimus along with rituximab. Limiting our analysis to those cases staining positive for phospho-Akt (pAkt), we found a strong trend towards UCH-L1 expression associated with a worse prognosis, despite the small numbers of patients (n = 10 pAkt+/UCH-L1– v. 5 pAkt+/UCH-L1+, median overall survival 913 v. 201 days, respectively; p=0.08). We conclude that UCH-L1 is a physiological regulator of mTOR/Akt signaling and that this function is co-opted in the pathogenesis of B-cell malignancies. Our data suggest that UCH-L1 expression is a negative predictor for responsiveness to mTOR inhibition in B-cell lymphoma, a finding that warrants further evaluation in larger cohorts. Disclosures: Off Label Use: Results from a clinical trial using the drug temsirolimus will be discussed.

Abstract Abstract 4182 De-ubiquitinating enzymes (DUBs) are important regulators of many cellular processes through their ability to reverse the modifications installed by ubiquitin ligases. Several members of the DUB family are implicated in human cancers, where they may act as oncogenes or tumor suppressors. There is little direct in vivo evidence, however, for their roles in cancer. We recently reported that the DUB UCH-L1 is a potent oncogene that drives the development of spontaneous B-cell lymphoma in transgenic mice, and is required for the survival of malignant B-cells in vitro. The mechanism of these effects involves the strong induction of Akt signaling that is observed in culture and in lymphoid tissues of Uchl1 transgenic mice. Akt is phosphorylated on two distinct sites by the kinases PDK1 and mTOR, with both phosphorylation events required for maximal signaling. The activity of mTOR towards its substrates is dependent on the binding partners to which it is bound. When mTOR binds to raptor, it forms mTORC1, an important regulator of nutrient sensitive protein translation, whereas when complexed with rictor and SIN1, it forms mTORC2, a critical regulator of Akt signaling. It is unclear how the cell regulates the formation of mTORC1/2. Here we show a novel mechanism by which UCH-L1 regulates mTOR-dependent Akt signaling. By co-immunoprecipitating mTOR and its binding partners, we find that UCH-L1 expression promotes the binding of mTOR with rictor at the expense of raptor, without changing the level of either protein. These data suggest a novel ubiquitin-dependent regulatory step in mTOR complex assembly. In addition, UCH-L1 expression leads to a reduction in the levels of the Akt antagonist phosphatase PHLPP1 in vitro and in vivo. This decline is completely dependent on UCH-L1 DUB activity as expression of a catalytic mutant does not affect levels of PHLPP1. We find that the mechanism of this decline is not due to proteasome degradation, nor transcriptional control. We therefore hypothesize that the UCH-L1 associated shift in cellular mTOR activities leads to reduced mTORC1-dependent translation of PHLPP1. To test this, we inhibited mTORC1 activity by depleting raptor using shRNA and found a dramatic decline in PHLPP1 levels, whereas depletion of rictor had no effect on PHLPP1. We conclude that UCH-L1 boosts Akt signaling through a novel two-step mechanism by which it promotes the generation of mTORC2, reduces the levels of mTORC1, and thereby impairs the translation of PHLPP1. These data have important implications for understanding the roles of ubiquitin, mTOR, and Akt, in lymphoma biology and may lead to more intelligent use of next generation mTOR inhibitors in the treatment of lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract The success of proteasome inhibition in multiple myeloma highlights the critical role for the ubiquitin-proteasome system (UPS) in this disease. However, there has been little progress in finding more specific targets within the UPS involved in myeloma pathogenesis. In an unbiased screen of de-ubiquitinase activity, we previously found the ubiquitin hydrolase UCH-L1 to be frequently over-expressed in B-cell malignancies, including myeloma. Using a transgenic mouse model, we also found that aberrant expression of UCH-L1 is sufficient to drive the development of spontaneous B-cell lymphomas and plasmacytomas, demonstrating its oncogenic activity. Whether and how UCH-L1 affects the clinical behavior of myeloma, however, is not known. Here we show that UCH-L1 is a potent negative prognostic factor that is essential for the dissemination and progression of myeloma. We found high levels of UCHL1 to predict early progression in newly diagnosed patients; a finding reversed in studies including bortezomib. We also found high UCHL1 levels to be a critical factor in the superiority of bortezomib over high-dose dexamethasone in relapsed patients. High UCHL1 partially overlaps with, but is distinct from, known genetic risks including 4p16 rearrangement and 1q21 amplification. Using an orthotopic mouse model, we found UCH-L1 depletion delays myeloma dissemination and causes regression of established disease. We conclude that UCH-L1 is a biomarker of aggressive myeloma that may be an important marker of bortezomib response, and may itself be an effective target in disseminated disease. Disclosures Galardy: Mission Therapeutics: Research Funding.

Abstract Abstract 29 Acute lymphoblastic leukemia (ALL) cells are frequently characterized by abnormal numbers of chromosomes, a condition known as aneuploidy. Aneuploidy in ALL is not just a curiosity, but impacts on the prognosis and risk stratification in children. Additionally, aneuploidy itself contributes to the development of cancer by dysregulating gene expression, and driving the loss of heterozygosity of mutated tumor suppressor genes. The molecular underpinnings of aneuploidy in ALL are largely unknown. The mitotic checkpoint is a complex pathway that acts in mitosis to prevent the premature onset of anaphase that leads to improper segregation of chromosomes and aneuploidy. Through the concerted action of many intermediates, the mitotic checkpoint functions to inhibit the activity of a large ubiquitin E3 ligase complex known as the anaphase promoting complex/cyclosome (APC/C) that drives the separation of paired sister chromatids by effecting the ubiquitin-dependent degradation of cyclin B1 and securin. The de-ubiquitinating enzyme USP44 was recently identified as an important regulator of mitotic progression that assists in maintaining mitotic checkpoint signaling until all chromosomes are properly attached to the mitotic spindle. Microarray studies of childhood acute leukemias have observed high levels of USP44 in T-cell ALL, but the significance of this observation is unclear. Here we show that USP44 is indeed over-expressed in T-ALL in children and that its presence may contribute to aneuploidy that is frequently observed in this disease. We compared the expression of USP44 in a series of 24 samples from patients with T-ALL to that found in ten samples of peripheral T-cells isolated from healthy donors, using quantitative real-time PCR. There was a dramatic increase in the expression of USP44 in T-ALL with 18 out of 24 cases having at least 3-fold increase, with an average of 16-fold increase, in USP44 mRNA. We next studied the consequences of USP44 over-expression in primary cells using live-cell microscopy. We find that excess USP44 leads to dramatic and significant increase in chromosome missegregation with most defects involving lagging chromosomes due to improper chromosome attachment to the mitotic spindle. We observe that these mitotic errors lead to the development of aneuploidy, through a mechanism that requires USP44 catalytic activity. These effects are accompanied by reinforced mitotic checkpoint signaling that leads to delayed progression through mitosis, and a prolonged duration of mitotic arrest in cells exposed to spindle poisons such as nocodazole or paclitaxel. At the molecular level, we observe that excess USP44 leads to enhanced binding of the mitotic checkpoint effector Mad2 to the APC/C and increased levels of cyclin B1 in early mitosis. Taken together, we conclude that USP44 is a chromosome instability gene that is frequently expressed at high levels in T-ALL cells, and that this may contribute to the pathogenesis of T-ALL by leading to chromosome shuffling and aneuploidy. These data have important implications in our understanding of the pathogenesis of this disease and may contribute to understanding the therapeutic effectiveness of novel aurora inhibitors in cancer. Disclosures: No relevant conflicts of interest to declare.

Abstract DUB-2 is a T-cell specific de-ubiquitinating enzyme whose expression is induced by IL-2. Expression of DUB-2 results in prolonged STAT5 phosphorylation, enhanced IL-2 induced gene expression, and resistance to apoptosis resulting from cytokine withdrawal. The molecular basis of DUB-2s effects, however, is not understood. In order to better understand the role of DUB-2 in the IL-2 response, we performed a systematic analysis of DUB-2 catalytic activity, subcellular localization, and domain organization. Subcellular localization of DUB-2 was examined using a C-terminal DUB-2-Cherry (enhanced red fluorescence) fusion and live-cell imaging. When expressed in HeLa cells, wild-type DUB-2-Cherry is predominantly localized to the cytoplasm, although 8–10% of cells have a strong nuclear accumulation. Strikingly, the nuclear localization is entirely eliminated by a mutation in the catalytic cysteine (c60s). This effect on DUB-2s localization is not due to changes in cell cycle progression, as we observe identical cell cycle profiles (DNA content) in wt or c60s expressing cells. Conversely, enhanced DUB-2 nuclear localization is seen following nocodazole-induced cell cycle synchronization and with mutant DUB-2-Cherry fusions lacking a duplicated 8-residue sequence within the C-terminal domain. We next analyzed the catalytic activity of DUB-2 toward ubiquitin and other ubiquitin-like (UbL) molecules using a series of semi-synthetic activity-based probes. These probes consist of full-length Ub or UbL molecules equipped with a C-terminal electrophilic group. These probes act as suicide substrates, forming a covalent bond with the active site cysteine of catalytically active (but not inactive) Ub(L) isopeptidases. Unexpectedly, DUB-2 forms adducts not only with the Ub-based probe, but also the probe derivative of the UbL molecule ISG15, which suggests that it has dual-specificity. The activity towards Ub and ISG15 is abrogated by a mutation in the catalytic cysteine (c60s) of DUB-2, but is not affected by deletions of the poly-lysine or hypervariable domains. Taken together, these results suggest that DUB-2 is a bi-specific (Ub and ISG15) isopeptidase which shuttles between the cytoplasm and the nucleus in a manner dependent on its catalytic activity. This work, and the ultimate elucidation of the molecular target of DUB-2, will further increase our understanding of DUB-2’s role in IL-2-dependent T-cell proliferation and immunologic tolerance.

Abstract BACKGROUND: Primary Mediastinal B-Cell Lymphoma (PMBL) is a rare form of B-cell non-Hodgkin lymphoma (Lones/Cairo et al, JCO 2000). We have previously reported a significant decrease in EFS among pediatric and adolescent patients with PMBL compared with other stage III non-PMBL diffuse large B-cell lymphoma (DLBCL) patients following FAB/LMB 96 therapy, suggesting that new therapeutic targeted agents are needed (Gerrard/Cairo et al, Blood 2013). Activation of the B-cell receptor (BCR) signaling pathway has now emerged as a central oncogenic pathway that promotes growth and survival in both normal and malignant B-cells. The cascade of signaling pathways downstream of the BCR includes BTK, MAPK, NF-κB and AKT. PMBL has a constitutively activated NF-kB pathway (Rosenwald et al, JEM 2003). Ibrutinib is a selective and covalent BTK inhibitor that inhibits chronic active BCR signaling and downstream signaling including the NF-kB pathway (Herman et al, Blood 2014) and is an active agent in ABC-DLBCL, which also has an activated NF-kB pathway (Griner et al, PNAS 2014). Preclinical studies of ibrutinib in CLL and MCL suggested that the inhibitory effects on cell proliferation were seen in the range of 1.0uM to 25.0uM (Herman et al, Blood 2011; Cinar et al, Leuk. Res 2013). Despite these relatively high IC50 values in vitro, ibrutinib has been highly effective in the treatment of patients with refractory CLL and MCL (Byrd et al, NEJM 2013 and Wang et al, NEJM 2013). Ibrutinib was approved by the FDA (IMBRUVICA, USPI) for patients with MCL in November 2013 or CLL in February 2014, who have received at least one prior therapy. However, the antitumor activity against PMBL of ibrutinib alone and in combination with other agents is currently unknown. OBJECTIVES: We hypothesize that ibrutinib in combination with other agents may be a future targeted agent in the treatment of PMBL. Therefore, we investigated the effect of ibrutinib alone and in combination with dexamethasone, rituximab and carfilzomib on cell proliferation and apoptosis in a PMBL cell line. METHODS: The Karpas-1106P PMBL cell line (DSMZ, Germany) was treated with ibrutinib alone (0-10uM, generously provided by Janssen R&D LLC) and in combination with dexamethasone (1uM), rituximab (20ug/ml) and carfilzomib (5nM) for 5 days treatment. The MTS cell proliferation assay (Promega) and western blot analysis were performed. Significant differences were determined by Student's t -test.The IC50 values were determined with CompuSyn software (Chou and Martin, ComboSyn, 2005). RESULTS: We observed a significant down-regulation of phosphorylated BTK after 5 days with ibrutinib treatment in Karpas-1106P cell line with 1uM ibrutinib (0.178 ± 1.63, p<0.0001) and the expression of total BTK protein was also significantly decreased with 1uM ibrutinib following 5 days of treatment (0.803±0.190, p<0.005) compared to control (1.000±0.00). We also observed a significant dose-dependent decrease in cell proliferation (0-10uM, p<0.0001) in ibrutinib-treated Karpas-1106P PMBL cell line. In combination with dexamethasone, the ibrutinib IC50 values for inhibition of cell proliferation were significantly decreased from 0.71uM ± 0.172 (ibrutinib alone) to 0.04uM ± 0.007 (p<0.005) with 1.0uM dexamethasone combination following 5 days of treatment. Second, in combination with rituximab experiment, the ibrutinib IC50 values was significantly decreased from 0.845uM ± 0.289 (ibrutinib alone) to 0.37uM ± 0.056 (p<0.05) with 20ug/ml rituximab following 5 days of treatment. Lastly, the ibrutinib IC50 values were significantly decreased from 0.72uM ± 0.185 (ibrutinib alone) to 0.002uM ± 0.001 (p<0.005) with 5nM carfilzomib combination following 5 days of treatment. CONCLUSIONS: Ibrutinib alone and in combination with dexamethasone, rituximab and carfilzomib significantly inhibited cell proliferation in the Karpas-1106P PMBL cell line. Ibrutinib's potential as an adjuvant agent in combination treatment in patients with PMBL is supported by these experiments. Future studies will focus on the in vivo effects of ibrutinib alone and in combination with dexamethasone and carfilzomib in a NOD/SCID PMBL xenograft mouse model. Disclosures Galardy: Mission Therapeutics: Research Funding.

AbstractThe deubiquitinase (DUB) ubiquitin C‐terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small‐molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine‐based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

SummaryIn the modern era, clinicians and pathologists increasingly make challenging diagnoses in patients with suspected lymphoma using minimal amounts of diagnostic material. The increase in utilization of minimally invasive procedures, such as fine needle aspiration or needle core biopsies, lead to challenges in our ability to make accurate histopathological assessments of disease, including the integration of new diagnostic and prognostic testing, with smaller amounts of material. The trend towards minimally invasive diagnostics is also often in conflicting interest with researchers seeking to study tissue specimens to better understand the biology and genetics of these diseases to move the field forward. Thankfully, there are emerging fields which seek to extract large amounts of diagnostic and prognostic data out of material that is circulating in the blood of patients with lymphoma. Here we will review recent exciting data regarding the use of circulating tumour cells, circulating tumour DNA, and the detection and utility of circulating exosomes and how it can assist in diagnosis, prognosis and therapeutic monitoring. These advances hold the promise to enable continued safe patient care while also advancing discovery, translational and clinical research.