Kuranz, Maja

Background The ubiquitin proteasome system (UPS) maintains homeostasis in eukaryotic cells by degrading and recycling proteins associated with DNA transcription and cell cycle regulation. In tumor cells with a high protein turnover rate, particularly in immunoglobulin secreting cancers such as Waldenströms Macroglobulinemia (WM), proteasomes are critical for sustained cell proliferation. Proteasome inhibitors (PI) such as bortezomib and carfilzomib target the B5 subunit of the proteasome catalytic core and disrupt its activity leading to accumulation of polyubiquitinated proteins. Although highly effective in WM, persistent use of bortezomib eventually leads to emergence of a bortezomib resistant (BR) subclone. Point mutation in the PSMB5 gene (encoding the B5 subunit) leading to altered binding kinetics is identified as a potential underlying mechanism of PI resistance. Despite these adaptive modifications that support resistance to PI resistance, the UPS as a whole remains a relevant target with vulnerable sites that can be therapeutically exploited. The 19S cap of the proteasome contains deubiquitinating enzymes (DUBS) that function to deubiquitinate proteins prior to their entry into the proteasomal core. Using a novel DUB-inhibitor b-AP15, which blocks the UCHL5 and USP14 DUBS, D’Arcy et al demonstrated that disruption of these DUBS does not alter proteasomal activity yet induces robust tumor cell death in various cancers. Aim Investigation of b-AP15 in preclinical models of BR WM and characterization of associated downstream signaling interactions. Methods For these studies, we developed and used BR variants of the most commonly used WM cell lines (variants termed BCWM.1/BR, RPCI-WM1/BR and MWCL-1/BR). Proteasomal activity was measured in these cells using synthetic fluorogenic peptide substrates and apoptosis was measured by annexin-v/PI staining. For gene expression profiling (GEP), the Nanostring nCounter mRNA quantification assay was utilized. Results We treated all BR WM clones with either single agent bortezomib (10nM), carfilzomib (10nM), b-AP15 (100nM) or all three agents in combination and assessed their effect on chymotrypsin-like, caspase-like and trypsin-like proteasomal activity. b-AP15, either alone or in combination with the other PI, did not alter proteasomal activity, nor did it abrogate the inhibition of chymotrypsin-like activity of other PI. Although proteasomal activity was not disrupted, we observed an increase in high molecular weight ubiquitinated proteins on immunoblot analysis. We hypothesized that despite the BR cells resistance to B5-targeting PI, they would remain sensitive to UPS inhibition if targeted through DUB inhibition. We treated BR cells with b-AP15 at concentrations ranging from 50nM - 1.25uM and observed that b-AP15 induced robust WM cell death in a dose and time dependent manner. Apoptosis was more pronounced in MWCL-1/BR cells and optimal cell death (55%) was observed at 500nM; however a commitment towards cellular demise was evident by PARP-1 and caspase cleavage as early as 3 hrs. Use of a pan-caspase inhibitor resulted in rescuing of the BR WM cells from b-AP15 mediated death and demonstrated a caspase-dependent death process. In order to gain further mechanistic insight into the processes that regulates response to b-AP15, we performed GEP and immunoblot of BR WM cells treated with 50nM of b-AP15. We observed an increase in phospho-p38 protein, which belongs to the MAPK family and regulates IL8 secretion. When BR WM cells were treated with a p38 inhibitor in combination with b-AP15, a significant decrease in cell viability was noted relative to b-AP15 (100nM) alone. Furthermore, GEP and network analysis identified key regulator genes associated with p53, cell cycle and MAPK signaling. Summary We provide first evidence that inhibition of the DUBS, UCHL5 and USP14 with b-AP15 results in caspase-dependent apoptosis of BR WM cells. Our investigation uncovers important molecular interactions that may dictate the BR cells responsiveness to DUB-inhibition by b-AP15. These observations validate the pervasive role of the UPS and confirm alternate therapeutics targets using our unique BR WM models. We are grateful to the Waterfall Waldenström Macroglobulinemia Research Fund, the Leukemia and Lymphoma Society and the International Waldenström Macroglobulinemia Foundation for their continued support. Disclosures: Martin: Teva: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech: Speakers Bureau; Millennium: Research Funding; Seattle Genetics: Consultancy, Speakers Bureau.

Abstract Background Waldenstršms 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. Aim The 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. Methods The 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. Results After 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. Summary Thus 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.

Abstract Background Multiple 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. Aim Since 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. Methods Human 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. Results Gene 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. Summary In 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.

Abstract Background In the U.S. over 14,000 new cancer cases occur annually in people under 21 years of age; approximately 21% of cases are acute lymphoblastic leukemia (ALL) and ∼ 30% classify as Burkitt lymphoma (BL). Most cases of ALL and BL are due to malignant B-cells that have acquired disease-defining oncogenic lesions that facilitate their rapid proliferation. To maintain a state of homeostasis, the malignant cells rely heavily on the ability of the internal degradation and recycling systems to match the high rate of protein turnover. As such, the ubiquitin proteasome system (UPS) is central in this operation where it prevents buildup of misfolded proteins while also regulating the availability of anti-apoptotic proteins such as BCL2 and MCL1. These observations coupled with the observation that UPS disruption leads to cellular demise have resulted in the development of several proteasome inhibitors (PI). These PI have primarily been examined in adult B-cell malignancies. The feasibility of a bortezomib-containing regimen for use in pediatric malignancies has been examined by the TACL consortium and demonstrated a 73% response rate in relapsed/refractory ALL patients. However, a major limitation of bortezomib-based therapy is an increased incidence of peripheral neuropathy (PN), which was also noted in the TACL study. As such, more potent and reportedly less toxic PI have been developed that seem to carry considerably lower risk for development of PN. Such agents that maintain a high response rate and carry reduced long-term adverse effects are highly desirable in pediatric age patients. Aim Investigation of the novel proteasome inhibitors carfilzomib and MLN9708 (Millennium Pharmaceuticals) in preclinical models of pediatric B-lymphocytic cancer. Methods Representative preclinical models of BL (Raji cell line) and B-ALL (ALL-1 cells) were used in this study. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Cell viability was measured by MTS assay. Apoptosis was determined by annexin-v/PI staining and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiling was performed via immunoblot. Results In an effort to understand the effect of carfilzomib and MLN9708 in our pediatric cancer models, we first assessed their ability to inhibit proteasomal activity. While chymotrypsin-like activity, which is the target of both PIs, is inhibited by more than 90% in both cell lines, inhibition (∼ 35%) of caspase-like function conferred by the b1 subunit was more significantly pronounced in MLN9708 treated Raji and ALL-1 cells. Next we performed an MTS assay to determine the growth inhibitory effects of these PI in vitro. Both PI inhibited the growth of the Raji and ALL-1 cells in a dose dependent manner. Carfilzomib showed a more potent effect (IC50 ∼1nM in both cell lines) as compared to MLN9708 (IC50 ∼30nM in ALL-1 cells and ∼50nM in Raji cells). To further understand mechanisms of growth inhibition we examined both tumor cell lines for induction of apoptosis along with corresponding markers. As compared to Raji cells, we observed ALL-1 cells to be more sensitive to the effects of both PI, albeit with MLN9708 (50nM) inducing more cell death (65%) as compared to carfilzomib (10nM, 50% cell death). Further, we observed altered MOMP, cleavage of caspases 9, 3 and PARP-1 were found to occur in both cell lines in presence of carfilzomib or MLN9708. These changes indicate the apoptotic effects of novel PI in ALL-1 and Raji cells are via mitochondrial destabilization and caspase activation. As inhibition of the UPS aids NOXA and BIM (pro-apoptotic BCL2 proteins) mediated cell death, we examined the expression of BCL2 family proteins changes in Raji and ALL-1 cells in response to novel PI. Indeed protein levels of both BCL2 and MCL1 were reduced in carfilzomib and MLN9708 treated cells. Reduced BCL2 expression was more pronounced in ALL-1 cells as compared to Raji cells and downregulation was noted to be dose-dependent. Summary Our data show that both carfilzomib and MLN9708 potently inhibit the viability of malignant ALL and BL cells in vitro. We conclude that PI are promising anti-neoplastic therapeutics that target not only the UPS but also modulate the expression of critical anti-apoptotic proteins. Data from mechanistic studies conducted herein underscore the need for further research in in vivo models of these diseases. Disclosures: Foran: Celgene: Research Funding.

Abstract Background Waldenströms 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. Aim To define the anti-WM activity of the novel DUB inhibitor, b-AP15 in preclinical models of WM. Methods The 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. Results As 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. Conclusion b-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.

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.