Selective Inhibition of the Chymotrypsin-Like Activity of the Immunoproteasome and Constitutive Proteasome Represents a Valid Anti-Tumor Strategy in Waldenstrom Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4911-4911
Author(s):  
Aldo M. Roccaro ◽  
Antonio Sacco ◽  
Monette Aujay ◽  
Hai Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Parp Cleavage ◽  
Low Grade ◽  
Employment Equity ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Synergistic Cytotoxicity ◽  
Equity Ownership

Abstract Abstract 4911 Introduction Proteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom macroglobulinemia (WM), where single-agent Bortezomib has been tested in phase 2 clinical trials, achieving 40% to 80% responses. Nevertheless, a significant fraction of patients relapse, or develop significant neuropathy. Therefore preclinical evaluation of new proteasome inhibitor is needed in order to improve patient outcome. We tested PR-047, a new orally bioavailable analog of carfilzomib which selectively inhibits the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome, in WM. Methods WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity, DNA synthesis, cell cycle and apoptosis were measured by thymidine uptake, MTT, PI staining/flow cytometry analysis and DNA fragmentation, respectively. NF-kB activity has been evaluated on nuclear proteins using a DNA-binding ELISA-based assay. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method. Results Primary bone-marrow derived WM cells are characterized by higher expression of the immunopreoteasome as compared to the constitutive proteasome. PR-047 inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, leading to induction of cytotoxicity in primary WM cells; as well as to programmed cell death in a caspase-dependent and -independent manner, as shown by activation of c-jun-N-terminal kinase; inhibition of NF-kB; and initiation of the unfolded protein response. PR-047 induced cytotoxicity and inhibited DNA synthesis in primary WM cells (IC50: 50-80nM), as well as in IgM secreting low grade lymphoma cells (IC50: 30-50nM). Importantly, PR-047 exerted cytotoxicity in WM cells, even in the context of bone marrow milieu, by inhibiting IL-6- and IGF1-BMSC secretion and BMSCs-induced phosphorylation of Akt and ERK in WM cells. Moreover, combination of PR-047 and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced caspases-, PARP-cleavage; NF-kB inhibition; and synergy in inhibiting the chymotrypsin- and caspase-like activities of the immunoproteasome and constitutive proteasome. Conclusion These preclinical findings demonstrate that PR-047 targets WM cells, due to its anti-CT-L activity of both immunoproteasome and constitutive proteasome, providing the framework for testing PR-047 in this disease. Disclosures Aujay: Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Carfilzomib Exerts Anti-Neoplastic Activity in Waldenstrom Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4916-4916
Author(s):  
Antonio Sacco ◽  
Aldo M. Roccaro ◽  
Monette Aujay ◽  
Hai Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Lines ◽  
Low Grade ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Employment Equity ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Synergistic Cytotoxicity ◽  
Equity Ownership ◽  
Dose Dependent

Abstract Abstract 4916 Introduction Proteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom's macroglobulinemia (WM), where single-agent Bortezomib has been successfully tested in phase 2 clinical trials. Nevertheless, a significant fraction of patients relapse, or develop significant toxicity due to high toxicity in non-transformed cells. Therefore preclinical evaluation of new proteasome inhibitor with a more selective inhibition of neoplastic cells is needed in order to increase efficacy and improve patient outcome. We tested Carfilzomib, a tetrapeptide epoxyketone selective inhibitor of the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in WM. Methods WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity and DNA synthesis were measured by thymidine uptake and MTT, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method. Results Primary CD19 bone-marrow derived WM cells express higher level of the immunopreoteasome as compared to the constitutive proteasome. Carfilzomib inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, in a dose-dependent manner; leading to inhibition of proliferation (IC50: 5nM; 48h) and induction of cytotoxicity (IC50: 7.5nM; 48h) in WM cells. Carfilzomib mediated apoptosis in WM by increasing PARP-, caspase-9- and -3-cleavage; as well as by inducing activation of c-jun-N-terminal kinase and ER-stress in a dose-dependent manner. Moreover, combination of Carfilzomib and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced PARP-, caspase-9- and -3-cleavage; and synergy in inhibiting the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome. Conclusion Taken together, these findings provide the pre-clinical rational for testing Carfilzomib in Waldenstrom Macroglobulinemia. Disclosures Aujay: Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

The Combination of Bortezomib and NPI-0052 Exerts Anti-Tumor Activity in Waldenstrom Macroglobulinemia (WM).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1516-1516 ◽  
Author(s):  
Aldo Roccaro ◽  
Xavier Leleu ◽  
Antonio Sacco ◽  
Xiaoying Jia ◽  
Anne-Sophie Moreau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Parp Cleavage ◽  
Caspase 8 ◽  
Low Grade ◽  
Dependent Manner ◽  
Secreting Cell ◽  
Akt Phosphorylation

Abstract Background: WM is an incurable low-grade lymphoplasmacytic lymphoma. Bortezomib has recently demonstrated about 50% ORR in patients with relapsed WM. We therefore investigated the in vitro effect of the new proteasome inhibitor NPI-0052 (N) alone and in combination with Bortezomib (B). Methods: WM cell lines (BCWM1,WSU-WM) and IgM secreting cell lines (MEK1, Namalwa) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured using MTT assay and [3H]-thymidine uptake. Determination of the synergistic effect [combination index (CI)] of combination was calculated using the CalcuSyn software. Cell signaling and apoptotic pathways were determined by Western Blot. We also tested the effect of N on WM cells in the co-culture with BMSCs. Activity of the 20S proteasome was determined by detecting the release of the fluorophore AMC, after cleavage from the labeled substrates specific for each enzymatic activity. Results: N induced cytotoxicity and inhibition of DNA synthesis (IC50 15nM) in BCWM.1 (48 h). Similar effects were demonstrated in IgM secreting cell lines and primary CD19+ WM cells (IC50 18–30nM). No cytotoxicity was observed on peripheral blood mononuclear cells. The combination of N+B significantly inhibited BCWM.1 proliferation compared to each agent alone: B (5nM) induced cytotoxicity in 8.5%, which increased to 26%, 40% and 53% in the presence of N 2.5nM (CI:0.83), 5nM (CI:0.72) and 10nM (CI:0.7) respectively, indicating synergism. To determine the mechanism of synergy, we investigated the effect of the two agents and their combination on proteasome activity and on signaling pathways, specficially the Akt pathway. Both N and B inhibited the three proteasome activities: the combination of N+B was increased compared to the effect of each agent alone on the caspase-like (C-l) activity of the proteasome. B and N used as single agents induced 29% and 34% inhibition of the C-l activity, respectively, compared to 60% when B and N were used in combination. The C-l activity preferentially cleaves substrates with an aspartic residue in P1 position, like the substrates of the caspases. N induced caspase-8, PARP cleavage and increase of Smac as well as down-modulation of the anti-apoptotic proteins c-IAP, XIAP, survivin, Bcl-2, Mcl-1. The combination of N+B induced a stronger and more significant induction of caspase-8, -PARP cleavage, as well as caspase-3 and -9, which were not affected by using N alone. Similarly, Smac modulation resulted in a more significant induction when cells were exposed to both proteasome inhibitors. N inhibited Akt phosphorylation in BCWM.1 cells (6h) in a dose-dependent manner. GSK3 phosphorylation and ribosomal protein-S6, Akt-downstream target proteins, were also markedly inhibited. Importantly, N inhibited Akt phosphorylation and Akt activity in BCWM.1, even when combined with B, which induced increase of Akt phosphorylation. Lastly, neither exposure to IL-6 nor adherence to BMSCs conferred protection to WM cells against NPI-induced cytotoxicity. Conclusion: NPI-0052 has significant antitumor activity in WM in vitro especially in combination with Bortezomib. These results provide the framework for clinical trials in WM.

scholarly journals Dual targeting of the proteasome regulates survival and homing in Waldenström macroglobulinemia

Blood ◽  
2008 ◽  
Vol 111 (9) ◽  
pp. 4752-4763 ◽  
Author(s):  
Aldo M. Roccaro ◽  
Xavier Leleu ◽  
Antonio Sacco ◽  
Xiaoying Jia ◽  
Molly Melhem ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Proteasome Inhibitors ◽  
B Cell Lymphoma ◽  
Parp Cleavage ◽  
Low Grade ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Synergistic Cytotoxicity

AbstractWaldenström macroglobulinemia (WM) is an incurable low-grade B-cell lymphoma characterized by high protein turnover. We dissected the biologic role of the proteasome in WM using 2 proteasome inhibitors, NPI-0052 and bortezomib. We found that NPI-0052 inhibited proliferation and induced apoptosis in WM cells, and that the combination of NPI-0052 and bortezomib induced synergistic cytotoxicity in WM cells, leading to inhibition of nuclear translocation of p65NF-κB and synergistic induction of caspases-3, -8, and -9 and PARP cleavage. These 2 agents inhibited the canonical and noncanonical NF-κB pathways and acted synergistically through their differential effect on Akt activity and on chymotrypsin-like, caspaselike, and trypsinlike activities of the proteasome. We demonstrated that NPI-0052–induced cytotoxicity was completely abrogated in an Akt knockdown cell line, indicating that its major activity is mediated through the Akt pathway. Moreover, we demonstrated that NPI-0052 and bortezomib inhibited migration and adhesion in vitro and homing of WM cells in vivo, and overcame resistance induced by mesenchymal cells or by the addition of interleukin-6 in a coculture in vitro system. Theses studies enhance our understanding of the biologic role of the proteasome pathway in WM, and provide the preclinical basis for clinical trials of combinations of proteasome inhibitors in WM.

scholarly journals Waldenström Macroglobulinemia: Unusual Presentation With Cast Nephropathy/Light Chain Tubulopathy

2019 ◽  
Vol 12 ◽  
pp. 117954761982870
Author(s):  
Muddasir Ashraf ◽  
Prerna Rastogi
Keyword(s):  
Bone Marrow ◽  
Light Chain ◽  
Case Reports ◽  
Interesting Case ◽  
Medical Attention ◽  
Low Grade ◽  
Waldenström Macroglobulinemia ◽  
Cast Nephropathy ◽  
Waldenstrom Macroglobulinemia ◽  
Total Bone Marrow

Cast Nephropathy/Light chain tubulopathy is usually present in patients with multiple myeloma and is very rare in patients with Waldenstrom Macroglobulinemia. There are very few case reports mentioned in the literature. We present an interesting case of Cast Nephropathy and light chain tubulopathy in an 81-year-old female patient with Waldenstrom Macroglobulinemia who required medical attention for worsening renal failure. Serum protein electrophoresis/Immunofixation showed IgM Kappa monoclonal gammopathy. Renal biopsy was remarkable for cast nephropathy and light chain tubulopathy. Furthermore on bone marrow biopsy a low grade B cell lymphoproliferative disorder with plasmacytic differentiation was present. This was most consistent with lymphoplasmacytic lymphoma, accounting for 50-60 percent of total bone marrow cellularity, in a hypercellular (60-80 percent) bone marrow.

Resveratrol Plus Simvastatin Are Effective in Decreasing IgM Secretion in Asymptomatic Waldenstrom Macroglobulinemia. One Year Follow-up.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4401-4401
Author(s):  
Francesco Iuliano ◽  
Stefania Infusino ◽  
Alessia Perricelli ◽  
Massimo Di Maio ◽  
Angelo Pomillo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Vitro Activity ◽  
Bone Marrow Infiltration ◽  
In Vitro Activity ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Asymptomatic Patients

Abstract Abstract 4401 Background Waldenström macroglobulinemia (WM) is a distinct B-cell lymphoproliferative disorder characterized by lymphoplasmacytic bone marrow infiltration along with an immunoglobulin M (IgM) monoclonal gammopathy. Asymptomatic patients with monoclonal IgM and without morphologic evidence of bone marrow infiltration < 10% clonal marrow cells) are classified as having IgM-MGUS.Therapy is postponed for asymptomatic patients, and progressive anemia is the most common indication for initiation of treatment. Resveratrol (3,4',5-tri-hydroxy-trans-stilbene) is an antioxidant constituent of a wide variety of plant species including grapes. It has gained considerable attention because of its anticancer properties, as shown in solid and hematologic malignancies. Published data show that resveratrol has significant antitumor activity in WM cells line. Moreover, simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, induced inhibition of proliferation, cytotoxic effect and apoptosis in IgM secreting cell lines as well as in primary CD19(+) WM cells. With this background we have treated 4 patients with asymptomatic WM with an association of simvastatin and resveratrol to test the efficacy of such of drugs in asymptomatic Waldenstrom macroglobulinemia Methods 4 pts (3 males and 1 female), median age 42,3 yrs (range, 42–73) and asymptomatic WM were treated with a schedule containing resveratrol 40 mg/die and simvastatin 20 mg/die for at least 90 days. At enrollment patients characteristic were hemoglobin level median, 12.1 g/dL,serum beta(2)-microglobulin level median, 2.4 mg/L, and IgM peaks median, 1.8 g/dL. All patients have taken regularly the drugs and there have been no adverse events.CK and LDH serum levels were kept in the normal range. Results In all IgM-MGUS patients a reduction of more than 50% of the IgM peak was observed after 3 months of therapy and it was still maintained at 12 months of follow-up. In SWM patient the reduction was about 25% and it was manteined over time. Striking, another patient with Waldentrom disease resistant to the previous therapy with EDX and anti-CD20 MoAb achieved a CR only after adding resveratrol and simvastatin. Conclusions Our data demonstrate clearly that the association between resveratrol with simvastatin decreases IgM secretion in Waldenstrom macroglobulinaemia and can be useful in asymptomatic or low risk patients not having any adverse effects. Disclosures: Off Label Use: Simvastatin showed in vitro activity on waldentrom cell lines Resveratrol showed in vitro activity on waldenstro cell lines.

Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5028-5028 ◽  
Author(s):  
Deepak Sampath ◽  
Elizabeth Punnoose ◽  
Erwin R. Boghaert ◽  
Lisa Belmont ◽  
Jun Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Single Agent ◽  
Employment Equity ◽  
Bone Marrow Biopsies ◽  
Xenograft Models ◽  
Equity Ownership

Abstract Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

The Proteasome Inhibitor NPI-0052 in Combination with Bortezomib Induces Antitumor Activity in Waldenstrom Macroglobulinemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4746-4746
Author(s):  
Xiaoying Jia ◽  
Xavier Leleu ◽  
Anne-Sophie Moreau ◽  
Evdoxia Hatjiharisi ◽  
Hai Ngo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Single Agent ◽  
Low Grade ◽  
Inhibition Of Proliferation ◽  
Inhibition Of Dna Synthesis ◽  
Vitro Effect

Abstract Background: Waldenstrom’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma. Recently, Bortezomib has demonstrated clinical activity in patients with relapsed WM. The purpose of this study was to investigate the in vitro effect of the new proteasome inhibitor NPI-0052 (Nereus Pharmaceuticals, CA), alone and in combination with Bortezomib, in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa) were used. Bone marrow primary CD19+ malignant cells were obtained from WM patients after informed consent. Inhibition of growth was measured using MTT assays. DNA synthesis was measured using the thymidine incorporation assay. Apoptosis was determined using Apo2.7 staining, followed by flow cytometric analysis. Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method with the combination index (CI)<1.0 indicating synergism. Results: NPI-0052 induced cytotoxicity and inhibition of DNA synthesis, with an IC50 of 20–30nM in all cell lines tested at 24 hrs. NPI-0052 induces 50% apoptosis at 48 hrs. Similar effects were demonstrated in primary CD19+ WM cells. Bortezomib induced cytotoxicity and inhibition of DNA synthesis with an IC50 of 20–30nM in all cell lines tested. Importantly, the combination of NPI-0052 and Bortezomib induced significant inhibition of proliferation compared to each agent alone, specifically at the combination of 2.5nM and 5nM of Bortezomib and 10nM of NPI-0052. For example, Bortezomib single agent at 2.5nM and 5 nM induced 8% and 13% cytotoxicity in BCWM.1 in vitro, respectively, while the combination Bortezomib and NPI-0052 10nM induced a synergistic cytotoxic effect, 44% (CI=0.7) and 58% (CI=0.47), respectively. The combination effect of NPI-0052 20 nM with Bortezomib 2.5nM and 5nM was also synergistic, with 61% (CI=0.7) and 66% (CI=0.7) cytotoxicity, respectively. Similar effects were observed in 3 primary CD19+ WM cells obtained from patients’ bone marrow. Conclusion: NPI-0052 has significant anti-tumor activity against WM in vitro, especially in combination with Bortezomib. These results provide the framework to further study the potential therapeutic role of NPI-0052 in WM. *XJ and XL are co-first authors.

Regulation of Histone Deacetylase in Waldenström Macroglobulinemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2617-2617
Author(s):  
Xiaoying Jia ◽  
Aldo M. Roccaro ◽  
Abdel Kareem Azab ◽  
Hai T. Ngo ◽  
Antonio Sacco ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Western Blot ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Parp Cleavage ◽  
Low Grade ◽  
Dependent Manner ◽  
Apoptotic Pathways ◽  
Dose Dependent

Abstract Background: Waldenström’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Histone deacetylase (HDAC) inhibitors represent promising new treatment strategy in B cell malignancies. We therefore investigated the in vitro effect of the novel hydroxamic acid derivative HDAC inhibitor LBH589 in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTS assay and thymidine uptake assay. Cell signaling and apoptotic pathways were determined by Western Blot and immunofluorescence. Results: LBH589 induced a significant decrease of proliferation and triggered cytotoxicity in all cell lines tested and primary CD19+ WM cells (IC50 of 20–40nM), even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, LBH589 did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells. LBH589 induced both intrinsic and extrinsic apoptotic pathways, with caspase-9, caspase-8, caspase-3, and PARP cleavage in a dose-dependent manner. We also demonstrated significant upregulation of the proapoptotic transcription factor p53 and down-regulation of the anti-apoptotic proteins BclxL, Mcl-1 and c-myc. We then demonstrated that LBH589 induced apoptosis in WM cells in a caspase-independent manner through induction of autophagy, as shown by upregulation of LC3B and Rab7 expression. We further determined the mechanism of action of LBH589 in WM, investigating the effect of LBH589 on histone acetylation and NF-kB pathways. We found that LBH589 induced a dose-dependent increase in histone H3-H4 acetylation; and inhibited both canonical and non-canonical pathways of NF-κB, as shown by western blot and immunofluorescence. In addition, LBH589 augmented rituximab, fludarabine, bortezomib, and perifosine-induced cyotoxicity in WM cells. Conclusion: LBH589 has significant antitumor activity in WM in vitro, providing the framework for clinical trials evaluating LBH589 as a new therapeutic agent in patients with WM.

AMG 900, a Potent and Highly Selective Aurora Kinase Inhibitor Shows Promising Preclinical Activity Against Acute Myeloid Leukemia Cell Lines In Vitro and In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3823-3823 ◽  
Author(s):  
Kam Cheung ◽  
Gloria Juan ◽  
William Wayne ◽  
Kelly Hanestad ◽  
Kathleen Keegan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Phase 1 ◽  
Tumor Burden ◽  
Drug Holiday ◽  
Vehicle Group ◽  
Employment Equity ◽  
Aurora Kinases ◽  
Equity Ownership

Abstract Aurora kinases A and B play essential roles in multiple stages of mitosis and are frequently overexpressed in a subset of human cancers, including acute myeloid leukemia (AML) (Ikezoe T et al, 2007). AMG 900, a potent and highly selective small molecule inhibitor of aurora kinases, is currently in Phase 1 clinical testing in adult patients with AML. In this study, we report the preclinical effects of AMG 900 in AML cell lines. We show that AMG 900 inhibits the phosphorylation of Histone H3 on serine-10 (a proximal substrate of aurora-B) leading to aborted cell division, apoptosis and/or polyploidy. We evaluated the activity of AMG 900 and two other well-characterized aurora kinase inhibitors [AZD1152-hQPA (B-selective AKI) and MLN8054 (A-selective AKI)] in a panel of AML cell lines. AMG 900 inhibited proliferation in all 10 cell lines at single-digit nanomolar concentrations. At effective concentrations, AMG 900 and AZD1152-hQPA showed similar cellular phenotypes, indicating that the activity of AMG 900 may occur through inhibition of aurora-B. A subset of cell lines sensitive to AMG 900 and MLN8054 were insensitive to AZD1152-hQPA, suggesting that AMG 900 may be less susceptible to resistance mediated by drug-efflux (Grundy M et al, 2011). Two AMG 900 oral dosing schedules are being evaluated in the ongoing AML clinical trial; patients receive either 4 or 7 consecutive daily doses followed by a drug holiday in 14-day cycles. In this study, we evaluated the in vivo anti-proliferative effects of AMG 900 using the two dose schedules in the skeleton of NOD/SCID IL2γnull mice bearing MOLM-13 (AML) cells expressing luciferase. To assess tumor cell proliferation in vivo, we used 18FLT (radioactive thymidine analog) PET/CT imaging, a technique that has been used to monitor early treatment response in the bone marrow of AML patients (Vanderhoek M et al, 2011). Mice were imaged for luciferase activity and 18FLT uptake before treatment and at multiple time-points during the drug holiday phase within the 14-day cycle. While the two AMG 900 dosing schedules resulted in a similar decrease in tumor burden across study time points (as measured by luciferase activity), they differed in the timing of skeletal 18FLT responses. Mice administered AMG 900 showed an attenuated skeletal 18FLT uptake compared with the vehicle group, followed by an 18FLT flare. This 18FLT flare event is notably higher using the AMG 900 4-day schedule, although the cumulative dose is similar for both schedules. This difference in 18FLT flaring may indicate the schedules differ in the duration and/or level of target inhibition in the skeletal tumor and bone marrow cells. Mice treated with sunitinib (positive control agent) did not show a skeletal 18FLT flare during the drug holiday, suggesting its mode of action is distinct from that of AMG 900. At the end of the study, mouse bone marrow was assessed for tumor burden by flow cytometry. Mice treated with AMG 900 showed a significant decrease in tumor burden compared with the vehicle group. Interestingly, the mice administered AMG 900 7-day schedule showed the most suppression of tumor growth compared with either AMG 900 4-day schedule or sunitinib. Together, our preclinical studies demonstrate that AMG 900 is a potent inhibitor of aurora kinases that robustly suppresses the growth of AML cells in vitro and in vivo. Furthermore, we highlight the utility of in vivo imaging to monitor AMG 900 drug action, which may help to inform future dose scheduling and drug combination studies. Disclosures: Cheung: Amgen Inc: Employment, Equity Ownership. Juan:Amgen Inc.: Employment, Equity Ownership. Wayne:Amgen Inc.: Employment, Equity Ownership. Hanestad:Amgen Inc.: Employment, Equity Ownership. Keegan:Amgen Inc.: Employment, Equity Ownership. Huard:Amgen Inc.: Employment, Equity Ownership. McElroy:Amgen Inc.: Employment, Equity Ownership. Stanton:Amgen Inc.: Employment, Equity Ownership. Bush:Amgen Inc.: Employment, Equity Ownership. Kendall:Amgen Inc.: Employment, Equity Ownership. Radinsky:Amgen Inc.: Employment, Equity Ownership. Abella:Amgen Inc. : Employment, Equity Ownership. Pieslor:Amgen Inc.: Employment, Equity Ownership. Friberg:Amgen Inc.: Employment, Equity Ownership. Coxon:Amgen Inc.: Employment, Equity Ownership. Gamelin:Amgen Inc: Employment, Equity Ownership. Payton:Amgen Inc.: Employment, Equity Ownership. Off Label Use: AMG 900 is currently in phase 1 clinical development, there is no approved label.

Lymphoplasmacytic Lymphoma and Waldenström Macroglobulinemia

2013 ◽  
Vol 137 (4) ◽  
pp. 580-585 ◽  
Author(s):  
Nadia Naderi ◽  
David T. Yang
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Bone Marrow Involvement ◽  
Accurate Diagnosis ◽  
Immunoglobulin M ◽  
Low Grade ◽  
Lymphoplasmacytic Lymphoma ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Definition Of

Lymphoplasmacytic lymphoma (LPL) is a low-grade, B-cell neoplasm composed of small lymphocytes, plasmacytoid lymphocytes, and plasma cells that typically involve the bone marrow, and it is associated with an immunoglobulin M (IgM) gammopathy. The definition of Waldenström macroglobulinemia (WM) and its relationship to LPL has been confusing in the past. In addition, the diagnosis of LPL itself can be challenging because LPL lacks disease-specific morphologic, immunophenotypic, and genetic features to differentiate it from other mature B-cell neoplasms. Accurate diagnosis of LPL/WM rests on recognition of the differential diagnostic features between LPL and other diagnostic possibilities and the use of the recently refined definition of WM and its relationship with LPL: The presence of an IgM monoclonal gammopathy of any level in the setting of bone marrow involvement by LPL. This review summarizes the clinical, laboratory, and histologic features of LPL/WM, with particular emphasis on unique aspects of LPL/WM that may aid in accurate diagnosis.

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