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Summary: Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy. [Copyright &y& Elsevier]

Summary SIRT1 belongs to the silent information regulator 2 (Sir2) protein family of enzymes and functions as a NAD+-dependent class III histone deacetylase. Here, we examined the anti-multiple myeloma (MM) activity of a novel oral agent, SRT1720, which targets SIRT1. Treatment of MM cells with SRT1720 inhibited growth and induced apoptosis in MM cells resistant to conventional and bortezomib therapies without significantly affecting the viability of normal cells. Mechanistic studies showed that anti-MM activity of SRT1720 is associated with: (i) activation of caspase-8, caspase-9, caspase-3, poly(ADP) ribose polymerase; (ii) increase in reactive oxygen species; (iii) induction of phosphorylated ataxia telangiectasia mutated/checkpoint kinase 2 signalling; (iv) decrease in vascular endothelial growth factor-induced migration of MM cells and associated angiogenesis; and (v) inhibition of nuclear factor-κB. Blockade of ATM attenuated SRT1720-induced MM cell death. In animal tumour model studies, SRT1720 inhibited MM tumour growth. Finally, SRT1720 enhanced the cytotoxic activity of bortezomib or dexamethasone. Our preclinical studies provide the rationale for novel therapeutics targeting SIRT1 in MM. [ABSTRACT FROM AUTHOR]

Summary: Multiple myeloma (MM) remains incurable despite novel therapies, suggesting the need for further identification of factors mediating tumorigenesis and drug resistance. Using both in vitro and in vivo MM xenograft models, we show that plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) microenvironment both mediate immune deficiency characteristic of MM and promote MM cell growth, survival, and drug resistance. Microarray, cell signaling, cytokine profile, and immunohistochemical analysis delineate the mechanisms mediating these sequelae. Although pDCs are resistant to novel therapies, targeting Toll-like receptors with CpG oligodeoxynucleotides both restores pDC immune function and abrogates pDC-induced MM cell growth. Our study therefore validates targeting pDC-MM interactions as a therapeutic strategy to overcome drug resistance in MM. [Copyright &y& Elsevier]

Summary: Bortezomib therapy has proven successful for the treatment of relapsed and/or refractory multiple myeloma (MM); however, prolonged treatment is associated with toxicity and development of drug resistance. Here, we show that the novel proteasome inhibitor NPI-0052 induces apoptosis in MM cells resistant to conventional and Bortezomib therapies. NPI-0052 is distinct from Bortezomib in its chemical structure, effects on proteasome activities, mechanisms of action, and toxicity profile against normal cells. Moreover, NPI-0052 is orally bioactive. In animal tumor model studies, NPI-0052 is well tolerated and prolongs survival, with significantly reduced tumor recurrence. Combining NPI-0052 and Bortezomib induces synergistic anti-MM activity. Our study therefore provides the rationale for clinical protocols evaluating NPI-0052, alone and together with Bortezomib, to improve patient outcome in MM. [Copyright &y& Elsevier]

PS-341 (bortezomib, Velcade™) is a promising novel agent for treatment of advanced multiple myeloma (MM); however, 65%of patients with relapsed refractory disease in a phase II study do not respond to PS-341. We have previously shown that lysophosphatidic acid acyltransferase (LPAAT)-ßinhibitor CT-32615 triggers caspase-dependent apoptosis, and can overcome resistance to conventional therapeutics (i.e., dexamethasone, doxorubicin, melphalan) in MM cells. In this study, we therefore determined whether CT-32615 could also overcome resistance to PS-341. We first characterized molecular mechanisms of resistance to PS-341 in DHL-4 cells. DHL-4 cells express low levels of caspase-3 and caspase-8; furthermore, no cleavage in caspase-8, caspase-9, caspase-3, poly ADP-ribose polymerase (PARP), or DNA fragmentation factor 45 was triggered by PS-341 treatment. We have previously shown that PS-341 treatment triggers phosphorylation of c-Jun NH2-terminal kinase (JNK), which subsequently induces caspase-dependent apoptosis; conversely, JNK inhibition blocks PS-341-induced apoptosis. We here show that phosphorylation of SEK-1, JNK, and c-Jun are not induced by PS-341 treatment, suggesting that PS-341 does not trigger a stress response in DHL-4 cells. Importantly, CT-32615 inhibits growth of DHL-4 cells in a time- and dose-dependent fashion: a transient G2/M cell cycle arrest induced by CT-32615 is mediated via downregulation of cdc25c and cdc2. CT-32615 triggered swelling and lysis of DHL-4 cells, without caspase/PARP cleavage or TUNEL-positivity, suggesting a necrotic response. Our studies therefore demonstrate that LPAAT-ßinhibitor CT-32615 triggers necrosis, even in PS-341-resistant DHL-4 cells, providing the framework for its evaluation to overcome clinical PS-341 resistance and improve patient outcome.Oncogene (2005) 24, 3121-3129. doi:10.1038/sj.onc.1208522 Published online 21 February 2005 [ABSTRACT FROM AUTHOR]

Normal cellular functioning requires processing of proteins regulating cell cycle, growth, and apoptosis. The ubiquitin-proteasome pathway (UBP) modulates intracellular protein degradation. Specifically, the 26S proteasome is a multienzyme protease that degrades misfolded or redundant proteins; conversely, blockade of the proteasomal degradation pathways results in accumulation of unwanted proteins and cell death. Because cancer cells are more highly proliferative than normal cells, their rate of protein translation and degradation is also higher. This notion led to the development of proteasome inhibitors as therapeutics in cancer. The FDA recently approved the first proteasome inhibitor bortezomib (Velcade ™ ), formerly known as PS-341, for the treatment of newly diagnosed and relapsed/refractory multiple myeloma (MM). Ongoing studies are examining other novel proteasome inhibitors, in addition to bortezomib, for the treatment of MM and other cancers. [ABSTRACT FROM AUTHOR]

The ubiquitin-conjugating enzyme CDC34 (UBC3) is linked to cell cycle progression in diverse cell types; however, its role in multiple myeloma (MM) pathogenesis is unclear. Here, we show that CDC34 is highly expressed in patient MM cells and MM cell lines versus normal cells. Blocking CDC34 using a dominant-negative strategy enhances the anti-MM activity of Bortezomib/ Proteasome inhibitor PS-341, dexamethasone (Dex) and 2-Methoxyestradiol (2ME2). The expression of wild-type CDC34 reduces Dex-induced cytotoxicity in MM cells. Moreover, inhibition of CDC34 enzymatic activity abrogates interleukin-6-induced protection against Dex- induced apoptosis. Together, these findings provide evidence that (1) CDC34 expression is associated with growth and survival of MM cells and (2) blocking CDC34 activity not only enhances anti-MM activity of Bortezomib and 2ME2 but also overcomes IL-6-triggered Dex- resistance. [ABSTRACT FROM AUTHOR]

Proteasome inhibitor PS-341 is one of the most promising novel agents against multiple myeloma (MM). We have previously shown that PS-341 inhibits IL-6 triggered phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 (also known as p42/44 mitogen-activated protein kinases) in MM cells. In this study, we further examined whether clinically achievable concentrations of PS-341 could inhibit IL-6 triggered signaling cascades in MM. We found that PS-341 inhibited not only ERK, but also signal transducers and activators of transcription (STAT) 3 as well as Akt phosphorylation. Since gp130 (CD130) dimerizes and is phosphorylated after IL-6 binding to gp80 (IL-6 receptor), we hypothesized that gp130 could be involved in PS-341-induced blockade of signaling cascades mediating MM cell growth, survival, and drug resistance in the bone marrow (BM) microenvironment. In this study, we first demonstrate that PS-341 induces downregulation of gp130 in a time- and dose-dependent manner in vitro, prior to MM cell death. Conversely, downregulation of gp130 is completely abrogated by the pan-caspase inhibitor Z-VAD-FMK, suggesting that downregulation of gp130 is mediated via caspase activation. Z-VAD-FMK also abrogates the inhibitory effect of PS-341 on IL-6-triggered signaling cascades. Importantly, we demonstrate that phosphorylation of ERK, STAT3, and Akt in MM.1S cells induced by either exogenous IL-6 or by binding of MM cells to BM stromal cells is abrogated by PS-341. These studies, therefore, define another novel mechanism whereby PS-341 can overcome the growth and survival advantage in MM cells conferred by the BM milieu. Importantly, this effect on cytokine-induced gp130 signaling cascades may account, at least in part, for the remarkable preclinical sensitivity and clinical responses achieved in MM with PS-341 treatment.Oncogene (2003) 22, 8386-8393. doi:10.1038/sj.onc.1207170 [ABSTRACT FROM AUTHOR]

Superoxide (O2-) radicals have been linked to apoptosis. Here, we show that 2-methoxyestradiol (2ME2)-induced apoptosis in multiple myeloma (MM) cells is associated with O2- generation, whereas dexamethasone (Dex)-induced apoptosis occurs without concurrent increase in O2-. In contrast, both these agents decrease mitochondrial transmembrane potential (??m). Treatment of MM cells with an antioxidant N-acetyl-L-cysteine blocks 2ME2, but not Dex-induced apoptosis as well as release of mitochondrial proteins cytochrome c (cyto c) and Smac. Taken together, these results demonstrate that there are at least two distinct apoptotic pathways: one dependent on O2-, which is induced by 2ME2 and is associated with release of cyto c and Smac; and the other an independent of O2-, which is triggered by Dex and associated with Smac release.Oncogene (2003) 22, 6296-6300. doi:10.1038/sj.onc.1206734 [ABSTRACT FROM AUTHOR]

Discusses a study indicating that the induction of apoptosis in multiple myeloma cells is associated with activation of c-Jun NH[sub 2]-terminal kinase (JNK), translocation of JNK from cytosol to mitochondria and release of second mitochondria-derived activator of capcases (Smac) from mitochondria to cytosol. JNK blocking by dominant-negative mutant or cotreatment with specific JNK inhibitors, SP600125.

Presents a study that determined the gene expression profiles of dexamethasone-treated multiple myeloma cells using oligonucleotide arrays. Results; Discussion; Methodology.

In this study we demonstrate that tumor necrosis factor α (TNFα) triggers only modest proliferation, as well as p44/p42 mitogen-activated protein kinase (MAPK) and NF-κB activation, in MM.1S multiple myeloma (MM) cells. TNFα also activates NF-κB and markedly upregulates (fivefold) secretion of interleukin-6 (IL-6), a myeloma growth and survival factor, in bone marrow stromal cells (BMSCs). TNFα in both a dose and time dependent fashion induced expression of CD11a (LFA-1), CD54 (intercellular adhesion molecule-1, ICAM-1), CD106 (vascular cell adhesion molecule-1, VCAM-1), CD49d (very late activating antigen-4, VLA-4), and/or MUC-1 on MM cell lines; as well as CD106 (VCAM-1) and CD54 (ICAM-1) expression on BMSCs. This resulted in increased (2–4-fold) per cent specific binding of MM cells to BMSCs, with related IL-6 secretion. Importantly, the proteasome inhibitor PS-341 abrogated TNFα-induced NF-κB activation, induction of ICAM-1 or VCAM-1, and increased adhesion of MM cells to BMSCs. Agents which act to inhibit TNFα may therefore abrogate the paracrine growth and survival advantage conferred by MM cell adhesion in the BM microenvironment. Oncogene (2001) 20, 4519–4527. [ABSTRACT FROM AUTHOR]

Related Adhesion Focal Tyrosine Kinase (RAFTK; also known as Pyk2), is a member of the Focal Adhesion Kinase (FAK) subfamily and is activated by TNFα, UV light and increases in intracellular calcium levels. However, the function of RAFTK remains largely unknown. Our previous studies demonstrated that treatment with dexamethasone (Dex), ionizing radiation (IR), and anti-Fas mAb induces apoptosis in multiple myeloma (MM) cells. In the present study, we examined the potential role of RAFTK during induction of apoptosis in human MM cells triggered by these three stimuli. Dex-induced apoptosis, in contrast to apoptosis triggered by anti-Fas mAb or IR, is associated with activation of RAFTK. Transient overexpression of RAFTK wild type (RAFTK WT) induces apoptosis, whereas transient overexpression of Kinase inactive RAFTK (RAFTK K-M) blocks Dex-induced apoptosis. In contrast, transient overexpression of RAFTK K-M has no effect on apoptosis triggered by IR or Fas. In Dex-resistant cells, Dex does not trigger either RAFTK activation or apoptosis. Finally, interleukin-6 (IL-6), a known survival factor for MM cells, inhibits both activation of RAFTK and apoptosis of MM.1S cells triggered by Dex. Our studies therefore demonstrate Dex-induced RAFTK-dependent, and IR or Fas induced RAFTK-independent apoptotic signaling cascades in MM cells. [ABSTRACT FROM AUTHOR]

The stress-activated protein kinases (SAPKs), also known as c-Jun amino-terminal kinases (JNKs), are activated in response to diverse stimuli including DNA damage, heat shock, interleukin-1, tumor necrosis factor-α and Fas. Although all these inducers cause apoptosis, whether SAPK/JNK activation is required for apoptosis is controversial. In this study, we demonstrate that ionizing radiation (IR) and dexamethasone (Dex) induce apoptosis in multiple myeloma (MM) derived cell lines, as well as in patient cells. IR-induced apoptosis is associated with activation of SAPK/JNK and p38 kinase, in contrast to Dex-induced apoptosis, which is not associated with activation of stress kinases. Moreover, Dex-induced apoptosis is associated with a significant decrease in the activities of mitogen activated protein kinase (MAPK) and p70S6K, whereas IR-treatment does not alter the activity of these kinases. Both IR and Dex induce poly (ADP ribose) polymerase (PARP) cleavage, a signature event of apoptosis. Finally, interleukin-6 (IL-6) inhibits Dex-induced apoptosis, downregulation of MAP and p70S6K growth kinases and PARP cleavage; in contrast, IL-6 does not inhibit IR-induced apoptosis, activation of SAPK/JNK, and PARP cleavage. Taken together, our findings suggest that SAPK/JNK activation is not required for apoptosis in MM cells, and that there are at least two distinct apoptotic signaling pathways: (i) SAPK/JNK-associated, which is induced by IR and unaffected by IL-6; and (ii) SAPK/JNK-independent, which is induced by Dex, associated with downregulation of MAPK and p70S6K and inhibited by IL-6. [ABSTRACT FROM AUTHOR]

Global metabolomics relies on highly reproducible and sensitive detection of a wide range of metabolites in biological samples. Here we report the optimization of metabolome analysis by nanoflow ultraperformance liquid chromatography coupled to high-resolution orbitrap mass spectrometry. Reliable peak features were extracted from the LC-MS runs based on mandatory detection in duplicates and additional noise filtering according to blank injections. The run-to-run variation in peak area showed a median of 14%, and the false discovery rate during a mock comparison was evaluated. To maximize the number of peak features identified, we systematically characterized the effect of sample loading amount, gradient length, and MS resolution. The number of features initially rose and later reached a plateau as a function of sample amount, fitting a hyperbolic curve. Longer gradients improved unique feature detection in part by time-resolving isobaric species. Increasing the MS resolution up to 120000 also aided in the differentiation of near isobaric metabolites, but higher MS resolution reduced the data acquisition rate and conferred no benefits, as predicted from a theoretical simulation of possible metabolites. Moreover, a biphasic LC gradient allowed even distribution of peak features across the elution, yielding markedly more peak features than the linear gradient. Using this robust nUPLC-HRMS platform, we were able to consistently analyze ~6500 metabolite features in a single 60 min gradient from 2 mg of yeast, equivalent to ~50 million cells. We applied this optimized method in a case study of drug (bortezomib) resistant and drug-sensitive multiple myeloma cells. Overall, 18% of metabolite features were matched to KEGG identifiers, enabling pathway enrichment analysis. Principal component analysis and heat map data correctly clustered isogenic phenotypes, highlighting the potential for hundreds of small molecule biomarkers of cancer drug resistance. [ABSTRACT FROM AUTHOR]

Objectives: Alkyl-lysophospholipids are a novel class of antitumor agents. Perifosine is a novel alkyl-lysophospholipid that can induce apoptosis in multiple myeloma (MM) tumor cells, both in vitro and in vivo. We investigated the effects of perifosine on the peripheral blood, bone marrow, and spleen of mice inoculated with subcutaneous plasmacytomas. Methods: Immunocompromised mice were inoculated with myeloma cell lines and treated with oral perifosine in either a daily or weekly schedule, or with vehicle only. When plasmacytomas reached 2 cm, mice were sacrificed. Terminal blood was analyzed with a Coulter counter, and counts were confirmed by light microscopy. Marrow and spleen were also analyzed by light microscopy. Results: In control mice, mean hemoglobin was 12 g/dL, white blood cell (WBC) count 7 × 109/L, and mean platelet count was 292 × 109/L. In contrast, the respective values for mice treated with perifosine weekly were 11 g/dL, 9 × 109/L, and 944 × 109/L; and for mice treated with perifosine daily were 10 g/dL, 11 × 109/L, and 752 × 109/L. The increase in WBCs was due, predominantly, to a neutrophilia. Compared to control mice, perifosine treatment induced marrow hypercellularity and splenic white pulp expansion. Conclusions: These findings have clinical relevance because myeloid suppression is a dose-limiting toxicity of many cytotoxic agents, and myeloid hyperplasia is usually only observed in the setting of growth factor stimulation. Coupled with its remarkable in vitro MM cytotoxicity, these results strongly support the use of perifosine in clinical trials for patients with MM. [Copyright &y& Elsevier]

Although PS-341 (bortezomib) is a promising agent to improve multiple myeloma (MM) patient outcome, 65%of patients with relapsed and refractory disease do not respond. We have previously shown that heat shock protein (Hsp)27 is upregulated after PS-341 treatment, that overexpression of Hsp27 confers PS-341 resistance, and that inhibition of Hsp27 overcomes PS-341 resistance. Since Hsp27 is a downstream target of p38 mitogen-activated protein kinase (MAPK)/MAPK-mitogen-activated protein kinase-2 (MAPKAPK2), we hypothesized that inhibition of p38 MAPK activity could augment PS-341 cytotoxicity by downregulating Hsp27. Although p38 MAPK inhibitor SCIO-469 (Scios Inc, CA, USA) alone did not induce significant growth inhibition, it blocked baseline and PS-341-triggered phosphorylation of p38 MAPK as well as upregulation of Hsp27, associated with enhanced cytotoxicity in MM.1S cells. Importantly, SCIO-469 enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK) and augmented cleavage of caspase-8 and poly(ADP)-ribose polymerase. Moreover, SCIO-469 downregulated PS-341-induced increases in G2/M-phase cells, associated with downregulation of p21Cip1 expression. Importantly, SCIO-469 treatment augmented cytotoxicity of PS-341 even against PS-341-resistant cell lines and patient MM cells. These studies therefore provide the framework for clinical trials of SCIO-469 to enhance sensitivity and overcome resistance to PS-341, thereby improving patient outcome in MM.Oncogene (2004) 23, 8766-8776. doi:10.1038/sj.onc.1208118 Published online 11 October 2004 [ABSTRACT FROM AUTHOR]

Although c-Jun NH2-terminal kinase (JNK) is activated by treatment with therapeutic agents, the biologic sequelae of inhibiting constitutive activation of JNK has not yet been clarified. In this study, we examine the biologic effect of JNK inhibition in multiple myeloma (MM) cell lines. JNK-specific inhibitor SP600125 induces growth inhibition via induction of G1 or G2/M arrest in U266 and MM.1S multiple myeloma cell lines, respectively. Neither exogenous IL-6 nor insulin-like growth factor-1 (IGF-1) overcome SP600125-induced growth inhibition, and IL-6 enhances SP600125-induced G2/M phase in MM.1S cells. Induction of growth arrest is mediated by upregulation of p27Kip1, without alteration of p53 and JNK protein expression. Importantly, SP600125 inhibits growth of MM cells adherent to bone marrow stromal cells (BMSCs). SP600125 induces NF-?B activation in a dose-dependent fashion, associated with phosphorylation of I?B kinase a (IKKa) and degradation of I?Ba. In contrast, SP600125 does not affect phosphorylation of STAT3, Akt, and/or ERK. IKK-specific inhibitor PS-1145 inhibits SP600125-induced NF-?B activation and blocks the protective effect of SP600125 against apoptosis. Our data therefore demonstrate for the first time that inhibiting JNK activity induces growth arrest and activates NF-?B in MM cells.Oncogene (2003) 22, 8797-8801. doi:10.1038/sj.onc.1206919 [ABSTRACT FROM AUTHOR]

Previous studies demonstrate that interleukin-6 (IL-6) mediates growth and survival in human multiple myeloma (MM) cells via the MEK/MAPK and JAK/STAT signaling pathways, respectively. IL-6 also confers protection against Dexamethasone (Dex)-induced apoptosis via activation of protein tyrosine phosphatase (SHP2). In the current study, we characterized IL-6 triggered phophatidylinositol-3 kinase/Akt kinase (PI3-K/Akt) signaling in MM cells. IL-6 induces Akt/PKB phosphorylation in a time and dose dependent manner in MM.1S MM cells. IL-6 also induced phosphorylation of downstream targets of Akt, including Bad, GSK-3β, and FKHR, confirming Akt activation. Inhibition of Akt activation by the PI3-K inhibitor LY294002 partially blocked IL-6 triggered MEK/MAPK activation and proliferation in MM.1S cells, suggesting cross-talk between PI3-K and MEK signaling. We demonstrate that Dex-induced apoptosis in MM.1S cells is mediated by downstream activation of caspase-9, with resultant caspase-3 cleavage; and conversely, that IL-6 triggers activation of PI3-K and its association with SHP2, inactivates caspase-9, and protects against Dex-induced apoptosis. LY294002 completely abrogates this signaling cascade, further confirming the importance of PI3-K/Akt signaling in conferring the protective effect of IL-6 against Dex-induced apoptosis. Finally, we show that IL-6 triggered PI3-K/Akt signaling in MM.1S cells inactivates forkhead transcriptional factor (FKHR), with related G1/S phase transition, whereas LY294002 blocks this signaling, resulting in upregulation of p27KIP1 and G1 growth arrest. Our data therefore suggest that PI3-K/Akt signaling mediates growth, survival, and cell cycle regulatory effects of IL-6 in MM. Oncogene (2001) 20, 5991–6000. [ABSTRACT FROM AUTHOR]

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A. [ABSTRACT FROM AUTHOR]

Marizomib ( MRZ) is an irreversible, pan-subunit proteasome inhibitor ( PI) in clinical development for relapsed/refractory multiple myeloma ( RRMM) and glioma. This study analysed MRZ, pomalidomide ( POM) and low-dose dexamethasone (Lo- DEX) [ PMD] in RRMM to evaluate safety and determine the maximum tolerated dose ( MTD) and/or recommended Phase 2 dose ( RP2D). Intravenous MRZ (0·3-0·5 mg/m2) was administered over 2 h on days 1, 4, 8, 11; POM (3-4 mg) on days 1-21; and Lo- DEX (5 or 10 mg) on days 1, 2, 4, 5, 8, 9, 11, 12, 15, 16, 22 and 23 of every 28-day cycle. Thirty-eight patients were enrolled that had received a median of 4 (range 1-10) prior lines of therapy; all patients received prior lenalidomide and bortezomib. No dose-limiting toxicities ( DLTs) were observed and 0·5 mg/m2 MRZ was determined to be the RP2D. The most common treatment-related ≥Grade 3 adverse events were: neutropenia (11/38 patients: 29%), pneumonia (4/38 patients 11%), anaemia (4/38 patients; 11%) and thrombocytopenia (4/38 patients; 11%). The overall response rate and clinical benefit rate was 53% (19/36) and 64% (23/36), respectively. In conclusion, PMD was well tolerated and demonstrated promising activity in heavily pre-treated, high-risk RRMM patients. [ABSTRACT FROM AUTHOR]

p53-related protein kinase (TP53RK, also known as PRPK) is an upstream kinase that phosphorylates (serine residue Ser15) and mediates p53 activity. Here we show that TP53RK confers poor prognosis in multiple myeloma (MM) patients, and, conversely, that TP53RK knockdown inhibits p53 phosphorylation and triggers MM cell apoptosis, associated with downregulation of c-Myc and E2F-1-mediated upregulation of proapoptotic Bim. We further demonstrate that TP53RK downregulation also triggers growth inhibition in p53-deficient and p53-mutant MM cell lines and identify novel downstream targets of TP53RK including ribonucleotide reductase-1, telomerase reverse transcriptase, and cyclin-dependent kinase inhibitor 2C. Our previous studies showed that immunomodulatory drugs (IMiDs) downregulate p21 and trigger apoptosis in wild-typep53 MM.1S cells, Importantly, we demonstrate by pull-down, nuclear magnetic resonance spectroscopy, differential scanning fluorimetry, and isothermal titration calorimetry that IMiDs bind and inhibit TP53RK, with biologic sequelae similar to TP53RK knockdown. Our studies therefore demonstrate that either genetic or pharmacological inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and axis-independent pathways, validating TP53RK as a novel therapeutic target in patients with poor-prognosis MM. [ABSTRACT FROM AUTHOR]

p53-related protein kinase (TP53RK, also known as PRPK) is an upstream kinase that phosphorylates (serine residue Ser15) and mediates p53 activity. Here we show that TP53RK confers poor prognosis in multiple myeloma (MM) patients, and, conversely, that TP53RK knockdown inhibits p53 phosphorylation and triggers MM cell apoptosis, associated with downregulation of c-Myc and E2F-1-mediated upregulation of pro-apoptotic Bim. We further demonstrate that TP53RK downregulation also triggers growth inhibition in p53-deficient and p53-mutant MM cell lines and identify novel downstream targets of TP53RK including ribonucleotide reductase-1, telomerase reverse transcrip-tase, and cyclin-dependent kinase inhibitor 2C. Our previous studies showed that immunomodulatory drugs (IMiDs) downregulate p21 and trigger apoptosis in wild-type-p53 MM. 1S cells, Importantly, we demonstrate by pull-down, nuclear magnetic resonance spectroscopy, differential scanning f luorimetry, and isothermal titration calorimetry that IMiDs bind and inhibit TP53RK, with biologic sequelae similar to TP53RK knockdown. Our studies therefore demonstrate that either genetic or pharmacological inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and axis-independent pathways, validating TP53RK as a novel therapeutic target in patients with poor-prognosis MM. [ABSTRACT FROM AUTHOR]

Proteasome inhibitors ( PIs) are highly active in multiple myeloma ( MM) but resistance is commonly observed. All clinical stage PIs effectively inhibit chymotrypsin-like ( CT-L) activity; one possible mechanism of resistance is compensatory hyperactivation of caspase-like (C-L) and trypsin-like (T-L) subunits, in response to CT-L blockade. Marizomib ( MRZ), an irreversible PI that potently inhibits all three 20S proteasome subunits with a specificity distinct from other PIs, is currently in development for treatment of MM and malignant glioma. The pan-proteasome pharmacodynamic activity in packed whole blood and peripheral blood mononuclear cells was measured in two studies in patients with advanced solid tumours and haematological malignancies. Functional inhibition of all proteasome subunits was achieved with once- or twice-weekly MRZ dosing; 100% inhibition of CT-L was frequently achieved within one cycle at therapeutic doses. Concomitantly, C-L and T-L activities were either unaffected or increased, suggesting compensatory hyperactivation of these subunits. Importantly, this response was overcome by continued administration of MRZ, with robust inhibition of T-L and C-L (up to 80% and 50%, respectively) by the end of Cycle 2 and maintained thereafter. This enhanced proteasome inhibition was independent of tumour type and may underlie the clinical activity of MRZ in patients resistant to other PIs. [ABSTRACT FROM AUTHOR]

Our prior study utilized both in vitro and in vivo multiple myeloma ( MM) xenograft models to show that a novel alkylator melphalan-flufenamide (Melflufen) is a more potent anti- MM agent than melphalan and overcomes conventional drug resistance. Here we examined whether this potent anti- MM activity of melflufen versus melphalan is due to their differential effect on DNA damage and repair signalling pathways via γ-H2 AX/ ATR/ CHK1/Ku80. Melflufen-induced apoptosis was associated with dose- and time-dependent rapid phosphorylation of γ-H2 AX. Melflufen induces γ-H2 AX, ATR, and CHK1 as early as after 2 h exposure in both melphalan-sensitive and -resistant cells. However, melphalan induces γ-H2 AX in melphalan-sensitive cells at 6 h and 24 h; no γ-H2 AX induction was observed in melphalan-resistant cells even after 24 h exposure. Similar kinetics was observed for ATR and CHK1 in meflufen- versus melphalan-treated cells. DNA repair is linked to melphalan-resistance; and importantly, we found that melphalan, but not melflufen, upregulates Ku80 that repairs DNA double-strand breaks. Washout experiments showed that a brief (2 h) exposure of MM cells to melflufen is sufficient to initiate an irreversible DNA damage and cytotoxicity. Our data therefore suggest that melflufen triggers a rapid, robust, and an irreversible DNA damage which may account for its ability to overcome melphalan-resistance in MM cells. [ABSTRACT FROM AUTHOR]

Marizomib (MRZ) is a novel, irreversible proteasome inhibitor in clinical development for the treatment of relapsed or relapsed and refractory multiple myeloma (RRMM). MRZ inhibits the 3 proteolytic activities of the 20S proteasome with specificity distinct from bortezomib and carfilzomib. StudyNPI-0052-101 Part 1 enrolled relapsed or RRMMpatients into an open-label, dose-escalation design to determine the maximum tolerated dose and recommended phase 2 dose (RP2D) of MRZ administered intravenously on 2 different schedules: scheduleA (0.025-0.7mg/m2 once weekly on days 1, 8, and 15 of 4-week cycles) and schedule B (0.15-0.6 mg/m2 twice weekly on days 1, 4, 8, and 11 of 3-week cycles; concomitant dexamethasone was allowed with schedule B). Patients had received an average of 4.9 and 7.3 prior treatment regimens (schedules A and B, respectively). MRZ schedule A was administered to 32 patients, and theRP2D was established as 0.7mg/m2 infused over 10 minutes. ScheduleBwas administered to 36 patients, and the RP2D was determined to be 0.5 mg/m2 infused over 2 hours. The most common (>20% of patients) related adverse events were fatigue, headache, nausea, diarrhea, dizziness, and vomiting.Six patients achieved clinical benefit responses (defined as minimal response or better), including 5 partial responses (1 patient on schedule A and 4 on schedule B; 3 of these 4 patients received concomitant dexamethasone). MRZ was generallywell tolerated, and results suggest activity in previously treated RRMM vpatients. Combination studies using pomalidomide and dexamethasone are now underway. The trial was registered at www.clinicaltrials.gov as #NCT00461045. [ABSTRACT FROM AUTHOR]

The proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity, peripheral neuropathy and drug resistance. Our earlier studies showed that the novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action and effects on proteasomal activities, and that it can overcome bortezomib resistance. Pomalidomide, like lenalidomide, has potent immunomodulatory activity and has been approved by the US Food and Drug Administration for the treatment of RRMM. Here, we demonstrate that combining low concentrations of marizomib with pomalidomide induces synergistic anti-MM activity. Marizomib plus pomalidomide-induced apoptosis is associated with: (i) activation of caspase-8, caspase-9, caspase-3 and PARP cleavage, (ii) downregulation of cereblon (CRBN), IRF4, MYC and MCL1, and (iii) suppression of chymotrypsin-like, caspaselike, and trypsin-like proteasome activities. CRBN-siRNA attenuates marizomib plus pomalidomide-induced MM cells death. Furthermore, marizomib plus pomalidomide inhibits the migration of MM cells and tumour-associated angiogenesis, as well as overcomes cytoprotective effects of bone marrow microenvironment. In human MM xenograft model studies, the combination of marizomib and pomalidomide is well tolerated, inhibits tumour growth and prolongs survival. These preclinical studies provide the rationale for on-going clinical trials of combined marizomib and pomalidomide to improve outcome in patients with RRMM. [ABSTRACT FROM AUTHOR]

Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM. [ABSTRACT FROM AUTHOR]

Proteasome inhibitors have demonstrated that targeting protein degradation is effective therapy in multiple myeloma (MM). Here we show that deubiquitylating enzymes (DUBs) USP14 and UCHL5 are more highly expressed in MM cells than in normal plasma cells. USP14 and UCHL5 short interfering RNA knockdown decreases MM cell viability. A novel 19S regulatory particle inhibitor b-AP15 selectively blocks deubiquitylating activity of USP14 and UCHL5 without inhibiting proteasome activity. b-AP15 decreases viability in MM cell lines and patient MM cells, inhibits proliferation of MM cells even in the presence of bone marrow stroma cells, and overcomes bortezomib resistance. Anti-MM activity of b-AP15 is associated with growth arrest via downregulation of CDC25C, CDC2, and cyclin B1 as well as induction of caspase-dependent apoptosis and activation of unfolded protein response. In vivo studies using distinct human MM xenograft models show that b-AP15 is well tolerated, inhibits tumor growth, and prolongs survival. Combining b-AP15 with suberoylanilide hydroxamic acid, lenalidomide, or dexamethasone induces synergistic anti-MM activity. Our preclinical data showing efficacy of b-AP15 in MM disease models validates targeting DUBs in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance and provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in MM. [ABSTRACT FROM AUTHOR]

We recently demonstrated that Nicotinamide phosphoribosyltransferase (Nampt) inhibition depletes intracellular NAD+ content leading, to autophagic multiple myeloma (MM) cell death. Bortezomib has remarkably improved MM patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here we observed higher Nampt messenger RNA levels in bortezomib-resistant patient MM cells, which correlated with decreased overall survival. We demonstrated that combining the NAD+ depleting agent FK866 with bortezomib induces synergistic anti-MM cell death and overcomes bortezomib resistance. This effect is associated with (1) activation of caspase-8, caspase-9, caspase-3, poly (ADP-ribose) polymerase, and downregulation of Mcl-1; (2) enhanced intracellular NAD+ depletion; (3) inhibition of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities; (4) inhibition of nuclear factor κB signaling; and (5) inhibition of angiogenesis. Furthermore, Nampt knockdown significantly enhances the anti-MM effect of bortezomib, which can be rescued by ectopically overexpressing Nampt. In a murine xenograft MM model, low-dose combination FK866 and Bortezomib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Taken together, these findings indicate that intracellular NAD+ level represents a major determinant in the ability of bortezomib to induce apoptosis in MM cells and provide proof of concept for the combination with FK866 as a new strategy to enhance sensitivity or overcome resistance to bortezomib. [ABSTRACT FROM AUTHOR]

miRs play a critical role In tumor patho-genesis as either oncogenes or tumor-suppressor genes. However, the role of miRs and their regulation in response to proteasome inhibitors in multiple my-eloma (MM) Is unclear. In the current study, miR profiling in proteasome inhibi-tor MLN2238-treated MM.1S MM cells shows up-regulation of miR33b. Mecha-nistic studies indicate that the induction of miR33b is predominantly via transcrip-tional regulation. Examination of miR33b in patient MM cells showed a constitutively low expression. Overexpression of miR33b decreased MM cell viability, migra-tion, colony formation, and increased ap-optosis and sensitivity of MM cells to MLN2238 treatment. In addition, overex-pression of miR33b or MLN2238 expo-sure negatively regulated oncogene PIM-1 and blocked PIM-1 wild-type, but not PIM-1 mutant, luciferase activity. Moreover, PIM-1 overexpression led to significant abrogation of miR33b- or MLN2238-induced cell death. SGI-1776, a biochemi-cal inhibitor of PIM-1, triggered apoptosis in MM. Finally, overexpression of miR33b Inhibited tumor growth and prolonged survival In both subcutaneous and dis-seminated human MM xenograft models. Our results show that miR33b is a tumor suppressor that plays a role during MLN2238-induced apoptotic signaling in MM cells, and these data provide the basis for novel therapeutic strategies targeting miR33b In MM. [ABSTRACT FROM AUTHOR]

Malignant cells have a higher nicotinamide adenine dinucleotlde (NAD⁺) turnover rate than normal cells, making this blosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD⁺ synthesis, Nampt, in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 trig-gered cytotoxicity in MM cell lines and patient MM cells, but not normal donor as well as MM patients PBMCs. Importantly, FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role In maintenance of both MM cell viability and intracellular NAD⁺ stores. Interestingly, cytotoxicity of FK866 triggered autophagy, but not apoptosis. A transcriptlonal-dependent (TFEB) and independent (PI3K/mTORCl) activation of autophagy mediated FK866 MM cytotoxicity. Finally, FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model, associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleav-age of LC3 in tumor cells. Our data there-fore define a key role of Nampt in MM biology, providing the basis for a novel targeted therapeutic approach. [ABSTRACT FROM AUTHOR]

Summary Recent advances regarding the introduction of anti-adhesion strategies as a novel therapeutic concept in oncology hold great promise. Here we evaluated the therapeutic potential of the new-in-class-molecule selective-adhesion-molecule (SAM) inhibitor Natalizumab, a recombinant humanized IgG4 monoclonal antibody, which binds integrin-α4, in multiple myeloma (MM). Natalizumab, but not a control antibody, inhibited adhesion of MM cells to non-cellular and cellular components of the microenvironment as well as disrupted the binding of already adherent MM cells. Consequently, Natalizumab blocked both the proliferative effect of MM-bone marrow (BM) stromal cell interaction on tumour cells, and vascular endothelial growth factor (VEGF)-induced angiogenesis in the BM milieu. Moreover, Natalizumab also blocked VEGF- and insulin-like growth factor 1 (IGF-1)-induced signalling sequelae triggering MM cell migration. In agreement with our in vitro results, Natalizumab inhibited tumour growth, VEGF secretion, and angiogenesis in a human severe combined immunodeficiency murine model of human MM in the human BM microenvironment. Importantly, Natalizumab not only blocked tumour cell adhesion, but also chemosensitized MM cells to bortezomib, in an in vitro therapeutically representative human MM-stroma cell co-culture system model. Our data therefore provide the rationale for the clinical evaluation of Natalizumab, preferably in combination with novel agents (e.g. bortezomib) to enhance MM cytotoxicity and improve patient outcome. [ABSTRACT FROM AUTHOR]

Summary Novel therapies have transformed the treatment paradigm for multiple myeloma with significant improvements in survival now seen in both younger and older patients. Nonetheless, the disease is heterogeneous and high-risk patients in particular continue to have poor outcome. Moreover, the disease remains incurable. Efforts to refine risk stratification and disease characteristics continue with the use of cytogenetics, enhanced imaging techniques and other new technologies, such as genomics. The integration of novel therapies into induction therapy, consolidation and maintenance continues to evolve, and the appropriate use of combination strategies including proteasome inhibition and immunomodulatory treatment is emerging as a platform with application across the disease spectrum. Despite these advances, resistance to novel agents occurs and so the identification of new targets and the recognition of clonal heterogeneity are especially important as improvements to current treatment strategies are developed, with the goal of further improving patient outcome. [ABSTRACT FROM AUTHOR]

PR-924 is an LMP-7-selective tripeptide epoxyketone proteasome inhibitor that covalently modifies proteasomal N-terminal threonine active sites. In the present study, we show that PR-924 inhibits growth and triggers apoptosis in multiple myeloma (MM) cell lines and primary patient MM cells, without significantly affecting normal peripheral blood mononuclear cells. PR-924-induced apoptosis in MM cells is associated with activation of caspase-3, caspase-8, caspase-9, BID, PARP and cytochrome-c release. In vivo administration of PR-924 inhibits tumour growth in human plasmacytoma xenografts. Results from SCID-hu model show a significant reduction in the shIL-6R levels in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated as evidenced by the lack of weight loss. Importantly, treatment of tumour-bearing mice with PR-924, but not vehicle alone, prolonged survival. Our preclinical findings therefore validate immunoproteasome LMP-7 subunit as a novel therapeutic target in MM. [ABSTRACT FROM AUTHOR]

This study investigated the cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026 inhibited growth and survival of MM cells and cytokine-induced osteoclast differentiation more potently (9- to 10-fold) than AZD6244. Inhibition of proliferation induced by AS703026 was mediated by G0-G1 cell cycle arrest and was accompanied by reduction of MAF oncogene expression. AS703026 further induced apoptosis via caspase 3 and Poly ADP ribose polymerase (PARP) cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. Significant tumour growth reduction in AS703026- vs. vehicle-treated mice bearing H929 MM xenograft tumours correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nmol/l) was cytotoxic against the majority of tumour cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC-induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti-MM agents, to improve patient outcome. [ABSTRACT FROM AUTHOR]

Our previous study showed that the novel proteasome inhibitor NPI-0052 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib (Velcade™, Takeda, Boston, MA, USA) therapies. In vivo studies using human MM-xenografts demonstrated that NPI-0052 is well tolerated, prolongs survival, and reduces tumour recurrence. These preclinical studies provided the basis for an ongoing phase-1 clinical trial of NPI-0052 in relapsed/refractory MM patients. Here we performed pharmacodynamic (PD) studies of NPI-0052 using human MM xenograft murine model. Our results showed that NPI-0052: (i) rapidly left the vascular compartment in an active form after intravenous (i.v.) administration, (ii) inhibited 20S proteasome chymotrypsin-like (CT-L, β5), trypsin-like (T-L, β2), and caspase-like (C-L, β1) activities in extra-vascular tumours, packed whole blood (PWB), lung, liver, spleen, and kidney, but not brain and (iii) triggered a more sustained (>24 h) proteasome inhibition in tumours and PWB than in other organs (<24 h). Tissue distribution analysis of radiolabeled compound (3H-NPI-0052) in mice demonstrated that NPI-0052 left the vascular space and entered organs as the parent compound. Importantly, treatment of MM.1S-bearing mice with NPI-0052 showed reduced tumour growth without significant toxicity, which was associated with prolonged inhibition of proteasome activity in tumours and PWB but not normal tissues. [ABSTRACT FROM AUTHOR]

Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-β family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated iNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distalless homeobox-5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials. [ABSTRACT FROM AUTHOR]

This study characterized the preclinical anti-myeloma activity of VE465, a low molecular weight pan-Aurora kinase inhibitor. After 96-h drug exposure, several multiple myeloma (MM) cell lines were more sensitive to VE465 compared to non-malignant cells. The anti-MM activity of VE465 was maintained in the presence of interleukin-6 and, interestingly, enhanced by co-culture with stromal cells. However, primary MM cells were less responsive than cell lines. Combinations with dexamethasone (Dex), doxorubicin (Doxo) and bortezomib showed no antagonism. Our study highlights the potential role of the tumour microenvironment in modulating the activity of this drug class. [ABSTRACT FROM AUTHOR]

The interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment induces proliferation and survival of MM cells, as well as osteoclastogenesis. This study investigated the therapeutic potential of novel p38 mitogen-activated protein kinase (p38MAPK) inhibitor LY2228820 (LY) in MM. Although cytotoxicity against MM cell lines was modest, LY significantly enhanced the toxicity of bortezomib by down-regulating bortezomib-induced heat shock protein 27 phosphorylation. LY inhibited interleukin-6 secretion from long term cultured-BM stromal cells and BM mononuclear cells (BMMNCs) derived from MM patients in remission. LY also inhibited macrophage inflammatory protein-1α secretion from patient MM cells and BMMNCs as well as normal CD14 positive osteoclast precursor cells. Moreover, LY significantly inhibited in vitro osteoclastogenesis from CD14 positive cells induced by macrophage-colony stimulating factor and soluble receptor activator of nuclear factor-κB ligand. Finally, LY also inhibited in vivo osteoclatogenesis in a severe combined immunodeficiency mouse model of human MM. These results suggest that LY represents a promising novel targeted approach to improve MM patient outcome both by enhancing the effect of bortezomib and by reducing osteoskeletal events. [ABSTRACT FROM AUTHOR]

Osteolytic bone disease in multiple myeloma (MM) is associated with upregulation of osteoclast (OCL) activity and constitutive inhibition of osteoblast function. The extracellular signal-regulated kinase 1/2 (ERK1/2) pathway mediates OCL differentiation and maturation. We hypothesized that inhibition of ERK1/2 could prevent OCL differentiation and downregulate OCL function. It was found that AZD6244, a mitogen-activated or extracellular signal-regulated protein kinase (MEK) inhibitor, blocked OCL differentiation and formation in a dose-dependent manner, evidenced by decreased αV β3-integrin expression and tartrate-resistant acid phosphatase positive (TRAP+) cells. Functional dentine disc cultures showed inhibition of OCL-induced bone resorption by AZD6244. Major MM growth and survival factors produced by OCLs including B-cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL), as well as macrophage inflammatory protein (MIP-1 α), which mediates OCL differentiation and MM, were also significantly inhibited by AZD6244. In addition to ERK inhibition, NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1) and c-fos were both downregulated, suggesting that AZD6244 targets a later stage of OCL differentiation. These results indicate that AZD6244 inhibits OCL differentiation, formation and bone resorption, thereby abrogating paracrine MM cell survival in the bone marrow microenvironment. The present study therefore provides a preclinical rationale for the evaluation of AZD6244 as a potential new therapy for patients with MM. [ABSTRACT FROM AUTHOR]

Akt mediates growth and drug resistance in multiple myeloma (MM) cells in the bone marrow (BM) microenvironment. We have shown that a novel Akt inhibitor Perifosine induces significant cytotoxicity in MM cells in the BM milieu. This study further delineated molecular mechanisms whereby Perifosine triggered cytotoxicity in MM cells. Neither the intensity of Jun NH2-terminal kinase phosphorylation nor caspase/poly (ADP-ribose) polymerase cleavage correlated with Perifosine-induced cytotoxicity in MM.1S, INA6, OPM1 and OPM2 MM cells. However, survivin, which regulates caspase-3 activity, was markedly downregulated by Perifosine treatment, without changes in other anti-apoptotic proteins. Downregulation of survivin by siRNA significantly inhibited OPM1 MM cell growth, confirming that survivin mediates MM cell survival. Perifosine significantly downregulated both function and protein expression of β-catenin. Co-culture with BM stromal cells (BMSCs) upregulated both β-catenin and survivin expression in MM cells, which was blocked by Perifosine. Importantly, Perifosine treatment also downregulated survivin expression in human MM cells grown in vivo in a severe combined immunodeficient mouse xenograft model. Finally, Perifosine inhibited bortezomib-induced upregulation of survivin, associated with enhanced cytotoxicity of combined bortezomib and Perifosine treatment. These preclinical studies provide the framework for clinical trials of bortezomib with Perifosine to improve patient outcome in MM. [ABSTRACT FROM AUTHOR]

A critical role for vascular endothelial factor (VEGF) has been demonstrated in multiple myeloma (MM) pathogenesis. Here, we characterized the effect of the small-molecule VEGF receptor inhibitor pazopanib on MM cells in the bone marrow milieu. Pazopanib inhibits VEGF-triggered signaling pathways in both tumor and endothelial cells, thereby blocking in vitro MM cell growth, survival, and migration, and inhibits VEGF-induced up-regulation of adhesion molecules on both endothelial and tumor cells, thereby abrogating endothelial cell-MM cell binding and associated cell proliferation. We show that pazopanib is the first-in-class VEGF receptor inhibitor to inhibit in vivo tumor cell growth associated with increased MM cell apoptosis, decreased angiogenesis, and prolonged survival in a mouse xenograft model of human MM. Low-dose pazopanib demonstrates synergistic cytotoxicity with conventional (melphalan) and novel (bortezomib and immunomodulatory drugs) therapies. Finally, gene expression and signaling network analysis show transcriptional changes of several cancer-related genes, in particular c-Myc. Using siRNA, we confirm the role of c-Myc in VEGF production and secretion, as well as angiogenesis. These preclinical studies provide the rationale for clinical evaluation of pazopanib, alone and in combination with conventional and novel therapies, to increase efficacy, overcome drug resistance, reduce toxicity, and improve patient outcome in MM. [ABSTRACT FROM AUTHOR]

The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM). Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood. However, resistance to bortezomib as a single agent develops in the majority of patients, and activity in other malignancies has been less impressive. To elucidate mechanisms of bortezomib resistance, we compared differential gene expression profiles of bortezomib-resistant SUDHL-4 and bortezomib-sensitive SUDHL-6 diffuse large B-cell lymphoma lines in response to bortezomib. At concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in SUDHL-6 cells, but not in SUDHL-4 cells. We showed that overexpression of activating transcription factor 3 (ATF3), ATF4, ATF5, c-Jun, JunD and caspase-3 is associated with sensitivity to bortezomib-induced apoptosis, whereas overexpression of heat shock protein (HSP)27, HSP70, HSP90 and T-cell factor 4 is associated with bortezomib resistance. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is a malignancy of plasma cells within the bone marrow characterized by bone loss, renal disease, and immunodeficiency. Recent advances in the understanding of MM pathogenesis have improved established conventional cytotoxic therapy as well as transplantation regimens. Despite these advances, median overall survival is only 3–5 years. Therefore new therapies are urgently needed. Besides thalidomide, whose antimyeloma activity has only recently been defined, a plethora of novel agents, including the thalidomide-derived immunomodulatory drugs and bortezomib, have been identified to directly target the tumor cell or its microenvironment, and thereby inhibit MM cell growth and survival, and to overcome drug resistance. After validation of their preclinical anti-MM activity, several clinical trials are now ongoing to test the efficacy of these novel therapeutics, administered alone or in combination with conventional or other novel therapeutics and bone marrow transplantation. This article reviews the development of MM therapy, from its initial description approximately 150 years ago to the novel therapy regimens of recent years, highlighting that MM may soon become a chronic disease. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is a plasma cell malignancy that claims thousands of lives each year and has considerable morbidity. The disease remains incurable despite recent advances in the understanding of the disease biology and the introduction of more effective drugs is needed. This study evaluated the anti-MM activity of 3-(7-fluoro-4H-quinazolin-3-yl)-piperidine-2,6-dione, hydrochloride (FQPD), a novel immunomodulatory drug. FQPD inhibited the proliferation of multiple MM cell lines, including those resistant to conventional treatments, such as dexamethasone. It induced apoptosis in MM cell lines, as well as freshly isolated patient MM cells, without cytotoxicity on normal human lymphocytes. Moreover, it induced apoptosis in MM cells adherent to bone marrow (BM) stromal cells or in the presence of cytokines, such as interleukin-6 and vascular endothelial growth factor, confirming its ability to overcome the protective effects of the BM milieu. Apoptosis in the MM cells was mediated via poly-ADP ribose polymerase cleavage as well as cleavage of caspase 8 and caspase 9. Our studies therefore demonstrated in vitro anti-MM activity of FQPD and provide the rationale for its in vivo evaluation in animal models and derived clinical trials. [ABSTRACT FROM AUTHOR]

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) is a small-molecule, selective, noncompetitive inhibitor directed against human IMPDH. In this report, we show that VX-944 inhibits in vitro growth of human multiple myeloma (MM) cell lines via induction of apoptosis. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs) do not protect against VX-944-induced MM cell growth inhibition. VX-944 induced apoptosis in MM cell lines with only modest activation of caspases 3, 8, and 9. Furthermore, the pan-caspase inhibitor z-VAD-fmk did not inhibit VX-944-induced apoptosis and cell death. During VX-944-induced apoptosis, expressions of Bax and Bak were enhanced, and both apoptosis-inducing factor (AIF) and endonuclease G (Endo G) were released from the mitochondria to cytosol, suggesting that VX-944 triggers apoptosis in MM cells primarily via a caspase-independent, Bax/AIF/Endo G pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin and melphalan even in the presence of BMSCs. Taken together, our data demonstrate a primarily non-caspase-dependent apoptotic pathway triggered by VX-944, thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional agents which trigger caspase activation.Oncogene (2005) 24, 5888–5896. doi:10.1038/sj.onc.1208739; published online 6 June 2005 [ABSTRACT FROM AUTHOR]

We have shown that the proteasome inhibitor bortezomib (formerly known as P5-341) triggers significant antitumor activity in multiple myeloma (MM) in both preclinical models and patients with relapsed refractory disease. Recent studies have shown that unfolded and misfolded ubiquitinated proteins are degraded not only by proteasomes, but also by aggresomes, dependent on histone deacetylase 6 (HDAC6) activity. We therefore hypothesized that inhibition of both mechanisms of protein catabolism could induce accumulation of ubiquitinated proteins followed by significant cell stress and cytotoxicity in MM cells. To prove this hypothesis, we used bortezomib and tubacin to inhibit the proteasome and HDAC6, respectively. Tubacin specifically triggers acetylation of u-tubulin as a result of HDAC6 inhibition in a dose- and time-dependent fashion. It induces cytotoxicity in MM cells at 72 h with an IC50 of 5-20 pM, which is mediated by caspase-dependent apoptosis; no toxicity is observed in normal peripheral blood mononuclear cells. Tubacin inhibits the interaction of HDAC6 with dynein and induces marked accumulation of ubiquitinated proteins. It synergistically augments bortezomib-induced cytotoxicity by c-Jun NH2-terminal kinase/caspase activation. Importantly, this combination also induces significant cytotoxicity in plasma cells isolated from MM patient bone marrow. Finally, adherence of MM cells to bone marrow stromal cells confers growth and resistance to conventional treatments; in contrast, the combination of tubacin and bortezomib triggers toxicity even in adherent MM cells. Our studies therefore demonstrate that tubacin combined with boil-ezomib mediates significant anti-MM activity, providing the frame- work for clinical evaluation of combined therapy to improve patient outcome in MM. [ABSTRACT FROM AUTHOR]

Thalidomide and immunomodulatory drugs (IMiDs), which target multiple myeloma (MM) cells and the bone marrow microenvironment, can overcome drug resistance. These agents also have immunomodulatory effects. Specifically, we have reported that thalidomide increased serum interleukin-2 (IL-2) levels and natural killer (NK) cell numbers in the peripheral blood of responding MM patients. In this study, we investigated the mechanisms whereby IMiDs augment NK cell cytotoxicity. NK cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) of peripheral blood mononuclear cells cultured with IMiDs were examined in the presence or absence of anti-IL-2 antibody, ciclosporin A or depletion of CD56-positive cells. IMiDs-induced signalling pathways, triggering IL-2 transcription in T cells, were also delineated. IMiDs facilitated the nuclear translocation of nuclear factor of activated T cells-2 and activator protein-1 via activation of phosphoinositide-3 kinase signalling, with resultant IL-2 secretion. IMiDs enhanced both NK cell cytotoxicity and ADCC induced by triggering IL-2 production from T cells. These studies defined the mechanisms whereby IMiDs trigger NK cell-mediated tumour-cell lysis, further supporting their therapeutic use in MM. [ABSTRACT FROM AUTHOR]

Interleukin-6 (LI-6) is a known growth and survival factor in multiple myeloma via activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling cascade. In this report we show that Grb2-associated binder (Gab) family adapter proteins Gab1 and Gab2 are expressed by multiple myeloma cells; and that interleukin-6 induces their tyrosine phosphorylation and association with downstream signaling molecules. We further demonstrate that these events are Src family tyrosine kinase-dependent and specifically identify the role of hematopoietic cell kinase (Hck) as a new Gab family adapter protein kinase. Conversely, inhibition of Src family tyrosine kinases by the pyrazolopyrimidine PP2, as in kinase-inactive Hck mutants, significantly reduces IL-6-triggered activation of extracellular signal-regulated kinase and AKT-1, leading to significant reduction of multiple myeloma cell proliferation and survival. Taken together, these results delineate a key role for Hck-mediated phosphorylation of Gab1 and Gab2 docking proteins in IL-6-induced proliferation and survival of multiple myeloma cells and identify tyrosine kinases and downstream adapter proteins as potential new therapeutic targets in multiple myeloma. [ABSTRACT FROM AUTHOR]