Btz Treatment Research Articles

WTAP and m6A-modified circRNAs modulation during stress response in acute myeloid leukemia progenitor cells

N6-methyladenosine (m6A) is one of the most prevalent and conserved RNA modifications. It controls several biological processes, including the biogenesis and function of circular RNAs (circRNAs), which are a class of covalently closed-single stranded RNAs. Several studies have revealed that proteotoxic stress response induction could be a relevant anticancer therapy in Acute Myeloid Leukemia (AML). Furthermore, a strong molecular interaction between the m6A mRNA modification factors and the suppression of the proteotoxic stress response has emerged. Since the proteasome inhibition leading to the imbalance in protein homeostasis is strictly linked to the stress response induction, we investigated the role of Bortezomib (Btz) on m6A regulation and in particular its impact on the modulation of m6A-modified circRNAs expression. Here, we show that treating AML cells with Btz downregulated the expression of the m6A regulator WTAP at translational level, mainly because of increased oxidative stress. Indeed, Btz treatment promoted oxidative stress, with ROS generation and HMOX-1 activation and administration of the reducing agent N-acetylcysteine restored WTAP expression. Additionally, we identified m6A-modified circRNAs modulated by Btz treatment, including circHIPK3, which is implicated in protein folding and oxidative stress regulation. These results highlight the intricate molecular networks involved in oxidative and ER stress induction in AML cells following proteotoxic stress response, laying the groundwork for future therapeutic strategies targeting these pathways.

Establishment of Patient-Derived Xenograft (PDX) Zebrafish Model of Multiple Myeloma and Its Application in Drug Screening

To establish a MM patient-derived tumor xenograft model (MM-PDX) in zebrafish, and to evaluate the anti-myeloma activity of indirubin-3'-monoxime(I3MO) using this model. Zebrafish embryos 2 days after fertilization were transplanted with fluorescence labeled myeloma primary tumor cells, the survival of primary tumor cells in zebrafish was observed at 0,16 and 24 hours after cell injection. The zebrafish embryos after tumor cell transplantation were randomly divided into control group, BTZ treatment and I3MO treatment group. Before and 24 hours after treatment with BTZ and I3MO, the positive area with calcein or Dil in zebrafish were observed under fluorescence microscope to reflect the survival of tumor cells, and it was verified. MM patient derived tumor cells survived in zebrafish. The construction of MM-PDX was successful. Compared with control group, the fluo- rescence area of the BTZ and I3MO treatment groups in zebrafish were significantly decreased(P<0.05), and BTZ and I3MO significantly inhibited the survival of MM cells in zebrafish. MM-PDX model was successfully established. Zebrafish model derived from tumor cells of MM patients can be used as a tool for drug screening of MM.

A Novel Indirubin-3-Monoxime Derivative Functions As Regulator of Proteostasis Via Targeting TRIM28 in Multiple Myeloma

Introduction: Multiple myeloma (MM) remains an incurable plasma cell malignancy. Agents disturbing the proteostasis play indispensable roles in MM treatment. Our previous studies have shown that the indirubin-3-monoxime (I3MO) works as a novel proteasome inhibitor that effectively inhibits MM cell growth. In this study, we aim to investigate the improvement of I3MO combined with histone deacetylase 6 (HDAC6) inhibitors, and clarified the molecular mechanisms. Methods: We constructed a compound 8b combined with I3MO and HADC6 inhibitor. The anti-MM effects of compound 8b were investigated in vivo and in vitro studies. Western blots, immunofluorescence, and proteasome activity assay were utilized to investigate the effects of 8b on MM cell proteasome activity, aggresome and autophagosome formation. RNA-seq and Dual-luciferase reporter gene assay were performed to identify the downstream targets of 8b treatment. We constructed MM cell lines with TRIM28 overexpression or shRNA knockdown. We further explored the function of TRIM28, one of the targets of 8b treatment, by ChIP-seq and IP-MS. Results: The novel I3MO derivative 8b more efficiently suppressed myeloma cell growth in vitro and in vivo. Compound 8b not decreased proteasome activity but inhibited the formation of aggresomes and autophagosomes. Interesting, we found that compound 8b efficiently inhibited TRIM28 promoter activity and repressed TRIM28 transcription level further. GEO data analysis demonstrated a positive correlation between TRIM28 and the expression of proteasome subunits as well as autophagy-related genes in MM. Further, we utilized ChIP-seq to confirm that TRIM28 could bind to the promoter region of multiple proteasome subunits-encoding genes, including PSMB1. Our data indicated that TRIM28 knockdown not only decreased the proteasome activity, but inhibited the formation of autophagosomes. Knockdown TRIM28 induced a higher sensitivity to BTZ treatment in MM cells. Furthermore, IP-MS with Co-IP analysis showed that TRIM28 interacts with 14-3-3ζ. TRIM28 acts as an E3 ligase in mediating the ubiquitination and degradation of 14-3-3ζ, a well-known negative regulator of autophagy. 14-3-3ζ degradation induced by TRIM28 activates autophagy in MM cells. These data suggested that TRIM28 suppressed by compound 8b would involve in proteostasis by regulation of proteasome activity and autophagy. Conclusions: Our study identified a novel compound 8b, I3MO combined with HDAC6i, efficiently suppressed MM cells survival. Additionally, we for the first time provided novel important insights for the roles of TRIM28 in MM pathobiology by regulation of proteasome activity and autophagy.

Proteasome inhibition protects blood–brain barrier P-glycoprotein and lowers Aβ brain levels in an Alzheimer’s disease model

BackgroundLoss of P-glycoprotein (P-gp) at the blood–brain barrier contributes to amyloid-β (Aβ) brain accumulation in Alzheimer’s disease (AD). Using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576), we previously showed that Aβ triggers P-gp loss by activating the ubiquitin–proteasome pathway, which leads to P-gp degradation. Furthermore, we showed that inhibiting the ubiquitin-activating enzyme (E1) prevents P-gp loss and lowers Aβ accumulation in the brain of hAPP mice. Based on these data, we hypothesized that repurposing the FDA-approved proteasome inhibitor, bortezomib (Velcade®; BTZ), protects blood–brain barrier P-gp from degradation in hAPP mice in vivo.MethodsWe treated hAPP mice with the proteasome inhibitor BTZ or a combination of BTZ with the P-gp inhibitor cyclosporin A (CSA) for 2 weeks. Vehicle-treated wild-type (WT) mice were used as a reference for normal P-gp protein expression and transport activity. In addition, we used the opioid receptor agonist loperamide as a P-gp substrate in tail flick assays to indirectly assess P-gp transport activity at the blood–brain barrier in vivo. We also determined P-gp protein expression by Western blotting, measured P-gp transport activity levels in isolated brain capillaries with live cell confocal imaging and assessed Aβ plasma and brain levels with ELISA.ResultsWe found that 2-week BTZ treatment of hAPP mice restored P-gp protein expression and transport activity in brain capillaries to levels found in WT mice. We also observed that hAPP mice displayed significant loperamide-induced central antinociception compared to WT mice indicating impaired P-gp transport activity at the blood–brain barrier of hAPP mice in vivo. Furthermore, BTZ treatment prevented loperamide-induced antinociception suggesting BTZ protected P-gp loss in hAPP mice. Further, BTZ-treated hAPP mice had lower Aβ40 and Aβ42 brain levels compared to vehicle-treated hAPP mice.ConclusionsOur data indicate that BTZ protects P-gp from proteasomal degradation in hAPP mice, which helps to reduce Aβ brain levels. Our data suggest that the proteasome system could be exploited for a novel therapeutic strategy in AD, particularly since increasing Aβ transport across the blood–brain barrier may prove an effective treatment for patients.

BORTEM-17: A phase IB/II single arm, multicentre study investigating the efficacy of sequential bortezomib and temozolomide in recurrent GBM with unmethylated MGMT promoter—The results of an interim analysis.

2019 Background: Glioblastoma (GBM) is the most frequent, lethal primary brain tumor in adults. Patients harboring tumors with functional O6 methylguanine DNA methyltransferase ( MGMT) DNA enzyme gain limited benefit from temozolomide therapy. As shown in preclinical studies, bortezomib depletes the MGMT enzyme, restoring the tumor ́s susceptibility to temozolomide, if administered in a precise schedule when the MGMT enzyme is depleted. We hypothesized that recurrent GBM patients with unmethylated MGMT promoter may obtain clinical benefit from sequential BTZ and TMZ treatment. Methods: BORTEM-17 is a multicenter, open label, single arm, non-randomized phase IB/II trial to investigate potential survival benefit for recurrent glioblastoma patients administered bortezomib 48hrs prior to temozolomide. Sample size is calculated to 63 patients, of whom 10 included in phase IB. The doses of bortezomib is 1.3mg/m2 intravenously days 1, 4, and 7 during 4-week cycle, and temozolomide 200mg/m2 days 5-7. The control group is a retrospective cohort of 467 patients treated at 2 referral hospitals in Norway from January 2015 to December 2017. For the survival analysis, the patients included in BORTEM-17 (n=44) were compared with MGMT unmethylated control, age matched patients (n=116). The pre-defined interim analysis was performed after inclusion of 15 patients and as more than 2 of 15 patients had clinical benefit, the study was continued. Results: Until January 2023, 44 patients with median age 55 years (range 25-69), 30 males and 14 females were treated. Median KPS was 90 (70-100) and median NANO score 1 (0-7). The interim analysis was performed after inclusion of 15 patients in the phase IB and II and clinical benefit was observed in 5 patients. Two of them had tumor volume reduction and three experienced stable disease. The study proceeded to the next step. No treatment related deaths were observed. The most common adverse effects included hematological toxicity, gastrointestinal symptoms, muscle weakness, lower back pain and fatigue. Patients that progressed during the BORTEM-17 trial and were fit for further therapy received treatment at their physician’s discretion. The preliminary data analysis after 44 of planned 63 patients were treated indicate prolonged median survival for BORTEM-17 patients 19.0 months vs 12.2 months of control MGMT unmethylated age matched patients. Median survival after recruitment is 5.5 months (1.0-23.8). Conclusions: The sequential BTZ+TMZ therapy is safe, feasible and effective as indicated by preliminary data when 70% of planned MGMT unmethylated patients have been included. Preliminary data indicate that the combination of BTZ and TMZ may offer an additional line of treatment with limited toxicity to the group of patients with particularly dismal prognosis. Clinical trial information: NCT03643549 .

Molecular characterization stratifies VQ myeloma cells into two clusters with distinct risk signatures and drug responses.

Multiple myeloma (MM) is a cancer of malignant plasma cells in the bone marrow and extramedullary sites. We previously characterized a VQ model for human high-risk MM. The various VQ lines display different disease phenotypes and survival rates, suggesting significant intra-model variation. Here, we use whole-exome sequencing and copy number variation (CNV) analysis coupled with RNA-Seq to stratify the VQ lines into corresponding clusters: Group A cells had monosomy chromosome (chr) 5 and overexpressed genes and pathways associated with sensitivity to bortezomib (Btz) treatment in human MM patients. By contrast, Group B VQ cells carried recurrent amplification (Amp) of chr3 and displayed high-risk MM features, including downregulation of Fam46c, upregulation of cancer growth pathways associated with functional high-risk MM, and expression of Amp1q and high-risk UAMS-70 and EMC-92 gene signatures. Consistently, in sharp contrast to Group A VQ cells that showed short-term response to Btz, Group B VQ cells were de novo resistant to Btz in vivo. Our study highlights Group B VQ lines as highly representative of the human MM subset with ultrahigh risk.

Neurofilament light chain levels indicate acute axonal damage under bortezomib treatment

IntroductionBortezomib (BTZ) is a selective and reversible proteasome inhibitor and first line treatment for multiple myeloma (MM). One of the side effects is BTZ-induced peripheral neuropathy (BIPN). Until now there is no biomarker which can predict this side effect and its severity. Neurofilament light chain (NfL) is a neuron specific cytoskeletal protein, of which higher levels can be detected in peripheral blood in case of axon damage. In this study, we aimed to evaluate the relationship between NfL serum levels and characteristics of BIPN.MethodsWe performed a first interim analysis of a monocentric, non-randomized, observational clinical trial including 70 patients (DRKS00025422) diagnosed with MM in the inclusion period of June 2021 until March 2022. Two groups of patients—one with ongoing BTZ treatment at the time of recruiting, and one with BTZ treatment in the past—were compared to controls. NfL in serum was analyzed via the ELLA™ device.ResultsBoth patients with previous and ongoing BTZ treatment had higher serum NfL levels than controls, and patients with ongoing BTZ treatment had higher NfL levels than patients with BTZ treatment in the past. Serum NfL levels correlated with electrophysiological measures of axonal damage in the group with ongoing BTZ treatment.ConclusionElevated NfL levels indicate acute axonal damage under BTZ in MM patients.

Liposomal Melphalan: Approach to obtain improved plasma stability, pharmacokinetics, and in vitro and in vivo anticancer efficacy in combination with liposomal simvastatin against mouse RPMI-8226 multiple myeloma model

Drug resistance and dose-limiting toxicities are the chief hurdles of multiple myeloma (MM) treatment. The targeted nanoparticulate drugs have shown increased specificity and efficacy and decreased toxicities than plain drugs. The present study was aimed to test the anticancer efficacy of Melphalan (MLN) in combination with an antihyperlipidemic drug (Simvastatin; SVN) and improve the treatment using liposomal drugs against mouse RPMI-8226 MM model. The Vitamin E oil (VEO)-composed and MLN- and SVN-loaded liposomes (MLs and SLs) surface coated with Pluronic F108 (PF-108) have shown a mean particle size below 200 nm. The in vitro cytotoxicity study results indicated improved anticancer activity of anticancer drug MLN in combination with SVN against human MM cells (RPMI-8226); whereas, the liposomal drugs showed the sustained anticancer effect. The in vitro plasma chemical stability and pharmacokinetic profiles of liposomal MLN have substantially improved. The liposomal combination (MLs + SLs) treatment has resulted in substantially improved tumor growth inhibition and reduced toxicity in comparison to plain drug combination (MLN + SVN) treatment that showed improved efficacy over standard BTZ treatment. Further, the BAX and Caspase-3 gene expressions are found moderately higher (1.98- and 1.62-fold higher) in tumor tissues treated with the liposomal combination. The above results indicate repurposing potential of SVN in amalgamation with MLN in MM treatment; however, the liposomal drugs could be used over plain drugs to achieve higher efficacy with decreased toxicity.

Caspase-8 Regulates the Antimyeloma Activity of Bortezomib and Lenalidomide.

Proteasome inhibitors and immunomodulatory drugs (IMiDs) are two major types of drugs for the treatment of multiple myeloma. Although different combination therapies for myeloma have been developed and achieved high responsive rate, these strategies frequently result in drug resistance. Therefore, it is necessary to explore new molecular mechanisms and therapeutic approaches to fulfill this unmet medical need. Here, we find that proteasome inhibitor bortezomib (Btz) causes cereblon (CRBN) cleavage and that caspase-8 (CASP-8) is responsible for this cleavage. Either inhibition or genetic depletion of CASP-8 decreased the CRBN cleavage upon Btz treatment, which could potentiate the antimyeloma activity of IMiD lenalidomide (Len). This work suggests that administration of CASP-8 inhibitors might enhance the overall effectiveness of Btz/Len-based therapeutic treatment of patients with myeloma. SIGNIFICANCE STATEMENT: Caspase-8 activation upon bortezomib treatment results in the cleavage of cereblon, a substrate receptor of the cullin-4 RING E3 ligase, which is responsible for the degradation of two transcription factors, Ikaros family zinc finger protein (IKZF) 1 and IKZF3, in the presence of immunomodulatory drugs including lenalidomide. The administration of caspase-8 inhibitor may enhance the antimyeloma activity of the combination therapy with bortezomib and lenalidomide.

Lipidomic Profiling of Plasma Samples in Patients with Newly Diagnosed Multiple Myeloma; A Biomarker Study for Predicting the Response and Toxicities of Melphalan, Prednisolone and Bortezomib (MPB) Regimen : An Ancillary Study of the JCOG1105 (JCOG1105A1)

Lipidomic Profiling of Plasma Samples in Patients with Newly Diagnosed Multiple Myeloma; A Biomarker Study for Predicting the Response and Toxicities of Melphalan, Prednisolone and Bortezomib (MPB) Regimen : An Ancillary Study of the JCOG1105 (JCOG1105A1)

Low expression of neural cell adhesion molecule, CD56, is associated with low efficacy of bortezomib plus dexamethasone therapy in multiple myeloma.

Bortezomib (Btz) is an active agent used to treat multiple myeloma (MM). Not all patients who receive Btz-containing therapy show a favorable response. Interaction of cellular adhesion molecules with MM and bone marrow stromal cells is crucial for the survival of MM cells. However, little is known about the role of these molecules in the sensitivity of MM to Btz-containing therapy. Thus, we evaluated the correlation between the level of cellular adhesion molecules in MM cells and the efficacy of Btz plus dexamethasone (Bd) therapy. The expression of the neural cell adhesion molecule gene (NCAM, also known as CD56), ITGA4, CXCR4, and other genes were analyzed in 74 samples of primary MM cells collected from patients before they received Bd therapy. Of the eight genes tested, expression of NCAM was lower among patients who responded poorly to Bd therapy. In vitro expression of NCAM induced by transfection of MM cells enhanced their sensitivity to Btz treatment by causing accumulation of polyubiquitinated proteins. Our results indicate that expression of NCAM is associated with better response to Btz treatment and is a promising candidate biomarker for predicting response to therapies involving Btz.

Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib.

The activation of Nrf2 has been demonstrated to play a crucial role in cancer cell resistance to different anticancer therapies. The inhibition of proteasome activity has been proposed as a chemosensitizing therapy but the activation of Nrf2 could reduce its efficacy. Using the highly chemoresistant neuroblastoma cells HTLA-230, here we show that the strong reduction in proteasome activity, obtained by using low concentration of bortezomib (BTZ, 2.5 nM), fails in reducing cell viability. BTZ treatment favours the binding of Nrf2 to the ARE sequences in the promoter regions of target genes such as heme oxygenase 1 (HO-1), the modulatory subunit of γ-glutamylcysteine ligase (GCLM) and the transporter for cysteine (x-CT), enabling their transcription. GSH level is also increased after BTZ treatment. The up-regulation of Nrf2 target genes is responsible for cell resistance since HO-1 silencing and GSH depletion synergistically decrease BTZ-treated cell viability. Moreover, cell exposure to all-trans-Retinoic acid (ATRA, 3 μM) reduces the binding of Nrf2 to the ARE sequences, decreases HO-1 induction and lowers GSH level increasing the efficacy of bortezomib. These data suggest the role of Nrf2, HO-1 and GSH as molecular targets to improve the efficacy of low doses of bortezomib in the treatment of malignant neuroblastoma.

Targeting Neddylation to Overcome Drug Resistance in Multiple Myeloma

Background: The overexpression of CKS1B resulting from chromosomal amplification of the 1q21 region identifies a subset of multiple myeloma (MM) patients with poor clinical outcomes. Bortezomib (Btz) is an FDA-approved first-in-class proteasome inhibitor that has greatly improved clinical outcomes in MM. However, toxicities affiliated with pan-proteasomal inhibition and resistant populations remain a problem, highlighting the need for improved therapies. We have previously demonstrated that elevated expression of CKS1B results in the destabilization of p21 and contributes to insensitivity to bortezomib. MLN4924 is an inhibitor of the NEDD8-Activating Enzyme E1 (NAE1) and prevents the neddylation and subsequent activation of Cullin-1, a crucial component in the activation of SCF-driven ubiquitin-mediated degradation. We therefore explored if MLN4924 might serve as an alternative inhibitor to proteasomal degradation in MM.Materials and Methods: CKS1B was overexpressed or knocked down in myeloma cell lines using lentiviral vectors and shRNA. Cell proliferation and viability were assessed by cell counts and using PrestoBlue reagent. Clonogenicity was assessed by colony formation in soft agar. SA-b-galactosidase staining was used to assess senescence. Gene expression profiling was performed using the publicly available databases Total Therapy 2 (TT2) and APEX trials.Results: To examine the efficacy of Btz and MLN4924 in elevated CKS1B expression environments, we compared treatment with the MLN4924 to Btz in CKS1B OE cells in vitro. Cells with basal levels of CKS1B were sensitive to treatment with either Btz or MLN4924, with treatment leading to decreased proliferation and cellular viability. In the CKS1B OE background, we found cells were resistant to Btz repression of proliferation but sensitive to MLN4924. We also found that MLN4924 could more potently reduce colony formation in soft agar and more potently induce senescence in CKS1B OE cells compared to treatment with Btz. Immunoblot analyses confirmed a correlation between CKS1B expression and Cullin-1 neddylation. Further immunoblotting of known SCF-mediated ubiquitin targets in cells with and without CKS1B OE demonstrated a stabilization of p21 in all cells upon MLN4924 treatment that was not exhibited upon Btz treatment. To investigate the role of p21 in CKS1BOE cell sensitivity to MLN4924, we stably knocked down expression of p21. We found the knockdown of p21 partially abrogated the sensitivity of CKS1B OE cells to MLN4924, increasing cell viability, increasing colony formation in soft agar, and decreasing senescence induction.The importance of neddylation in the clinic was confirmed using GEP. We found that expression of the neddylation-related genes (i.e. NAE1, UBA3, and UBC12) is significantly upregulated (p<0.05) in MM patients relative to patients in MGUS or to healthy donors. Expression of NEDD8 was also significantly elevated in patients unresponsive to either Btz treatment or treatment with the combination of Btz and dexamethasone compared to responsive patients. The elevated expression of CKS1B highlights a population of patients with poor clinical outcomes. Considering the elevated expression of neddylation-related genes in MM progression, we performed Kaplan Meier survival analyses looking at patients segregated on two axes by CKS1B expression levels (high/low) and neddylation-related gene expression levels (either UBA3 or UBC12). We found patients with high/high expression of CKS1B and neddylation-related genes had significantly decreased survival relative to patients with mixed high/low expression from either axis. Together, these data suggest the importance of neddylation contributions to MM progression and clinical outcomes.Conclusions: These results shed light on the subtle differences in the blockade of proteasomal inhibition, illustrating the distinct effects of pan inhibition from inhibition of SCF-driven ubiquitin-mediated degradation. Mechanistically, we demonstrate at least a partial role for p21 in mediating cell sensitivity to MLN4924 in CKS1B overexpressing cells. Our findings highlight the important contributions of neddylation to MM disease progression and suggest the utility of targeting neddylation as a means of overcoming drug resistance in MM. DisclosuresNo relevant conflicts of interest to declare.

Lower expression of activating transcription factors 3 and 4 correlates with shorter progression-free survival in multiple myeloma patients receiving bortezomib plus dexamethasone therapy.

Bortezomib (BTZ), a proteasome inhibitor, is widely used in the treatment of multiple myeloma (MM), but a fraction of patients respond poorly to this agent. To identify factors predicting the duration of progression-free survival (PFS) of MM patients on BTZ treatment, the expression of proteasome and endoplasmic reticulum (ER) stress-related genes was quantified in primary samples from patients receiving a combination of BTZ and dexamethasone (BD). Fifty-six MM patients were stratified into a group with PFS<6 months (n=33) and a second group with PFS⩾6 months (n=23). Of the 15 genes analyzed, the expression of activating transcription factor 3 (ATF3) and ATF4 was significantly lower in patients with shorter PFS (P=0.0157 and P=0.0085, respectively). Chromatin immunoprecipitation analysis showed that these ATFs bind each other and transactivate genes encoding the pro-apoptotic transcription factors, CHOP and Noxa, which promote ER stress-associated apoptosis. When either ATF3 or ATF4 expression was silenced, MM cells partially lost sensitivity to BTZ treatment. This was accompanied by lower levels of Noxa, CHOP and DR5. Thus low basal expression of ATF3 and ATF4 may attenuate BTZ-induced apoptosis. Hence, ATF3 and ATF4 could potentially be used as biomarkers to predict efficacy of BD therapy in patients with MM.

Prolonged therapy with single-agent bortezomib (btz) in four patients (pts) with relapsed primary systemic AL amyloidosis treated in the CAN2007 phase II study.

e19509 Background: CAN2007 was the first prospective study of single-agent btz in relapsed AL. We describe 4 pts in CAN2007 (NCT00298766) who received btz treatment for up to 5.6 yrs. Methods: 70 pts received btz 0.7–1.6 mg/m2, d 1, 8, 15, 22, 35-d cycles (QW), or 0.7–1.3 mg/m2, d 1, 4, 8, 11, 21-d cycles (BIW), for up to 8 cycles; prolonged treatment was permitted at the discretion of the treating physician for pts showing ongoing clinical benefit. Results: Pt #1 (male, age 53 yrs; IgA lambda AL with cardiac and renal involvement; 16 mos since diagnosis; prior therapy: melphalan plus ASCT) received btz 1.3 mg/m2 QW for 57 cycles (5.6 yrs). Cumulative dose was 190.5 mg/m2. He had a hematologic CR to initial btz therapy and a complete renal response to sustained therapy, without grade 3/4 adverse events (AEs). Pt #2 (male, age 53 yrs; IgG kappa AL with gastrointestinal [GI] involvement; 38 mos since diagnosis; prior therapy: ASCT) received btz 0.7 mg/m2 BIW for 47 cycles (4.3 yrs; cumulative dose 77.7 mg/m2), had a hematologic response of stable disease, and had a renal response, with no grade 3/4 AEs. The pt progressed and is in remission on alternative therapy. Pt #3 (female, age 55 yrs; IgG lambda AL with GI and other-site involvement; 14 mos since diagnosis; prior therapy: ASCT) received btz 1.6 mg/m2 QW for 39 cycles (3.7 yrs; cumulative dose 167.7 mg/m2). She had grade 3 paralytic ileus in cycle 8 with dose adjustment to 1.3 then 1.0 mg/m2. She had a hematologic PR. Pt #4 (female, age 54 yrs; lambda light-chain AL with cardiac and GI involvement; 44 mos since diagnosis; prior therapy: cardiac allograft followed by ASCT) received 57 cycles (3.5 yrs; cumulative dose 173.7 mg/m2); starting dose was 1.3 mg/m2 BIW, reduced to 1.0 (cycle 3) and 0.7 mg/m2 (cycle 16) due to grade 3 AEs of vasculitis and volvulus, respectively. Other grade 3 AEs included pneumonia, hemoptysis, and C. difficile diarrhea. Best hematologic response was PR; a renal response was also achieved. Conclusions: This case series indicates the feasibility and activity of btz for long-term disease control in relapsed AL, particularly in pts who did not achieve a CR to initial btz therapy. Pts 1, 3, 4 remain progression-free and are ongoing on btz. Clinical trial information: NCT00298766.

Efficacious proteasome/HDAC inhibitor combination therapy for primary effusion lymphoma

Primary effusion lymphoma (PEL) is a rare form of aggressive B cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Current chemotherapy approaches result in dismal outcomes, and there is an urgent need for new PEL therapies. Previously, we established, in a direct xenograft model of PEL-bearing immune-compromised mice, that treatment with the proteasome inhibitor, bortezomib (Btz), increased survival relative to that after treatment with doxorubicin. Herein, we demonstrate that the combination of Btz with the histone deacetylase (HDAC) inhibitor suberoylanilidehydroxamic acid (SAHA, also known as vorinostat) potently reactivates KSHV lytic replication and induces PEL cell death, resulting in significantly prolonged survival of PEL-bearing mice. Importantly, Btz blocked KSHV late lytic gene expression, terminally inhibiting the full lytic cascade and production of infectious virus in vivo. Btz treatment led to caspase activation and induced DNA damage, as evidenced by the accumulation of phosphorylated γH2AX and p53. The addition of SAHA to Btz treatment was synergistic, as SAHA induced early acetylation of p53 and reduced interaction with its negative regulator MDM2, augmenting the effects of Btz. The eradication of KSHV-infected PEL cells without increased viremia in mice provides a strong rationale for using the proteasome/HDAC inhibitor combination therapy in PEL.

Risk of Second Primary Malignancies (SPMs) Following Bortezomib (Btz)-Based Therapy: Analysis of Four Phase 3 Randomized Controlled Trials in Previously Untreated or Relapsed Multiple Myeloma (MM)

Abstract 2933 Background:MM patients have a significantly increased risk of developing certain SPMs subsequent to their initial diagnosis, including a 3.49-fold increased risk of leukemia and, specifically, an 8.32-fold increased risk of acute non-lymphocytic leukemia (but no overall increased risk of solid tumor SPMs; Surveillance, Epidemiology and End Results [SEER] data 1973–2000). The relative SPM risk increases with age and time after initial diagnosis; the risk of leukemia rises from a 1.22-fold increase within 1 year after diagnosis to 3.12-fold, 7.01-fold, and 5.45-fold increases at 1–4, 5–9, and ≥10 years, respectively. An elevated risk of SPMs may be particularly associated with the use of specific therapeutic agents, including conventional or high-dose cytotoxic chemotherapy. Here we report an analysis of data from four phase 3, randomized, controlled trials of Btz alone or in combination to determine whether Btz treatment is associated with an increased SPM risk. Methods:Data were reviewed from: 1) the APEX study of Btz versus high-dose dexamethasone (Dex), and 2) the MMY-3001 study of Btz plus pegylated liposomal doxorubicin (PLD) versus Btz in patients with relapsed or refractory MM after 1–3 prior therapies; 3) the VISTA study of Btz plus melphalan-prednisone (VMP) versus MP in previously untreated transplant-ineligible patients; and 4) the HOVON65/GMMG-HD4 study of Btz, doxorubicin, and Dex (PAD) induction plus Btz maintenance post-transplant versus vincristine, doxorubicin, and Dex (VAD) induction plus thalidomide (Thal) maintenance in previously untreated transplant-eligible patients. Planned duration of Btz therapy was 39 weeks in APEX, 24 weeks in MMY-3001, 54 weeks in VISTA, and 9 weeks induction plus 2 years of maintenance in HOVON65/GMMG-HD4. For APEX, MMY-3001, and VISTA, clinical trial databases were reviewed for events within the MedDRA system organ class of ‘neoplasms', and new malignancies developing during or after treatment were recorded (excluding non-melanomatous skin cancers and in situ malignancies). In addition, for VISTA, data were obtained from an SPM survey after a median follow-up of 5 years. For HOVON65/GMMG-HD4, data were prospectively collected; median follow-up was 42 months. The incidence rate (IR) of SPMs was expressed as the number per 100 patient-years (pt-yrs). Results:The risk of SPMs with Btz-based therapy appeared uniformly low across all four phase 3 studies in different MM patient populations (Table). A total of 25 SPMs were seen in 1718 Btz-treated patients, including three cases of acute myeloid leukemia/myelodysplastic syndromes, one B-cell malignancy, and one case of cutaneous T-cell lymphoma (in a patient with substantial prior alkylating agent exposure for treatment of MM), plus 20 reports of solid tumors (reflecting the higher overall incidence of these tumors). The IR ranged from 0 in the single-agent Btz arm of MMY-3001 to 1.66 with VMP in VISTA, with an IR of 0.88 in APEX and 0.3 in the PAD arm of HOVON65/GMMG-HD4; median age of Btz-treated patients was highest in VISTA, at 71 years. Overall rates (%) also appeared higher in VISTA, probably due to the longer follow-up and older population, plus the potential effects of concomitant chemotherapy. In the three studies with non-Btz control arms, the IR did not appear to be increased with Btz-based therapy versus the control arm, and across all Btz-containing arms in the four studies, the IRs for hematologic malignancies and solid tumors were consistent with SEER estimates (2004–2008 data) of the overall incidence of malignancies in the US population, of 1.1, 1.9, and 2.4 per 100 pt-yrs in individuals aged 55–64, 65–74, and 75–84 years, respectively. Conclusions:Btz-based therapy for MM does not appear to be associated with an increased risk of either hematologic or solid tumor SPMs, with IRs consistent with SEER data for IRs in the overall US population.SPMs in phase 3 studies of Btz-based therapyAPEXMMY-3001VISTAHOVON65/ GMMG-HD4ArmBtzDexBtz+PLDBtzVMPMPPAD/BtzVAD/ThalN331332318316340337413414Median age, yrs62616162717157*57*SPMs, n (%)1 (0.30)01 (0.31)019 (5.6)13 (3.9)4 (0.97)11 (2.7)Heme1 (0.30)0003 (0.88)3 (0.89)1 (0.24)4 (0.97)Solid001 (0.31)016 (4.71)10 (2.97)3 (0.73)7 (1.69)Pt-yrs113.994.2106.3104.51167100412391183IR0.8800.9401.661.300.30.9Heme0.880000.260.300.0750.33Solid000.9401.401.000.2250.57Heme, hematologic malignancies.*ASH 2010 data Disclosures:San Miguel:Janssen-Cilag: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc: Consultancy. Richardson:Celgene: Consultancy; Janssen Research & Development: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy. Orlowski:Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding; Novartis: Consultancy; Onyx: Consultancy; Cephalon: Consultancy; Centocor: Consultancy; Celgene: Consultancy, Research Funding. Goldschmidt:Janssen-Cilag: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy. Corzo:Millennium Pharmaceuticals, Inc.: Employment. Satler:Millennium Pharmaceuticals, Inc.: Employment. Esseltine:Millennium Pharmaceuticals, Inc.: Employment, Equity Ownership; Johnson & Johnson: Equity Ownership. Neuwirth:Millennium Pharmaceuticals, Inc.: Employment. Ponsillo:Millennium Pharmaceuticals, Inc.: Employment. Cakana:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. King:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Deraedt:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Desai:Janssen Global Services: Employment; Johnson & Johnson: Equity Ownership. Lutska:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Gifkins:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Liu:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership.

Potentiation of bortezomib's Anti-Myeloma Effects in Combination with Anti-DR5 Agonistic Antibody: The Role of ER Stress in DR5 Editing and DR-Mediated Apoptotic Signaling

Abstract 5104Bortezomib (BTZ) is widely used in the treatment of myeloma (MM) with marked response rates in both relapsed/refractory as well as newly diagnosed MM. However, significant numbers of patients still remain outside benefit of the BTZ treatment; and various combinatory treatments with BTZ have been implemented to improve BTZ's anti-MM effects. On the other hand, immunotherapies seem attractive for yet incurable malignancies by chemotherapeutic agents such as MM and their clinical application has been studied. One such approach is a TNF-related apoptosis-inducing ligand (TRAIL)-mediated immunotherapy. In the present study, we therefore explored the role of BTZ on TRAIL receptor editing and its downstream signaling with special reference to endoplasmic reticulum (ER) stress and the cytotoxic effects of BTZ and anti-TRAIL receptor agonistic antibody in combination on MM cells. Most MM cells expressed DR4 but weakly DR5, while normal peripheral blood mononuclear cells expressed neither DR4 nor DR5. BTZ at 10 nM markedly up-regulated the surface levels of DR5 and its mRNA expression but not those of DR4 in MM cell lines and primary MM cells. Furthermore, BTZ decreased the levels of c-FLIP, an inhibitor of DISC, along with activation of caspase-8 and caspase-3, suggesting potentiation of the DR-mediated extrinsic apoptotic pathway. Consistently, BTZ and anti-DR5 agonistic antibody cooperatively enhanced the cytotoxicity against MM cells. BTZ induced phosphorylation of eIF2alpha, ATF4 and CHOP, along with disappearance of anti-apoptotic proteins including Mcl-1 in MM cells, suggesting the enhancement of ER stress and subsequent suppression of protein translation by BTZ. However, such induction of ER stress by BTZ was not observed in BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, with a point mutation in BTZ-binding proteasome beta5 subunit (Ri et al. Leukemia 2010). In KMS-11/BTZ and OPM-2/BTZ, surface protein as well as mRNA levels of DR5 were not up-regulated by BTZ, suggesting a critical role of ER stress in up-regulation of DR5 expression by BTZ. Because DR5 expression has been shown to be transcriptionally up-regulated by CHOP, the up-regulation of DR5 mRNA and protein in MM cells by BTZ is suggested to be at least in part due to CHOP induced by BTZ-mediated ER stress. Although BTZ exerts its anti-MM effects through induction of ER stress, the present study demonstrates that induction of ER stress by BTZ is also able to sensitize MM cells to TRAIL-mediated immunotherapy. Therefore, the combination of BTZ and TRAIL-mediated immunotherapy is warranted for further study. Disclosures:No relevant conflicts of interest to declare.

PX-171-004, An Ongoing Open-Label, Phase II Study of Single-Agent Carfilzomib (CFZ) in Patients with Relapsed or Refractory Myeloma (MM); Updated Results From the Bortezomib-Treated Cohort.

Abstract 303 Background: Carfilzomib (CFZ) is a novel proteasome inhibitor that binds its target selectively and irreversibly, resulting in greater and more sustained proteasomal inhibition compared to BTZ (Demo et al, Cancer Res 2007). CFZ overcomes BTZ-resistance in vitro (Kuhn et al, Blood 2007) and in a previous Phase 2 study (PX-171-003), single-agent CFZ achieved durable responses and maintained disease control [e.g. ≥ Stable Disease (SD)] in patients with progressive multiple myeloma (MM) despite treatment with essentially all available agents. PX-171-004 is an ongoing Phase 2 study of CFZ monotherapy in MM patients with relapsed or refractory disease following 1–3 prior therapies. Here we report updated data for the BTZ-treated cohort. Methods: Patients with relapsed or refractory (defined as 2 IV was administered on Days 1, 2, 8, 9, 15 and 16 every 28 days, for up to 12 cycles. The primary endpoint was Overall Response Rate (ORR) [≥ Partial Response (PR)] by IMWG criteria. Secondary endpoints included Clinical Benefit Response (CBR = ORR + MR) and safety. Results: Thirty-five BTZ-treated patients were enrolled. Six (17%) patients had previously received BTZ exclusively as a single agent, 15 (43%) received BTZ as part of a chemotherapy combination and 10 (29%) received BTZ as part of a stem cell transplant (SCT) regimen. An additional 4 (11%) received BTZ in a chemotherapy combination as well as part of a separate transplant regimen. Other prior therapies included alkylators (89%), SCT (81%), thalidomide (69%), lenalidomide (37%), and anthracyclines (31%). Six (17%) patients had disease refractory to BTZ and 9 (26%) additional patients had discontinued BTZ due to toxicities. At baseline, 19 (54 %) patients had an ECOG score ≥ 1, 17 (49%) patients had neuropathy of Grade ≥ 1, 9 (26%) patients had impaired renal function (CrCl 19 cycles on a recently initiated extended treatment protocol. Conclusions: In a BTZ-exposed population that includes BTZ treatment failures and significant comorbidities (e.g. diabetes, renal insufficiency, etc), the 18% ORR (CBR 30%) is notable for this steroid- and anthracycline-sparing regimen. Single-agent CFZ is well tolerated, even in patients with renal insufficiency, and both myelosuppression and PN are uncommon. These data support the continuing evaluation of CFZ as a safe and effective treatment option in MM. Disclosures: Siegel: Celgene: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau. Wang: Proteolix, Inc.: Research Funding. Stewart: Genzyme, Celgene, Millenium, Proteolix: Honoraria; Takeda, Millenium: Research Funding; Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy. Kukreti: Celgene: Honoraria. Jakubowiak: Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Centocor Ortho Biotech: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Exelixis: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers-Squibb: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau. Jagannath: Millennium: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Celgene: Membership on an entity9s Board of Directors or advisory committees. McDonagh: Proteolix: Research Funding. Belch: Ortho Biotech: Honoraria, Research Funding. Bahlis: Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria, Speakers Bureau. Le: Proteolix, Inc.: Employment. Kunkel: Proteolix: Consultancy, Employment. Bennett: Proteolix: Employment. Kauffman: Proteolix, Inc.: Employment. Vij: Proteolix, Inc.: Consultancy, Research Funding.

Pre-Treatment p27 and Bcl-6 Staining Levels Correlate with Response to Bortezomib in Non-Hodgkin Lymphoma: Results from a Tissue Microarray Analysis

Background: Proteasome inhibitors (PI) affect a variety of intracellular processes, including the cell cycle, NF-kB and other signal transduction pathways, and the balance of bcl-2-related proteins, yet it remains unknown which of these targets are most crucial in determining response to PI treatment in non-Hodgkin Lymphoma (NHL). Responses to bortezomib (btz) have been seen in different NHL subtypes in several phase 2 studies. The consistency of response suggests that there may be biological characteristics that determine individual tumor responses to PIs. We used tissue microarray (TMA) technology to determine if pre-treatment tumor characteristics can predict outcome of btz treatment in NHL.Methods: TMAs were constructed using paraffin-embedded blocks of pre-treatment tissue from patients enrolled in a phase 2, CTEP-NCI sponsored trial of btz in NHL. Microsections were stained for 18 proteins with reported relevance to proteasome function and/or lymphoma prognosis. All stains were evaluated and scored by a central pathologist. Staining was correlated with quality and duration of response (DOR) or time to treatment failure (TTF). The best response was treated as a binary outcome (PR/CR vs SD/POD). Correlation between each marker and response was assessed by univariate analysis. The exact Wilcoxon rank-sum test was used to compare DOR and TTF with respect to the binary marker scores.Results: This analysis includes 35 of 75 patients treated with btz between 11/6/01 and 2/23/07 for whom pre-treatment tissue blocks could be obtained. Response data were available for 32 of these patients. Diagnoses include: FL (N=10), SLL (N=4), MCL (N=14) and MZL (N=4). Expression of the cell-cycle inhibitor p27 was more common in responding vs non-responding pts (p=0.024), and was also significantly associated with longer DOR (p=0.020) and longer TTF (p=0.021; figure). Presence of Bcl-6 was significantly associated with shorter DOR (p=0.049) and shorter TTF (p=0.048; figure), but not response.Conclusions: Using TMA analysis, we have shown that NHL patients with pre-treatment p27 staining were more likely to respond to btz, and had a longer DOR and TTF than those without baseline staining. Conversely, patients with baseline Bcl-6 expression had a shorter DOR and TTF than those without. Although the number of patients in this analysis is small, the data will help us generate hypotheses about disease and treatment biology that can be tested in larger studies. We are currently collecting samples from a broader pool of patients treated with btz at MSKCC to verify these results. TTF with bortezomib by (A) p27 and (B) Bcl-6 status. [Display omitted]