Second-generation Proteasome Inhibitor Research Articles

Highlights of This Issue

Curti and colleagues correlated the graft composition with the clinical outcome of 17 elderly patients with AML undergoing NK cell immunotherapy after induction/consolidation. AML patients received NK cells from haploidentical KIR-ligand mismatched donors after immunosuppression, followed by interleukin-2. The authors demonstrated that larger size of donor alloreactive NK cell repertoire correlates with better response to immunotherapy. Such finding represents a proof-of-concept of the antileukemic role of NK alloreactivity and has implications for the choice of the most suitable donor.Molecular predictors of response and mechanisms of resistance to azacytidine in Myelodysplastic syndrome (MDS) remain largely unidentified. Abnormal Stat3/5 signaling is implicated in clonal hematopoiesis. To explore the Stat3/5 signaling architecture in MDS, Miltiades and colleagues performed functional phenotyping in MDS patients undergoing azacytidine treatment. The Stat3/5 signaling biosignature identified patients in poor prognosis and marked a pathophysiologically and prognostically relevant, azacytidine resistant, CD34+ subset, whose kinetics paralleled the disease course and response to azacytidine. Their findings suggest that the aberrant Stat3/5 biosignature in MDS may represent a response predictor and also a therapeutic target to overcome azacytidine resistance.A mutation in NPM1 is one of the most common recurring genetic lesions in AML. To explore its role in rendering acute leukemia cells susceptible to ixazomib, a recently approved second-generation proteasome inhibitor for multiple myeloma, Garcia and colleagues evaluated the oxidative stress response in mediating cytotoxic activity. A direct association was observed between NPMc+ expression in AML, reduced antioxidant responses, and enhanced sensitivity to an oral proteasome inhibitor that induces oxidative stress. These data suggest that intracellular determinants of antioxidant responses may be good predictors of therapeutic response to ixazomib.The high frequency of the IGHV1-2*04 immunoglobulin gene in splenic marginal zone lymphoma (SMZL) strongly indicates antigen selection in disease development. IGHV1-2*04 expressing cases account for one-third of all SMZL and constitute a disease subgroup with a distinctive profile of genomic aberrations and an overall worse prognosis, indicating that immune signaling mediated by distinctive immunoglobulin receptors may impact on SMZL clonal behavior and eventual patient outcome. The present study advances the understanding of SMZL pathogenesis showing that ongoing antigen involvement is relevant throughout the natural history of SMZL, particularly IGHV1-2*04 cases, thus offering a strong rationale for signaling inhibition therapy.

A practical review on carfilzomib in multiple myeloma.

Carfilzomib, a second-generation proteasome inhibitor, has been increasingly used in relapsed/refractory multiple myeloma (MM) since its approval by the American food and drug administration (FDA) in the summer of 2012. The drug is active as a single agent and in combination with other antimyeloma agents. As a result of its efficacy and safety in the relapsed/refractory setting, carfilzomib is being evaluated in patients with newly diagnosed MM as well as in high-risk smoldering MM. This review will give a comprehensive summary of the drug, including its mechanism of action, the evaluated doses and schedules as well as a summary of the main clinical trials in the relapsed/refractory and newly diagnosed settings. A focus will be placed upon certain subgroups of interest as well as a description of the toxicity associated with carfilzomib use and a clinical perspective on toxicity management.

Carfilzomib potentiates CUDC-101-induced apoptosis in anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is one of the most aggressive human malignancies, with no effective treatment currently available. Previously, we identified agents active against ATC cells, both in vitro and in vivo, using quantitative high-throughput screening of 3282 clinically approved drugs and small molecules. Here, we report that combining two of these active agents, carfilzomib, a second-generation proteasome inhibitor, and CUDC-101, a histone deacetylase and multi-kinase inhibitor, results in increased, synergistic activity in ATC cells. The combination of carfilzomib and CUDC-101 synergistically inhibited cellular proliferation and caused cell death in multiple ATC cell lines harboring various driver mutations observed in human ATC tumors. This increased anti-ATC effect was associated with a synergistically enhanced G2/M cell cycle arrest and increased caspase 3/7 activity induced by the drug combination. Mechanistically, treatment with carfilzomib and CUDC-101 increased p21 expression and poly (ADP-ribose) polymerase protein cleavage. Our results suggest that combining carfilzomib and CUDC-101 would offer an effective therapeutic strategy to treat ATC.

Practical Approaches to the Management of Dual Refractory Multiple Myeloma.

The outcome of myeloma patients' dual refractory to lenalidomide and bortezomib is generally poor and represents a significant clinical challenge with a clear need for new therapeutic approaches. This has prompted the development of next-generation proteasome inhibitors and immunodulatory drugs (IMiDs), as well as new classes of drugs with novel mechanisms of action. As a result, several of these agents have received regulatory approval that have shown promising activity in the dual refractory setting including the second-generation proteasome inhibitor carfilzomib and third-generation IMiD pomalidomide. Moreover, the regulatory approval of several first-in-class drugs for myeloma such as the histone deacetylase (HDAC) inhibitor panobinostat and the anti-CD38 monoclonal antibody daratumumab has further broadened the therapeutic landscape for these patients. Collectively, these advances have provided new treatment strategies in dual refractory myeloma as well as important insights for the development of future studies with rationally designed drug combinations to target this challenging patient population.

Comparison of antiproliferative and apoptotic effects of a novel proteasome inhibitor MLN2238 with bortezomib on K562 chronic myeloid leukemia cells

Inhibition of the proteasome has emerged as a clinically effective anticancer therapeutic approach in recent years. Bortezomib (Velcade®) showed extremely high potency against a wide range of cancer cell lines. Ixazomib (MLN9708-MLN2238), the second-generation proteasome inhibitor, selectivity and potency were similar to that of bortezomib, is currently being investigated in phase I studies. It shows superior antitumor activity in hematologic malignancy, especially multiple myelomas. In this study, for the first time, we evaluated and compared the antiproliferative and apoptotic effects of the novel proteasome inhibitor MLN2238 (the active form of MLN9708) with bortezomib using in vitro chronic myeloid leukemia. Cytotoxic and apoptotic effects of MLN2238 and bortezomib were determined by trypan blue dye exclusion assays, WST-1 cell proliferation assay, increased AnnexinV-PI binding capacity, changes in caspase-3 activity and loss of mitochondrial membrane potential (JC-1). Associated with proteasome pathway NFκB1 and c-myc mRNA expression levels were examined by the qRT-PCR method. We observed that cytotoxic and apoptotic effects on K562 cells were started at 5 μm of MLN2238 and 1 μm of bortezomib after 24 and 48 h. Also, MLN2238 and bortezomib downregulated NFκB1 and c-myc mRNA expression at 24 h. Our result revealed that MLN22238 and bortezomib had significant cytotoxic and apoptotic effects on K562 cells. Here, we first demonstrate in vitro data that support the development of MLN2238, by direct comparison with bortezomib on K562 cells.

TOP2A Knockdown Resensitizes Carfilzomib-Resistant Hmcls to Carfilzomib

Introduction/background: Multiple myeloma (MM) remains incurable despite the introduction of novel therapeutic agents. Microarray-based technologies were adopted in our study to determine if a genetic signature associated with resistance to carfilzomib, a second-generation proteasome inhibitor already in use in clinical settings, could be identified.Materials and Methods: 18 genetically heterogeneous human myeloma cell lines (HMCLs) were treated with carfilzomib and a cell viability profile was assessed categorizing the HMCLs as sensitive, intermediate or resistant to carfilzomib. Following categorization gene expression profiling was performed and validated with q-RT-PCR and knockdown assays.Results: 29 genes were differentially regulated between the sensitive and resistant cell lines. Top genes based on intensity values and biological significance were: LOC731314, TSPAN13, APH1B, TSPYL5, COX7B2, PCSK1N, LRRC38, TCIRG1, TOP2A, ADM2, ITM2A, TSPAN13, STOM, UBE2C, SNHG8. Gene ontology (GO) enrichment analysis identified two pathways that were significantly different between the resistant and sensitive HMCLs; pathogenic escherichia coli infection (p=0.002) and lysosome (p=0.006). Eight GO terms were enriched: 4 related to biological processes and 4 related to cellular components.TOP2A, an enzyme that controls and alters the topologic states of DNA during transcription and is involved in cell cycle and proliferation, was identified to be overexpressed in resistant HMCLs. It functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. TOP2A may be used as a predictive factor for patient selection for specific protocols or as independent prognostic marker in solid tumors. TOP2A was also overexpressed in the 'proliferation cluster' associated with greater proliferation rate and poor outcome in newly diagnosed MM patients. Suppression of TOP2A by siRNA in carfilzomib-resistant HMCLs significantly resensitised the cell lines to carfilzomib.Conclusion: Our results suggest that TOP2A is overexpressed in carfilzomib-resistant HMCLs indicating a possible role as a predictive marker of response to carfilzomib in MM. DisclosuresNo relevant conflicts of interest to declare.

Carfilzomib Enhances Natural Killer Cell-Mediated Lysis of Myeloma Linked with Decreasing Expression of HLA Class I

Multiple myeloma (MM) is a malignant disorder characterized by uncontrolled monoclonal plasma cell proliferation. It accounts for 10% of all hematological malignancies and causes 15-20% of deaths from hematological malignancies. Although new therapies were introduced and overall survival of MM was improved in the last 10 years, MM still remains an incurable disease due to drug resistance. Natural killer (NK) cell-based treatments are promising therapies for multiple myeloma (MM). Carfilzomib (CFZ), a second-generation proteasome inhibitor, is used to treat patients with MM who are refractory or intolerant to both bortezomib and lenalidomide (or thalidomide). In this study, we determined that CFZ treatment enhanced the sensitivity of MM cells to NK cell-mediated lysis. Here, we report that CFZ decreased the expression of human leukocyte antigen (HLA) class I on MM cell lines and primary MM cells, the mean reduction was 47.7 ± 9.4% and 42.8 ± 12.4%, respectively. The down-regulation caused by CFZ occurred in a dose- and time- dependent manner. We compared the cell surface levels of HLA class I on MM cells in the presence or absence of CFZ after acid treatment. CFZ also down-regulated the expression of newly formed HLA class I on MM cells. CD107a expression levels were used to measure NK-cell degranulation. When NK cells were incubated with MM cells with CFZ treatment, the percentage of NK cells expressing CD107a on the surface greatly increased (mean ± SD: 33.6 ± 2.1%, for treated cells vs 16.7 ± 2.3%, for control cells, P < 0.05). We also showed that CFZ augmented NK-cell cytotoxity by a perforin/granzyme-mediated mechanism, because such enhancement was abolished when CMA, but not anti-TRAIL or anti-Fas-L antibodies, was added. Treatment of MM with CFZ significantly sensitized patients' MM cells to NK cell-mediated lysis (mean ± SD: 43.1 ± 6.4%, for treated cells vs 16.1 ± 4.0%, for control cells at effector/target (E/T) ratio of 10:1, n = 9, P < 0.01). Furthermore, the exogenous HLA-C binding peptides, used in the CFZ treated group rescued the down-regulation of HLA-C and reduced NK cell-mediated lysis to a similar level as in the untreated group. Blocking NKG2D, NCRs and TRAIL did not have a significant impact on NK cell lysis of myeloma cells. These implied the enhancement of NK cell-mediated lysis was mainly linked with the decreased expression of HLA class I. Our findings show a novel activity of CFZ as an immunomodulating agent and suggest a possible approach to therapeutically augment NK cell function in MM patients. DisclosuresNo relevant conflicts of interest to declare.

Carfilzomib Dosing Patterns and Survival in Patients with Relapsed and Refractory Multiple Myeloma: An Analysis from US Community Oncology Practices

IntroductionWhile novel agents have improved survival over the last decade, multiple myeloma (MM) remains incurable. Carfilzomib (CFZ), a second-generation proteasome inhibitor, was approved in July 2012 by the US Food and Drug Administration and had a label change in July 2015. During this study's data period, the label recommended Cycle 1 dose at 20 mg/m2/day and if tolerated increase Cycle 2 dose and subsequent cycles doses to 27 mg/m2/day. The purpose of this study is to assess baseline characteristics, CFZ dosing patterns and survival among MM patients in a US community oncology setting.MethodsA retrospective study of MM patients from US Oncology Network practices that fully implemented McKesson Specialty Health's iKnowMed (iKM) oncology-specific electronic health records database was conducted on patients whose first treatment of CFZ (index) occurred between Jul-2012 and Nov-2014. Patients were eligible if they had a documented initial MM diagnosis date and had their first CFZ cycle documented in the database. Additionally, patients were required, before Dec-2014, to have either another visit to the practice post-index or a record of death and not have participated in interventional clinical trials during the previous 6 years. Data on eligible patients were collected up to March 2015. The death event was defined by the Social Security Death Index, supplemented by iKM; patients without the event were censored at the date of last observed visit. To adjust for clinical practice variations, a 10% variability was allowed for the recommended daily dose levels of 20 mg/m2 and 27 mg/m2. A subgroup was defined for patients with a 2nd cycle: "escalators" if they received 20 mg/m2/day doses throughout Cycle 1 and increased to 27 mg/m2/day on the first dose of Cycle 2; "non-escalators" if they received only 20 mg/m2 doses throughout Cycle 1 and did not increase to 27 mg/m2 on the first dose of Cycle 2; receiving any dose not equal to 20 or 27 mg/m2 were classified into "other". Survival after index was estimated using the Kaplan-Meier method with 95% confidence intervals (CI). A multivariable Cox proportional hazards (PH) model was conducted to evaluate the impact of escalation on survival accounting for selected baseline demographic and clinical characteristics.ResultsThe cohort of 718 CFZ patients were identified with a median (interquartile range [IQR]) age of 68 (61-75) years at index, 57% (n=409) were male, and 12% (n=87) were Black and 77% (n=551) were Caucasian. At initial MM diagnosis, 19%, 27% and 42% were ISS Stage I, II, and III, respectively. Median (IQR) time from MM diagnosis to index was 3.6 (1.9-5.8) years. At index, 66% of patients had an ECOG performance status of 0-1, 21% of 2, and 2% of 3+; 54% (n=369) had moderate to severe renal impairment (eGFR<60 mL/min per 1.73 m2). Ninety percent (n=644) of patients started CFZ at 20 mg/m2, 4% (n=27) at 27 mg/m2 and 4% (n=25) at 15 mg/m2. Patients had a median (IQR) of 4 (2-7) cycles of CFZ and 45% (n=321) escalated to ≥27 mg/m2. Among these 321 patients, median (IQR) time to first escalation was 30 (28-56) days with 60% escalating in Cycle 2. The subgroup defined in "Methods" included 605 patients: 148 (24%) escalators, 342 (57%) non-escalators, and 115 (19%) other. Median (95% CI) duration from index to death was 21 (17.5-23.2) months. Unadjusted overall survival (OS) was significantly lower among non-escalators compared to escalators (log-rank p=0.024) [Figure 1]. Survival rates (95% CI) for non-escalators and escalators were 68% (62-74%) and 75% (66-82%) at year 1 and 42% (33-50%) and 61% (49-71%) at year 2, respectively. Within the multivariable Cox model, escalators showed a 33% significantly lower risk of death compared to non-escalators (HR=0.67, p=0.03) while also accounting for race, sex, age group, renal function per EGFR, and MM chain type. Other significant variables in this model were: EGFR < 15 and 15-29 vs 30-59 ml/min per 1.73m2 (HR=2.79, p<0.01; HR=1.64, p=0.04, respectively) and lambda vs kappa light chain (HR=1.55, p=0.03).ConclusionsThese results indicate escalation of CFZ at first dose of Cycle 2 is associated with better survival than dosing at 20 mg/m2 in Cycle 1 but not escalating at the start of Cycle 2. More research is needed to assess factors that impact physician decision-making on dose escalation to better inform physicians to improve the quality of multiple myeloma care. [Display omitted] DisclosuresRifkin:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Fonseca:McKesson Specialty Health, which received funding to conduct this research: Employment, Equity Ownership. Chen:Onyx Pharmaceuticals: Employment. Fox:McKesson Specialty Health, which received funding to conduct this research: Employment. Browning:Onyx Pharmaceuticals, An Amgen Subsidiary: Employment. Cong:Onyx Pharmaceuticals: Employment, Equity Ownership.

Marizomib Overcomes Compensatory Hyperactivation of Trypsin-like and Caspase-like Subunits to Provide Pan-Proteasome Subunit Inhibition in Patients with Multiple Myeloma and Solid Tumors

Background: Proteasome inhibitors (PIs) are a highly active drug class in multiple myeloma (MM), but development of resistance is commonly observed. Although all clinical-stage PIs are effective inhibitors of chymotrypsin-like (CT-L) proteasome subunit activity, a possible mechanism of resistance is compensatory hyperactivation of the caspase-like (C-L) and trypsin-like (T-L) subunits. Marizomib (MRZ) is a novel, irreversible, second-generation PI under development for the treatment of MM and malignant glioma. MRZ potently inhibits the 3 subunits of the 20S proteasome with a specificity and activity distinct from that of bortezomib and carfilzomib and their oral analogs ixazomib and oprozomib.Methods: In the clinical study NPI-0052-102, we evaluated the pharmacodynamic (PD) activity of MRZ against all three proteasome subunits in whole blood after single, or repeated administration, via two schedules in patients (pts) with previously treated advanced malignancies (solid tumors or hematologic). Pts received MRZ via Schedule A - weekly dosing for 3 doses in 4-week cycles by IV infusion over 1 to 10 min, or Schedule B - twice-weekly dosing for 4 doses in 3-week cycles by IV infusion over 10 min to 2 hr. Blood samples were collected on Days 1 and 15 (Schedule A) or 1 and 11 (Schedule B), pelleted by centrifugation and frozen within 48 hrs. Pellets were lysed and the activity of all 3 proteasomal subunits was assayed using specific fluorogenic substrates.Results: Partial or complete inhibition of all three proteasome subunits was achieved with both once-weekly and twice-weekly MRZ dosing. The rank order of sensitivity, CT-L > T-L > C-L, was as expected from the biochemical and cellular potencies of the drug. For CT-L activity, proteasome inhibition was dose dependent, with both the initial effect (on Cycle 1, Day 1 - C1D1) and the peak effect of proteasome inhibition increasing in a dose-dependent manner. CT-L inhibition was modest at the lowest dose levels (≤ 0.15 mg/m2), reaching moderate levels after repeat dosing. At intermediate dose levels of 0.3 to 0.55 mg/m2, CT-L inhibition was 31% to 75% on C1D1, rising to 70% to 93% after repeat dosing. At doses of 0.7 mg/m2 and above, inhibition of CT-L activity was usually complete within the first cycle. By contrast, C-L and T-L activities were unchanged or increased in the first cycle, suggesting compensatory hyperactivation in response to effective blockade of CT-L activity. Importantly, however, this response was overcome by repeat dosing with MRZ, and inhibition of T-L and C-L activity was noted across all dose levels. For C-L activity, treatment with MRZ at the recommended Phase 2 dose (RP2D) of 0.5 mg/m2 with twice-weekly dosing resulted in inhibition of up to 39% by Cycle 2 and was maintained, when tested, through Cycles 4 and 6. Treatment at the RP2D of 0.7 mg/m2 for once-weekly dosing resulted in C-L inhibition of 14% to 37% by the end of the first cycle, rising to 31% to 50% by the end of Cycle 2. Blockade of T-L activity was more robust after multiple cycles of MRZ therapy. Although inhibition of the T-L subunit was absent on C1D1 in patients receiving 0.5 to 0.55 mg/m2, inhibition of up to 80% was achieved by Cycle 2 and maintained for the duration of treatment. At the once-weekly RP2D of 0.7 mg/m2, T-L inhibition of 29% to 56% was achieved by the end of the first cycle, rising to 64% to 78% by the end of Cycle 2.Conclusions: The PD activity of MRZ against all three proteolytic subunits was assessed in patients with MM, solid tumors, and advanced lymphomas. At the twice-weekly and once-weekly RP2Ds, complete inhibition of CT-L activity was observed within 1-2 cycles of therapy, but accompanied by compensatory hyperactivation of the C-L and T-L subunits. This phenomenon in red cells suggests that it may be due to allosteric interactions within the catalytic core of the 20S proteasome rather than de novo synthesis of additional proteasomes. Ongoing MRZ therapy was able to overcome this adaptive response, resulting in robust pan-subunit proteasome inhibition within 2-6 cycles, most probably due to the cumulative effect of multiple exposures to MRZ. Due to their reversible binding mode (bortezomib, ixazomib) or monospecificity for the CT-L site (carfilzomib, oprozomib), other clinical PIs are predicted to lack this capability. This unique property of MRZ may explain in part its clinical activity in patients with MM resistant to both bortezomib and carfilzomib. DisclosuresOff Label Use: marizomib treatment for multiple myeloma and solid tumours.. Harrison:AbbVie: Research Funding; Janssen: Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding. Burrows:Triphase Accelerator Corporation: Consultancy. Reich:Triphase Accelerator Corporation: Consultancy. Trikha:Triphase Accelerator Corporation: Employment.

CBIO-14TARGETING PROTEASOME ACTIVITY WITH MARIZOMIB AS A THERAPEUTIC PERSPECTIVE FOR GLIOMA PATIENTS

Inhibition of the Ubiquitin-Proteasome pathway offers promise for the treatment of gliomas, but has yet to be tested because the approved drugs in this class don't penetrate the CNS. Marizomib is a novel second-generation proteasome inhibitor, with advantages over bortezomib and carfilzomib including irreversible inhibition of all three enzymatic activities of the proteasome and superior tolerability. While there are some promising data in preclinical models of solid tumors using proteasome inhibitors, only Marizomib has proven active in intracranial GBM xenografts, prompting the initiation of a Phase I trial in GBM in combination with bevacizumab. We aim to identify potential biomarkers predictive of response to Marizomib in GBM patients. Tumor tissues obtained from glioma patients at Toronto Western Hospital and utilized for proteasome activity assay. Glioma Stem Cells (GSC) isolated from freshly resected gliomas and used for in-vitro characterization of Marizomib treatment on GSCs. DNA and RNA were isolated from tumors of the patients (including those participating in the Phase I trial) for mutational analysis and expression of a cancer gene panel. Data shows significant increase in all three proteasome activities, Chymotrypsin-Like, Trypsin-Like and Caspase-Like, in GBM tumors compared to normal brain tissues, suggesting a role for the proteasome in GBM formation. Furthermore, progression of low-grade astrocytoma to GBM is associated with enhanced Chymotrypsin-Like and Trypsin-Like activities, indicating that targeting these subunits could potentially inhibit the progression of gliomas. In vitro analysis using GSC lines indicates that the GSC cells of proneural and mesenchymal origin differ in response to Marizomib, raising the possibility that molecular signatures associated with GBM subtypes could determine the therapeutic response to Marizomib in GBM patients. In conclusion, Marizomib represents as a potential therapeutic agent for GBM. Further investigation is necessary to assess the therapeutic benefits of Marizomib as a single agent or combined with other agents.

Novel agents in multiple myeloma treatment

The introduction of bortezomib and IMiDs has improved outcomes of patients with multiple myeloma (MM). Moreover, second-generation IMiDs (pomalidomide) have recently been approved in Japan. Due to the incurability of this disease, newer drugs with different mechanisms of action designed to prolong survival and achieve cure are urgently needed. There are novel agents which appear to be promising, such as monoclonal antibodies (elotuzumab, daratumumab), second-generation proteasome inhibitors (carfilzomib), deacethylase inhibitors (panobinostat), and the kinesin protein inhibitor (Arry-520). In the MM-003 trial, there was a significant advantage in the pomalidomide plus dexamethasone arm over high-dose dexamethasone in terms of ORR, PFS, and OS in MM patients who had failed both lenalidomide and bortezomib treatment. In addition, the ASPIRE trial showed carfilzomib in combination with lenalidomide plus dexamethasone to provide a survival advantage over lenalidomide plus dexamethasone for relapsed/refractory MM. These encouraging results indicate that the use of newer agents, alone or in combinations, may overcome double-refractory disease. Other agents such as kinase inhibitors or signaling pathway inhibitors are now under investigation. The use of cocktails of different agents with different mechanisms of action in an earlier stage of the disease may help to obtain durable remissions or even disease cure.

'Real-life' experience of preapproval carfilzomib-based therapy in myeloma - analysis of cardiac toxicity and predisposing factors.

Carfilzomib, the second-generation proteasome inhibitor, is still awaiting approval for the treatment of multiple myeloma in Europe. Results from clinical trials have demonstrated favorable efficacy in advanced disease but have opened an ongoing debate on cardiac complications related to carfilzomib treatment. 'Real-life' data are scarce. At our institution, 22 patients were registered within the European Carfilzomib Access Program for treatment with carfilzomib, dexamethasone, and a third combination partner depending on patient characteristics and prior treatment. Patients had received a median of six previous lines of therapy, and most were refractory to bortezomib (77%) and immunomodulatory drugs (95%). Overall response rate was 65% with a median progression-free survival of 6.0 months and a median duration of response of 6.8 months. Median overall survival was 14.9 months. Grade 3/4 adverse events (AEs) occurred in 50% of patients with 23% experiencing left ventricular failure. The risk of cardiac AEs was markedly increased after previous radiotherapy of the thoracic spine and concomitant administration of doxorubicin. Carfilzomib-based treatment is efficient in advanced multiple myeloma. In our 'real-life' patients, we found considerable cardiotoxic effects in some cases, indicating a need for careful patient selection and close monitoring of the at-risk population.

TOP2A knockdown resensitizes carfilzomib-resistant HMCLs to carfilzomib

Introduction/background: Multiple myeloma (MM) remains incurable despite the introduction of novel therapeutic agents. Microarray-based technologies were adopted in our study to determine if a genetic signature associated with resistance to carfilzomib, a second-generation proteasome inhibitor already in use in clinical settings, could be identified. Materials and Methods: 18 genetically heterogeneous human myeloma cell lines (HMCLs) were treated with carfilzomib and a cell viability profile was assessed categorizing the HMCLs as sensitive, intermediate or resistant to carfilzomib. Following categorization gene expression profiling was performed and validated with q-RT-PCR and knockdown assays. Results: 29 genes were differentially regulated between the sensitive and resistant cell lines. Top genes based on intensity values and biological significance were: LOC731314, TSPAN13, APH1B, TSPYL5, COX7B2, PCSK1N, LRRC38, TCIRG1, TOP2A, ADM2, ITM2A, TSPAN13, STOM, UBE2C, SNHG8. Gene ontology (GO) enrichment analysis identified two pathways that were significantly different between the resistant and sensitive HMCLs; pathogenic escherichia coli infection (p=0.002) and lysosome (p=0.006). Eight GO terms were enriched: 4 related to biological processes and 4 related to cellular components. TOP2A, an enzyme that controls and alters the topologic states of DNA during transcription and is involved in cell cycle and proliferation, was identified to be overexpressed in resistant HMCLs. It functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. TOP2A may be used as a predictive factor for patient selection for specific protocols or as independent prognostic marker in solid tumors. TOP2A was also overexpressed in the 9proliferation cluster9 associated with greater proliferation rate and poor outcome in newly diagnosed MM patients. Suppression of TOP2A by siRNA in carfilzomib-resistant HMCLs significantly resensitised the cell lines to carfilzomib. Conclusion: Our results suggest that TOP2A is overexpressed in carfilzomib-resistant HMCLs indicating a possible role as a predictive marker of response to carfilzomib in MM. Disclosures No relevant conflicts of interest to declare.

Polymer micelle formulations of proteasome inhibitor carfilzomib for improved metabolic stability and anticancer efficacy in human multiple myeloma and lung cancer cell lines.

Carfilzomib (CFZ) is a second-generation proteasome inhibitor drug approved for the treatment of multiple myeloma. Contrary to its excellent antimyeloma activity, CFZ has shown only limited efficacy in patients with solid malignancies. This lack of efficacy has been attributed in part to rapid degradation of CFZ in the body, possibly hindering the ability of CFZ to access the proteasome target in solid tumors. We hypothesized that polymer micelles, a currently Food and Drug Administration-approved nanoparticle drug delivery formulation, may protect CFZ from metabolic degradation and thus expand the clinical utility of the drug as an anticancer agent. To test our hypothesis, we prepared CFZ-entrapped polymer micelle particles with various compositions and drug release profiles and examined the extent of the CFZ metabolism in vitro using mouse liver homogenates. We also assessed the cytotoxic activities of the CFZ-entrapped micelle formulations in human cancer cell lines derived from B lymphocytes (RPMI-8226) and the lung (H460). Our data indicated that polymer micelle-based formulations can improve metabolic stability and cytotoxic effects of CFZ compared with free CFZ in human cancer cell lines tested. Taken together, these results suggest that polymer micelles may have potential as a delivery system for CFZ with an extended therapeutic utility for nonhematologic malignancies in the future.

Carfilzomib enhances natural killer cell-mediated lysis of myeloma linked with decreasing expression of HLA class I.

Natural killer (NK) cell-based treatments are promising therapies for multiple myeloma (MM). Carfilzomib (CFZ), is a second-generation proteasome inhibitor, used to treat relapsed and refractory MM. In this study, we determined that CFZ treatment enhanced the sensitivity of MM cells to NK cell-mediated lysis. Here, we report that CFZ decreased the expression of human leukocyte antigen (HLA) class I in a time- and dose-dependent manner. CFZ also down-regulated the expression of newly formed HLA class I on MM cells. Treatment of MM with CFZ enhanced NK cell degranulation and significantly sensitized patients' MM cells to NK cell-mediated lysis. Furthermore, the enhancement of NK cell-mediated lysis was linked with the decreased expression of HLA class I. Our findings show a novel activity of CFZ as an immunomodulating agent and suggest a possible approach to therapeutically augment NK cell function in MM patients.

Carfilzomib, a second-generation proteasome inhibitor, to enhance the activity of ibrutinib in rituximab-chemotherapy sensitive or resistant B-cell lymphoma cell lines and primary patient samples.

e19524 Background: Identification of critical signaling pathways required for the development, maintenance and progression of specific subtypes of B-cell lymphoma is essential in order to develop o...

Carfilzomib is an effective anticancer agent in anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is one of the most aggressive human malignancies. Currently, there is no standard or effective therapy for ATC. Drug repurposing for cancer treatment is an emerging approach for identifying compounds that may have antineoplastic effects. The aim of this study was to use high-throughput drug library screening to identify and subsequently validate novel therapeutic agents with anticancer effects in ATC. We performed quantitative high-throughput screening (qHTS) in ATC cell lines (SW-1736, 8505C, and C-643), using a compound library of 3282 drugs. qHTS identified 100 compounds that were active in all three ATC cell lines. Proteasome inhibitors were one of the most active drug categories according to enrichment analysis. Of the three proteasome inhibitors screened, a second-generation proteasome inhibitor, carfilzomib, was the most active. Treatment of ATC cells with carfilzomib significantly inhibited cellular proliferation and induced G2/M cell cycle arrest and caspase-dependent apoptosis. Mechanistically, carfilzomib increased expression of p27 (CDKN1B) and decreased expression of the anti-apoptotic protein ATF4. Pretreatment with carfilzomib reduced in vivo metastases (lung, bone, liver, and kidney) and disease progression, and decreased N-cadherin expression. Carfilzomib treatment of mice with established, widely metastatic disease significantly increased their survival, without significant toxicity. Our findings support the use or clinical study of carfilzomib as a therapeutic option in patients with advanced and metastatic ATC.

Carfilzomib: a second-generation proteasome inhibitor for the treatment of multiple myeloma.

The pharmacology, clinical efficacy, safety, cost, dosage and administration, and place in therapy of carfilzomib for the treatment of multiple myeloma (MM) are reviewed. Proteasome inhibition in MM has become a cornerstone in treatment regimens. Carfilzomib, a second-generation proteasome inhibitor, has demonstrated efficacy in patients with relapsed or refractory disease who have received at least two prior therapies including bortezomib and an immunomodulatory agent. Carfilzomib is an irreversible inhibitor and binds to a different site than bortezomib on the proteasome. A Phase II study evaluated 266 heavily pretreated patients with relapsed or refractory MM who had received at least two prior therapies, including bortezomib and either thalidomide or lenalidomide. The overall response rate was 23.7%, with a median duration of response of 7.8 months. The median overall survival time was 15.6 months. Carfilzomib has a similar adverse-effect profile to bortezomib, including anemia, thrombocytopenia, fatigue, dyspnea, and nausea; however, it does not result in the development or worsening of peripheral neuropathy. Carfilzomib is infused intravenously over 2-10 minutes for 2 consecutive days every week for three out of four weeks, with a 12-day rest period. Dosing is based on the patient's actual body surface area. Carfilzomib is available in 60-mg vials for single infusion. The total cost for a year of therapy is approximately $155,852. Carfilzomib, a second-generation proteasome inhibitor that irreversibly inhibits the 26S proteasome, has shown efficacy in clinical studies of patients with relapsed or refractory MM, though the drug's role in the management of MM is not yet clear.

Efficacy of panobinostat and marizomib in acute myeloid leukemia and bortezomib-resistant models

Current relapse rates in acute myeloid leukemia (AML) highlight the need for new therapeutic strategies. Panobinostat, a novel pan-histone deacetylase inhibitor, and marizomib, a second-generation proteasome inhibitor, are emerging as valuable therapeutic options for hematological malignancies. Here we evaluated apoptotic effects of this combinatorial therapy in AML models and report earlier and higher reactive oxygen species induction and caspase-3 activation and greater caspase-8 dependence than with other combinations. In a bortezomib refractory setting, panobinostat induced high levels of DNA fragmentation, and its action was significantly augmented when combined with marizomib. These data support further study of this combination in hematological malignancies.

How Should We Treat Elderly Newly Diagnosed Multiple Myeloma Patients?

Multiple myeloma is the second most frequent hematologic disease, which usually affects patients over 65 years of age. Treatment goals for these non-transplant-eligible patients should be to prolong survival by achieving the best response, while ensuring quality of life. Melphalan plus prednisone has been the classic backbone to which proteasome inhibitors and immunomodulatory drugs were added and, recently, lenalidomide plus low-dose dexamethasone emerged as a new standard of care, free of alkylator, and also as a backbone to which secondgeneration proteasome inhibitors are being added. Monoclonal antibodies will take part of these treatments regimens in the future. Prolonged treatment in elderly patients also improves the quality and duration of clinical responses, extending time to progression and progression-free survival; however, the optimal scheme, appropriate doses, and duration of long-term therapy have not yet been fully determined. Finally, elderly patients under treatment require close monitoring and individualized, dose-modified regimens to improve tolerability and treatment efficacy, while maintaining their quality of life.