Irreversible Proteasome Inhibitor Carfilzomib Research Articles

Elucidating the cardiac outcomes of irreversible proteasome inhibitor Carfilzomib in in vivo models of HFpEF and HFrEF. Preventive role of Metformin

Elucidating the cardiac outcomes of irreversible proteasome inhibitor Carfilzomib in in vivo models of HFpEF and HFrEF. Preventive role of Metformin

P-025: Investigating the effect of irreversible proteasome inhibitor carfilzomib in in vivo models of cardiometabolic syndrome and early stage HFrEFP: prophylactic role of metformin

P-025: Investigating the effect of irreversible proteasome inhibitor carfilzomib in in vivo models of cardiometabolic syndrome and early stage HFrEFP: prophylactic role of metformin

Relapsed or Refractory Multiple Myeloma Patients Treated with Second-Line Carfilzomib and Categorized By Prior Exposure to Bortezomib: A Subgroup Analysis of the Randomized Phase 3 Aspire and Endeavor Trials

Relapsed or Refractory Multiple Myeloma Patients Treated with Second-Line Carfilzomib and Categorized By Prior Exposure to Bortezomib: A Subgroup Analysis of the Randomized Phase 3 Aspire and Endeavor Trials

Single-Cell Transcriptomics of Human Plasma Cells from HLA-Sensitized Patients Reveals Population Heterogeneity and Drug Resistance Adaptations

Kidney transplantation improves the overall survival of end stage renal disease patients but the presence of preformed human leukocyte antigen (HLA)-specific antibodies often precludes finding an acceptable donor. Recent studies demonstrate that bone marrow plasma cells (BMPCs) are a major source of HLA-specific antibodies in highly sensitized transplant candidates. However, therapeutic strategies that target BMPCs and overcome HLA desensitization are only beginning to emerge. Proteasome inhibitor (PI) therapies effectively eliminate BMPCs, reduce HLA-antibody levels and have shown promise as an effective means of enhancing transplantation outcomes. However, PIs are frequently limited by de novo or therapy-induced drug resistance. In addition, BMPCs exhibit variable sensitivity to PI-based therapy. To identify potential mechanisms of drug resistance in BMPCs, we employed single-cell genomic profiling. Single-cell studies provide a powerful tool that allows us to study rare cell populations, such as BMPCs, and intermediate cell states that cannot be resolved at the population level. Here, we have performed single-cell genomic profiling of human BMPCs isolated from highly HLA-sensitized renal transplant candidates prior to and after desensitization therapy with the irreversible PI carfilzomib. HLA-sensitized transplant candidates received CFZ (20, 27, 36 mg/m2) with BM aspirates performed prior to and after CFZ therapy. CD138+BMPCs were isolated to >98% purity and individual cells analyzed using the recently developed method, Iterative Clustering and Guide-gene Selection (ICGS), which utilizes pair-wise correlation of dynamically expressed genes and iterative clustering with pattern-specific guide genes to delineate coherent gene-expression patterns. Strongly and differentially expressed gene sets were clustered and evaluated using gene enrichment, prior knowledge-based networks and ICGS. Each cluster corresponds to one or a group of closely related cell types that represent spatial or temporal distinct states. Single-cell profiling by the drop-seq method analyzed mRNA expression in 2,000 individual BMPCs isolated from the same HLA-sensitized patient prior to and after CFZ therapy. We used CellHarmony, a new bioinformatic approach that allows us to determine the frequency of all CFZ-treated cells in direct reference to the untreated cells and any altered gene expression profiles. In our first comparison of these cells, eight cell clusters were defined (Fig. 1A). Single-cell analysis of BMPCs obtained post-CFZ therapy indicated qualitative and quantitative changes in gene expression as well differences in the proportionality of existing cell clusters. To determine the frequency and expression of these genes in the CFZ-treated samples, we then performed classification of those samples using a k-nearest neighbor approach using all untreated cells as the reference. For example, protein catabolism, nonsense-mediated decay and integrin signaling were increased (clusters 1, 5 and 6) while clusters enriched in IL-12 signaling, interferon signaling and protein translation genes (2, 3 and 7) were reduced. Importantly, the state associated with the mitotic cell cycle was significantly upregulated in BMPCs that survived CFZ. BMPCs were analyzed from additional patients using a comparable approach identified common gene expression sets in pre-treatment samples as well as recurrent differences in post-treatment samples (Fig. 1B).Conclusions: The results presented here demonstrate the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering how cell states respond to therapeutic challenge. Our results indicate a shift toward highly mitotic states in BMPCs that survive CFZ therapy. Importantly, the single cell results also provide internal validation of our prior bulk RNA-seq studies on BMPCs. Our results suggest the feasibility of pathway-based secondary targeting of the recovering cell populations to enhance response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genomic and Biochemical Proteasomal Adaptations in Carfilzomib-Resistant Plasma Cells That Limit HLA-Desensitization in Transplantation Candidates

Bone marrow plasma cells (BMPCs) are the source of long-term humoral immunity including HLA antibodies, which promote organ rejection. End-stage renal disease (ESRD) affects more than 660,000 patients per year in the U.S. and is associated with significant morbidity and mortality. Renal transplantation remains the optimal treatment for a substantial proportion of ESRD patients and dramatically improves patient overall survival and quality-of-life. However, preformed human leukocyte antigen (HLA)-specific antibodies are associated with reduced transplantation rates, graft loss and poorer outcomes. Proteasome inhibitor (PI) therapies that target BMPCs to reduce HLA-antibody levels represent an effective means of enhancing transplantation outcomes but are limited by resistance mechanisms. Single antigen bead assays determined that the spectrum of antibody production by HLA-sensitized patient (BMPCs) was remarkably similar to that detected in matching serum samples, supporting the role of BMPCs as the primary source of circulating HLA antibodies. Desensitization therapy with the irreversible proteasome inhibitor carfilzomib reduced PCs in patient BM by ~70% and was associated with a profound reduction in HLA antibodies. We then used bulk RNA-seq transcriptomics to explore potential drug resistance mechanisms within carfilzomib-resistant BMPCs. Network-based pathway analysis revealed that genes encoding the immunoproteasome, a specialized proteasome variant with unique catalytic subunits, and the proteasome activator PA28 were upregulated in BMPCs that survived carfilzomib therapy. Native gel electrophoresis and in vitro functional studies confirmed that BMPCs isolated after carfilzomib therapy contained greater levels of immunoproteasomes as well as increased immunoproteasome-specific peptide-hydrolyzing activity relative to BMPCs from healthy donors or untreated HLA-sensitized patients. Supervised hierarchical clustering analysis (Fig. 1A) and western blotting (Fig. 1B) indicated that PSMB8, which encodes an immunoproteasome catalytic subunit, and PSME1 and PSME2 which encode the proteasome PA28 activator, were significantly upregulated in BMPCs isolated from patients following carfilzomib therapy. Assays then demonstrated that BMPCs that survived carfilzomib therapy were resistant to the FDA-approved PIs bortezomib, carfilzomib and ixazomib but were sensitive to an immunoproteasome-specific inhibitor KZR-616. Thus, we have uncovered proteasomal adaptations in BMPCs as a resistance mechanism underlying carfilzomib therapy and suggest novel strategies that could improve desensitization regimens and reduce antibody-mediated rejection in organ transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Glutamine-Dependence Targeting By Asparaginase Significantly Increases Anti-Myeloma Activity of Proteasome Inhibitors

Human tumors exhibit high dependence on Glutamine (Gln) for their metabolic processes and macromolecule synthesis, condition that has been defined as “Gln-addiction”. The role of such vulnerability has been recently investigated also in Multiple Myeloma (MM) cells, with Gln uptake exploited as novel therapeutic target. Indeed, MM cells by exhibiting significant lack of Gln synthetase become highly sensitive to Gln depletion. L-asparaginase (ASNase), a bacteric enzyme that by degradating circulating asparagine and glutamine leads to intracellular aminoacids depletion, has been reported to exert a certain degree of activity on human myeloma cell lines (HMLC).Based on these data, here we explored the anti-MM activity of Erwinia chrysantemi -derived L. Asparaginase (ASNase; 10-folder higher glutaminase activity than Escherichia Coli -derived formulation) in combination with the proteasome inhibitor (PI) carfilzomib (KAR), since PIs represent the backbone of current induction therapy for most MM patients.The IC50 value of both E. chrysantemi ASNase and KAR was evaluated in a panel of HMCL carrying a different genetic background. Next, by using Chou-Talalay method we found that low doses of KAR significantly enhances the anti-MM activity of ASNase. The effect of such combination was also tested in presence of IL6 and IGF-1, which mimics Bone Marrow Stromal Cells (BMSCs) milieu. Cell death analysis was measured with Annexin V/Propidium Iodide (AV/PI) staining followed by flow-cytometric analysis. The efficacy of co-treatment was confirmed by treating freshly isolated CD138 positive cells obtained from newly diagnosed MM patients (NDMM). Finally, western blot analysis was employed to fully elucidate mechanisms causing the observed synergism.ASNase treatment showed potent cytotoxic activity in all HMCL lines tested with an IC50 value ranging from 0,03 U/mL to 0.3 U/mL. High sensitivity was also observed on primary CD138+ MM cells obtained from NDMM (IC50 =0.3 U/mL). Importantly, anti-MM activity of ASNase was further increased by adding low doses of the irreversible proteasome inhibitor KAR with a Combination Index value less than 1 in almost all tested drug-concentrations. Similar data were observed in tumors cells obtained from NDMM patients. Of note, the combination ASNase plus KAR resulted more potent than single agent treatment also in culture condition of Gln deprivation, suggesting that ASNase toxicity is not only related to its glutaminase activity. IL6 or IGF-1 addition did not reduce anti-MM activity of ASNase plus KAR, indicating that BMSCs microenvironment may not influence its activity. A time and dose-dependent activity of co-treatment was also observed, with higher efficacy measured at 48 h compared with 24h of drugs exposure. The caspase-3 and PARP cleavage observed in co-treated cells, revealed the crucial role of apoptosis in the synergistic effect observed. Also authophagic features, including LC3II cleavage, were observed in tumor cells treated with KAR plus ASNase compared with single agent treatment.The effect of ASNase treatment on neoplastic cells metabolism has already showed potent antineoplastic activity in a wide range of hematologic malignancies including acute lymphoblastic leukemia and aggressive lymphomas. As result, the Gln dependence represents an attractive therapeutic target to be exploited also in plasma cells disorders. Our preliminary data show that E. chrysantemy derived asparaginase exerts a potent anti-MM activity, which is further increased by addition of the irreversible proteasome inhibitor carfilzomib through a mixed apoptotic/authophagic-mediated mechanism. DisclosuresNo relevant conflicts of interest to declare.

Carfilzomib in Relapsed or Refractory Multiple Myeloma Patients with Early or Late Relapse Following Prior Therapy: A Subgroup Analysis of the Randomized Phase 3 Aspire and Endeavor Trials

Carfilzomib in Relapsed or Refractory Multiple Myeloma Patients with Early or Late Relapse Following Prior Therapy: A Subgroup Analysis of the Randomized Phase 3 Aspire and Endeavor Trials

Retraction: In Vitro and In Vivo Interactions between the HDAC6 Inhibitor Ricolinostat (ACY1215) and the Irreversible Proteasome Inhibitor Carfilzomib in Non-Hodgkin Lymphoma Cells

Interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor carfilzomib were examined in non-Hodgkin lymphoma (NHL) models, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and double-hit lymphoma cells. Marked in vitro synergism was observed in multiple cell types associated with activation of cellular stress pathways (e.g., JNK1/2, ERK1/2, and p38) accompanied by increases in DNA damage (γH2A.X), G2–M arrest, and the pronounced induction of mitochondrial injury and apoptosis. Combination treatment with carfilzomib and ricolinostat increased reactive oxygen species (ROS), whereas the antioxidant TBAP attenuated DNA damage, JNK activation, and cell death. Similar interactions occurred in bortezomib-resistant and double-hit DLBCL, MCL, and primary DLBCL cells, but not in normal CD34+ cells. However, ricolinostat did not potentiate inhibition of chymotryptic activity by carfilzomib. shRNA knockdown of JNK1 (but not MEK1/2), or pharmacologic inhibition of p38, significantly reduced carfilzomib–ricolinostat lethality, indicating a functional contribution of these stress pathways to apoptosis. Combined exposure to carfilzomib and ricolinostat also markedly downregulated the cargo-loading protein HR23B. Moreover, HR23B knockdown significantly increased carfilzomib- and ricolinostat-mediated lethality, suggesting a role for this event in cell death. Finally, combined in vivo treatment with carfilzomib and ricolinostat was well tolerated and significantly suppressed tumor growth and increased survival in an MCL xenograft model. Collectively, these findings indicate that carfilzomib and ricolinostat interact synergistically in NHL cells through multiple stress-related mechanisms, and suggest that this strategy warrants further consideration in NHL. Mol Cancer Ther; 13(12); 2886–97. ©2014 AACR .

Retraction: Obatoclax Interacts Synergistically with the Irreversible Proteasome Inhibitor Carfilzomib in GC- and ABC-DLBCL Cells In Vitro and In Vivo.

Retraction: Obatoclax Interacts Synergistically with the Irreversible Proteasome Inhibitor Carfilzomib in GC- and ABC-DLBCL Cells In Vitro and In Vivo.

Irreversible proteasome inhibition with carfilzomib as first line therapy in patients with newly diagnosed multiple myeloma: Early in vivo cardiovascular effects

Patients who experienced cardiovascular side effects during cancer therapy with carfilzomib for multiple myeloma had relapsed multiple myeloma, so have be previously treated with other cancer therapies. The present is a single center cohort study to evaluate early cardiovascular effects of administration of irreversible proteasome inhibitor carfilzomib in naïve patients. We included 24 patients and collected cardiovascular side effects, echocardiographic parameters and endothelial function at baseline and after 4 cycles. At early follow up we observed increase in blood arterial pressure values (mean change in systolic pressure of 10 mmHg (P-value < 0.01; diastolic arterial pressure and mean arterial pressure of 3.3 mmHg and 5.4 mmHg, both P-value < 0.01). Reactive hyperemia PAT index was reduced in the whole cohort by a mean of 0.46 points (P-value < 0.01); diastolic function was changed: E-wave-deceleration-time (EDT) was reduced by 49,96 ± 31 ms, P-value < 0.05 and early diastolic tissue Doppler velocity (e′) by a mean value of 1.46 cm/s, P - value 0.04.At early follow up we did not observe events of grade 3 or 4. We observe correlation between events and endothelial dysfunction at baseline and age (OR 1.9, CI 95% 0.05–5.804, P- value: 0.038 for RHI<1.67; OR 1,4, CI 95%0.99–2.56, P- value: 0.04 for age). Our results suggest that therapy with carfilzomib when used as first line therapy is responsible for increase in systemic blood pressure, alteration of endothelium-mediated vascular dilatation and early myocardial diastolic dysfunction.

Abstract 2112: FoxO3a activation by HDAC class IIa inhibition induces cell cycle arrest in pancreatic cancer cells

Abstract Pancreatic cancer is highly chemo-resistant associated with oncogenic mutations such as KRAS and/or p53. The effect of conventional chemotherapy is not sufficient and new target and strategy is urgently needed. The forkhead box (Fox) proteins are multidirectional transcriptional factors strongly implicated in malignancies. Although Fox O (FoxO) protein, and particularly FoxO3a, works as negative regulator of cell proliferation by repressing cyclin proteins and inducing cell cycle inhibitors such as p21Waf1/Cip1, its expression is consistently suppressed by several oncogenic pathways including phosphatidylinositol-3 kinase (PI3K) / AKT pathway, constitutively activated in pancreatic cancer. Thus, upregulating FoxO3a activity could be a promising target of pancreatic cancer treatment without impact of underlying oncogenic mutations. Class IIa Histone deacetylase (HDAC) is a subgroup of HDAC. Though HDAC inhibitors (HDACi) have been extensively investigated as a cancer target, its action mechanism is considered due to histone modification by class I. Biological significance of class IIa HDACs which have minimal histone deacetylation activity have not been elucidated yet. Recent studies show class IIa HDACs act as a transcriptional regulator including FoxO3a. In this study, we investigate the biologic impact of HDAC class IIa inhibition on FoxO3a and anti-tumor effect against pancreatic cancer cell line using selective class IIa HDACi TMP269. TMP269 treatment showed increased FoxO3a expression in a dose dependent manner with immunoblotting and modest cell growth inhibition effect at 57.5 μM of IC50 dose for 48-hour treatment against AsPC-1 in MTT. G1/S arrest was observed with cell cycle assay. Upregulated p21Waf1/Cip1 and downregulated CDK2 and 4/6 and cyclin D1 and D2 expressions were further observed, consistent with inducing G1/S arrest and transcriptionally activated FoxO3a. Importantly, upregulated p21Waf1/Cip1 was observed in AsPC-1 p53 null cell line, suggesting independent with p53 pathway. These findings suggest upregulated FoxO3a induced by HDAC class IIa inhibition activated its transcription and resulted in cell growth inhibition. Because PI3K/AKT leads FoxO3a to the ubiquitylation-mediated proteasome degradation, we examined irreversible proteasome inhibitor carfilzomib (CFZ) combined with TMP269, aiming synergistic FoxO3a upregulating. As expected, FoxO3a expression was further increased in TMP269 combined with CFZ compared with TMP269 or CFZ alone. Following the activated FoxO3a, p21Waf1/Cip1 expression was upregulated and cell growth inhibition was dramatically enhanced. In conclusion, HDAC class IIa inhibition modified FoxO3a transcriptional activation and upregulating FoxO3 by dual inhibition of HDAC class IIa and proteasome is promising target against pancreas cancer. Citation Format: Makoto Usami, Shohei Kikuchi, Kohichi Takada, Yusuke Sugama, Yohei Arihara, Naotaka Hayasaka, Hajime Nakamura, Yuki Ikeda, Yusuke Kamihara, Masahiro Hirakawa, Makoto Yoshida, Masayoshi Kobune, Koji Miyanishi, Junji Kato. FoxO3a activation by HDAC class IIa inhibition induces cell cycle arrest in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2112. doi:10.1158/1538-7445.AM2017-2112

Abstract 3038: Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL)

Abstract Background: Molecular studies divide DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal lymphoma (PML). Florescence in situ hybridization (FISH) studies identified another subgroup of DLBCL, classified as DH-DLBCL, with a poor clinical outcome harboring concurrent gene rearrangements of the c-MYC, BCL2 and/or BCL6 proto-oncogenes, resulting in the over-expression of c-Myc, Bcl2 and Bcl6 proteins. Previously, our retrospective review from single institution series revealed that 30 out of 611 DLBCL patients had aberrations in c-MYC and BCL2 or BCL6 by FISH. These patients exhibited inferior response rates (RR) to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS), suggesting that newer therapies are in dire need. DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, resulting in rapid cellular proliferation and resistance to apoptotic stimuli. In ABC-DLBCL, anti-apoptotic factor MCL-1 is implicated in poor prognosis leading to resistance to standard chemotherapy. C-MYC transcriptionally upregulates Mcl1. Translocation of c-MYC in DH-DLBCL may contribute to the aggressive phenotype and chemotherapy resistance via the MCL-1 pathway. We hypothesize that dual inhibition of both anti-apoptotic proteins BCL2 and MCL1 is an effective strategy in inducing lymphoma cell death in DH-DLBCL. Materials &amp; Methods: At the pre-clinical level, we studied 3 novel therapeutic agents targeting BCL2 (ABT-199), c-MYC (JQ-1), and various cell cycle regulatory proteins (p21) and other BCL2 family members affecting ABT-199 activity (irreversible proteasome inhibitor carfilzomib(CFZ)) using DH lymphoma (DHL) cell lines (Val, DOHH-2, ROS-50). DHL cell lines were exposed to ABT-199 (0-10 uM), JQ-1 (0-100 uM) and carfilzomib (CFZ) (0-50 nM) at 24, 48 and 72 hours. Changes in cell viability were evaluated using Presto Blue assay. Subsequently, DHL cells were exposed to doublet combinations of ABT-199, JQ-1 and CFZ for 48 hours. Coefficient of synergy was calculated using CalcuSyn. Results: In vitro, ABT199, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining ABT199 with CFZ and to a lesser degree with JQ-1. Conclusion: ABT199 exhibited strong synergistic activity with CFZ. Dual targeting of BCL2 and c-MYC pathways results in synergistic activity in DHL cell lines. Of interest, this pharmacological interaction could be related to the effects of proteasome inhibition on MCL1 and p21 levels in lymphoma cells, further enhancing the activity of ABT199. Using combination therapy to inhibit c-MYC and the proteasome and in turn decreasing MCL1 will render ABT-199 more effective and be a more potent combination in causing apoptosis and lymphoma cell death. Further pre-clinical work is ongoing. Citation Format: Priyank P. Patel, Alison Zeccola, Juan Gu, Cory Mavis, Sheila N. J. Sait, Vishala Neppalli, Francisco J. Hernandez-Ilizaliturri. Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3038.

Synergistic Myeloma Cell Death via Novel Intracellular Activation of Caspase-10-Dependent Apoptosis by Carfilzomib and Selinexor.

Exportin1 (XPO1; also known as chromosome maintenance region 1, or CRM1) controls nucleo-cytoplasmic transport of most tumor suppressors and is overexpressed in many cancers, including multiple myeloma, functionally impairing tumor suppressive function via target mislocalization. Selective inhibitor of nuclear export (SINE) compounds block XPO1-mediated nuclear escape by disrupting cargo protein binding, leading to retention of tumor suppressors, induction of cancer cell death, and sensitization to other drugs. Combined treatment with the clinical stage SINE compound selinexor and the irreversible proteasome inhibitor (PI) carfilzomib induced synergistic cell death of myeloma cell lines and primary plasma cells derived from relapsing/refractory myeloma patients and completely impaired the growth of myeloma cell line-derived tumors in mice. Investigating the details of SINE/PI-induced cell death revealed (i) reduced Bcl-2 expression and cleavage and inactivation of Akt, two prosurvival regulators of apoptosis and autophagy; (ii) intracellular membrane-associated aggregation of active caspases, which depended on caspase-10 protease activity; and (iii) novel association of caspase-10 and autophagy-associated proteins p62 and LC3 II, which may prime activation of the caspase cascade. Overall, our findings provide novel mechanistic rationale behind the potent cell death induced by combining selinexor with carfilzomib and support their use in the treatment of relapsed/refractory myeloma and potentially other cancers.

Two-drug Combinations in the Treatment of Multiple Myeloma

A variety of two-drug (doublet) regimens are available for the treatment of multiple myeloma, with applicability from diagnosis to relapse. In newly diagnosed multiple myeloma, lenalidomide+dexamethasone and bortezomib+dexamethasone are two recommended regimens for induction therapy, the latter of which shows significantly improved response rates both post-induction and post-transplantation compared with vincristine+doxorubicin+ dexamethasone. However, bortezomib+dexamethasone is considered by some to be suboptimal for transplant-eligible patients. In patients ineligible for transplant due to comorbidities, frailty or older age, doublet regimens offer convenience and low intensity. Lenalidomide+low-dose dexamethasone is approved in this setting and has demonstrated better short-term overall survival (OS) and lower toxicity than lenalidomide+high-dose dexamethasone. For patients whose disease relapses following front-line treatment, there are a number of options available and in development, including doublet regimens with immunomodulatory drugs and proteasome inhibitors. The latest results of the FIRST trial support use of continuous lenalidomide+dexamethasone in relapsed multiple myeloma: continuous dosing significantly prolonged progression-free survival (PFS) compared with 18 cycles of treatment (26.0 vs 21.0 mo.). Lenalidomide+dexamethasone significantly prolonged time to progression and improved overall response rate (ORR) versus dexamethasone alone, and the investigational combination of pomalidomide+low-dose dexamethasone is associated with longer PFS and OS and higher ORR versus high-dose dexamethasone in patients refractory to lenalidomide and/or bortezomib. The widely used combination of bortezomib+dexamethasone was recently compared directly with the second-generation, irreversible proteasome inhibitor carfilzomib+dexamethasone in the ENDEAVOR trial. Treatment with carfilzomib+dexamethasone significantly improved PFS (18.7 vs 9.4 mo.; p<0.0001) and significantly increased ORR (76.9% vs 62.6%; p<0.0001) compared with bortezomib+dexamethasone. Carfilzomib+dexamethasone also exhibited a favourable benefit–risk profile, suggesting potential applicability as a future doublet regimen in relapsed multiple myeloma. With the many therapies available for multiple myeloma, it is important to make treatment decisions based on the individual needs of the patient, and doublet regimens offer useful options to balance efficacy and toxicity.

Synergistic Activity of Carfilzomib and Panobinostat in Multiple Myeloma Cells via Modulation of ROS Generation and ERK1/2.

Relapse of disease and subsequent resistance to established therapies remain as major challenges in the treatment of multiple myeloma (MM). New therapeutic options are needed for these extensively pretreated patients. To explore an optimized combinational therapy, interactions between the irreversible proteasome inhibitor carfilzomib exhibiting a well-tolerated side-effect profile and histone deacetylase inhibitor (HDACi) panobinostat (LBH589) were examined in MM cells. Coadministration of carfilzomib and LBH589 led to a synergistic inhibition of proliferation in MM cells. Further studies showed that the combined treatment synergistically increased mitochondrial injury, caspase activation, and apoptosis in MM cells. Lethality of the carfilzomib/LBH589 combination was associated with the reactive oxygen species (ROS) generation and ERK1/2 inactivation. In addition, the free radical scavenger N-acetylcysteine (NAC) could block carfilzomib and LBH589-induced oxidative stress and the subsequent apoptosis. Together, these findings argue that the strategy of combining carfilzomib and LBH589 warrants attention in MM.

Mucin 20 (MUC20) Modulates Proteasome Assembly Chaperones through the c-MET Pathway and Is a Biomarker of Proteasome Inhibitor Sensitivity in Myeloma

▪Background: Modulation of the activity of the ubiquitin-proteasome pathway with the proteasome inhibitor (PI) bortezomib is an established component of therapy for plasma cell disorders. More recently, the second-generation, irreversible proteasome inhibitor carfilzomib has been validated both pre-clinically and clinically, which can overcome bortezomib resistance in some patients. However, resistance emerges to carfilzomib as well, and the emergence of PI resistance in general is incompletely understood. Therefore, there is an urgent need to identify these mechanisms and develop biomarkers that could help guide therapy, and provide novel targets to overcome resistance.Methods: We generated carfilzomib-resistant (CR) myeloma cell lines by exposing drug-naive ANBL-6, KAS-6/1, U266, and OPM-2 cells to increasing concentrations of carfilzomib. These were then subjected to gene expression profiling (GEP) to identify prominent changes compared to their vehicle-treated counterparts. Molecular and pharmacologic approaches were then pursued to probe the significance of the observed changes to PI resistance in myeloma cell lines, murine models, and clinically annotated GEP databases.Results: Profiling and pathway analysis of CR myeloma cell lines identified suppressed expression of MUC20 as the most conserved and significant change compared to drug-naïve cells. Decreased levels of MUC20 in CR cells were confirmed by quantitative PCR and Western blotting, and also identified in bortezomib-resistant (BR) cell lines. Moreover, suppression of MUC20 with shRNAs was itself sufficient to confer carfilzomib resistance, which was associated with an increase in the proteasome chymotrypsin-like (ChT-L) activity. Reduced expression of MUC20 led to increased phosphorylation and activation of c-MET, STAT3, and ERK-1/2. In addition, exposure of drug-naïve cells to the c-MET ligand HGF induced activation of this pathway and of the proteasome ChT-L activity, but reduced carfilzomib sensitivity. Conversely, inhibition of c-MET with INCB28060, ARQ-197, or XL-184 restored carfilzomib sensitivity to the CR cell lines in culture, and ARQ-197 overcame resistance in a murine myeloma model. To explore the mechanisms behind the increase in ChT-L activity, we evaluated expression of proteasome components, and while 20S subunits were unchanged, Proteasome maturation protein (POMP), a key proteasome assembly chaperone, was more abundant. The POMP promoter has a consensus ETS-Like Gene 1 (ELK1) binding site, and ELK1 is a target for ERK-1/2. Therefore, we performed chromatin immunoprecipitation and gel mobility shift studies, which documented ELK1 binding, while mutation of this site reduced POMP expression in a reporter assay. Finally, analysis of the Millennium Pharmaceuticals GEP database of patients treated with bortezomib in the relapsed and relapsed/refractory settings showed patients who had higher expression of MUC20 had superior overall and progression-free survival compared to those who had lower expression.Conclusions: Taken together, our data support a role for signaling through the c-MET/ERK-1/2/ELK1/POMP axis in enhancing proteasome assembly and capacity, thereby reducing sensitivity to proteasome inhibitors like carfilzomib or bortezomib in myeloma. Also, they validate use of drugs suppressing c-MET as a potentially attractive strategy to overcome resistance to carfilzomib in the clinic. Finally, they indicate that MUC20 expression may be a viable biomarker to differentiate patients who have proteasome inhibitor-sensitive or relatively –resistant disease. DisclosuresOrlowski:Onyx Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

In vitro and in vivo interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor carfilzomib in non-Hodgkin lymphoma cells.

Interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor carfilzomib were examined in non-Hodgkin lymphoma (NHL) models, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and double-hit lymphoma cells. Marked in vitro synergism was observed in multiple cell types associated with activation of cellular stress pathways (e.g., JNK1/2, ERK1/2, and p38) accompanied by increases in DNA damage (γH2A.X), G2-M arrest, and the pronounced induction of mitochondrial injury and apoptosis. Combination treatment with carfilzomib and ricolinostat increased reactive oxygen species (ROS), whereas the antioxidant TBAP attenuated DNA damage, JNK activation, and cell death. Similar interactions occurred in bortezomib-resistant and double-hit DLBCL, MCL, and primary DLBCL cells, but not in normal CD34(+) cells. However, ricolinostat did not potentiate inhibition of chymotryptic activity by carfilzomib. shRNA knockdown of JNK1 (but not MEK1/2), or pharmacologic inhibition of p38, significantly reduced carfilzomib-ricolinostat lethality, indicating a functional contribution of these stress pathways to apoptosis. Combined exposure to carfilzomib and ricolinostat also markedly downregulated the cargo-loading protein HR23B. Moreover, HR23B knockdown significantly increased carfilzomib- and ricolinostat-mediated lethality, suggesting a role for this event in cell death. Finally, combined in vivo treatment with carfilzomib and ricolinostat was well tolerated and significantly suppressed tumor growth and increased survival in an MCL xenograft model. Collectively, these findings indicate that carfilzomib and ricolinostat interact synergistically in NHL cells through multiple stress-related mechanisms, and suggest that this strategy warrants further consideration in NHL.

The Irreversible Proteasome Inhibitor Carfilzomib Interacts Synergistically with the Selective HDAC6 Inhibitor ACY1215 in ABC- and GC-DLBCL and Mantle Cell Lymphoma Sensitive or Resistant to Bortezomib.

Abstract 2765The first FDA-approved proteasome inhibitor (PI), bortezomib, has shown limited single-agent activity in DLBCL but has been approved for the treatment of refractory mantle cell lymphoma (MCL). These considerations prompted effects to improve PI activity in these disorders by developing newer-generation PIs or by combining PIs with other targeted agents. Histone deacetylase (HDAC) inhibitors are epigenetic agents that kill transformed cells through diverse mechanisms, including induction of oxidative injury and DNA damage, and have been approved for the treatment of CTCL. Moreover, HDAC inhibitors that disrupt HDAC6 function induce cell death by interfering with aggresome function and protein disposition. We previously reported that the irreversible proteasome inhibitor carfilzomib (CFZ; Onyx), recently approved for the treatment of refractory multiple myeloma, interacted synergistically with the pan-HDAC inhibitor vorinostat in ABC- and GC- DLBCL cells as well as MCL cells.(Dasmahapatra et al. Blood 2010; 115(22):4478-87; Dasmahapatra et al. Mol Cancer Ther 2011: 10(9):1686-97). The purpose of the present study was to determine whether the selective HDAC6 inhibitor ACY-1215 (Acetylon) potentiated CFZ activity in DLBCL and MCL cells, and if so, by what mechanisms. Knock-down of HDAC6 protein by shRNA rendered SUDHL4 cells significantly more sensitive to CFZ compared to scrambled sequence control cells. Simultaneous treatment (24–48 hr) with sub-lethal CFZ concentrations (2.5–6 nM) and minimally toxic concentrations of either ACY-1215 (2.0–4.0μM) or Tubastatin A (5.0–7.5 μM) strikingly increased apoptosis in multiple DLBCL ( SUDHL16, SUDHL4, OCI-LY10) and MCL cells (e.g. Granta and Rec-1). Interactions were highly synergistic by Median Dose Effect analysis, with Combination Index (CI) values significantly less than 1.0 (e.g. 0.4 – 0.6). Other PIs (e.g. MG132) and commercially available HDAC6 inhibitors (e.g., Tubastatin A) also interacted synergistically. The CFZ/ACY1215 regimen also strikingly induced apoptosis in a primary DLBCL sample (ABC sub-type), but was relatively non-toxic to normal CD34+ bone marrow cells. Enhanced activity was associated with a sharp increase in caspase-3 activation, PARP cleavage, mitochondrial damage (loss of ΔΨm), induction of p21CIP1, NF-κB inactivation, ROS generation, induction/phosphorylation of the stress kinase JNK accompanied by down-regulation of phospho-ERK, and marked induction of γH2A.X, indicative of double-strand DNA breaks. Bortezomib- resistant cells (e.g. SUDHL16-10BR, SUDHL6–25BR, OCI-LY10–40BR and Granta-25BR) displayed partial but relatively modest cross-resistance to CFZ administered alone. However, co-administration of CFZ/ACY-1215 sharply increased lethality in all bortezomib-resistant cells. Genetic (e.g., shRNA knockdown or ectopic expression of dominant-negative JNK) interruption of JNK signaling significantly attenuated CFZ/ACY-1215-mediated lethality. Combined treatment with CFZ and ACY-1215 inhibitors triggered marked G2M arrest but did not induce further inhibition of proteasomal activity (CT-L) compared to CFZ treatment alone. However, ACY-1215/CFZ down-regulated the cargo protein HR23B, which is essential for shuttling ubiquitinated protein to the proteasome for degradation, potentially leading to protein overload and resulting lethality. Collectively, these findings indicate that combining the selective HDAC6 inhibitor ACY-1215 with CFZ synergistically induces apoptosis in multiple DLBCL and MCL cells through a JNK-dependent process in association with G2M arrest, down-regulation of HR23B, and induction of DNA damage. They also suggest that this strategy, which is active against sensitive as well as, bortezomib-resistant DLBCL and MCL cells, warrants further exploration in NHL. Supported by Lymphoma SPORE 1P50 CA130805. Disclosures:Off Label Use: We are investigating the pre-clinical use of carfilzomib and ACY1215 for treatment DLBCL and mantle cell lymphoma for which they are not yet approved. Grant:Onyx pharmacuticals and Acetylon Pharmaceuticals: Research Funding.

Obatoclax Interacts Synergistically with the Irreversible Proteasome Inhibitor Carfilzomib in GC- and ABC-DLBCL Cells In Vitro and In Vivo

Interactions between the irreversible proteasome inhibitor carfilzomib and the pan-BH3 mimetic obatoclax were examined in germinal center (GC)- and activated B-cell-diffuse large B-cell lymphoma (ABC-DLBCL) cells. Cotreatment with minimally toxic concentrations of carfilzomib (i.e., 2-6 nmol/L) and subtoxic concentrations of obatoclax (0.05-2.0 μmol/L) synergistically increased apoptosis in multiple DLBCL cell lines and increased lethality toward primary human DLBCL but not normal CD34(+) cells. Synergistic interactions were associated with sharp increases in caspase-3 activation, PARP cleavage, p-JNK induction, upregulation of Noxa, and AKT dephosphorylation. Combined treatment also diminished carfilzomib-mediated Mcl-1 upregulation whereas immunoprecipitation analysis revealed reduced associations between Bak and Mcl-1/Bcl-xL and Bim and Mcl-1. The carfilzomib/obatoclax regimen triggered translocation, conformational change, and dimerization of Bax and activation of Bak. Genetic interruption of c-jun-NH(2)-kinase (JNK) and Noxa by short hairpin RNA knockdown, ectopic Mcl-1 expression, or enforced activation of AKT significantly attenuated carfilzomib/obatoclax-mediated apoptosis. Notably, coadministration of carfilzomib/obatoclax sharply increased apoptosis in multiple bortezomib-resistant DLBCL models. Finally, in vivo administration of carfilzomib and obatoclax to mice inoculated with SUDHL4 cells substantially suppressed tumor growth, activated JNK, inactivated AKT, and increased survival compared with the effects of single-agent treatment. Together, these findings argue that a strategy combining carfilzomib and obatoclax warrants attention in DLBCL.

Highlights of This Issue

A gender bias exists in the development of gliomas. Females are at lower risk than males, implicating estrogen-mediated protective effects. Sareddy and colleagues observed decreased ERβ expression during glioma progression and examined potential use of ERβ agonists as a novel therapeutic to curb the growth of gliomas. Agonists promoted both expression and tumor suppressive functions of ERβ in gliomas. Liquiritigenin, a plant-derived ERβ agonist, significantly reduced in vivo growth of gliomas. ERβ agonists could represent a novel class of drugs to treat gliomas and provide an additional tool for enhancing survival.EGFR signaling is a key pathway in the growth control of many cancers, whose activity can be modulated by small molecule EGFR kinase inhibitors. This article describes the identification of proteins, through multiscale quantitative proteomics, whose presence or change in abundance is a biomarker of cancer cell response to the inhibition of EGFR tyrosine kinase. Kani and colleagues posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. This area of inquiry has significant biologic and clinical implication across many cancers, but most notably in lung cancer.To improve the activity of bortezomib against DLBCL cells, a novel strategy was developed combining the irreversible proteasome inhibitor carfilzomib with the pan-BH3 mimetic obatoclax. This regimen exhibited highly synergistic in vitro activity against both GC- and ABC-DLBCL cells as well as their bortezomibresistant counterparts. Notably, combined treatment resulted in significant activity and increased survival of tumor-bearing mice in vivo. Mechanisms underlying synergism were multifactorial, including JNK activation, Noxa upregulation, and untethering of Bak from Mcl-1/Bcl-xL. This dual-targeted approach has potentially important clinical implications for the treatment of DLBCL patients who might not otherwise respond to standard proteasome inhibitor therapy.Levallet and colleagues found an adverse prognostic effect of class III β-tubulin (TUBB3) tumor immunostaining in 412 NSCLC patients and TUBB3 expression associated with resistance to chemotherapy. TUBB3 content correlated with K-Ras mutations in a subset of 208 cryopreserved specimens. Human bronchial epithelial cells expressing a mutant form of K-Ras had a significantly higher content of TUBB3 protein as compared to their isogenic counterpart expressing wild-type K-Ras. Short interfering RNA K-Ras knockdown or inhibition of RAF/MEK/PI3K signaling pathways by pharmacological inhibitors downregulated TUBB3 expression in bronchial cells. These findings deserve further studies evaluating anti-MEK or anti-PI3K drugs in NSCLC patients with TUBB3-overexpressing tumors.