Aggresome Pathway Research Articles

Overcome the tumor immunotherapy resistance by combination of the HDAC6 inhibitors with antitumor immunomodulatory agents

Tumor immunotherapy is currently subject of intense scientific and clinical developments. In previous decade, therapists used natural immune system from the human body to treat several diseases. Although tumor immune disease is a big challenge, combinatorial therapeutic strategy has been succeeded to show the clinical significance. In this context, we discuss the HDAC6 and tumor immune diseases relationship. Also, we summarized the current state of knowledge that based on the combination treatments of the HDAC6 inhibitors (HDAC6is) with antitumor immunomodulatory agents. We observed that, the combination therapies slow down the tumor immune diseases by blocking the aggresome and proteasome pathway. The combination therapy was able to reduce M2 macrophage and increasing PD-L1 blockade sensitivity. Most importantly, multiple combinations of HDAC6is with other agents may consider as potential strategies to treat tumor immune diseases, by reducing the side effects and improve efficacy for the future clinical development.

Cauliflower mosaic virus P6 inclusion body formation: A dynamic and intricate process

During an infection, Cauliflower mosaic virus (CaMV) forms inclusion bodies (IBs) mainly composed of viral protein P6, where viral activities occur. Because viral processes occur in IBs, understanding the mechanisms by which they are formed is crucial. FL-P6 expressed in N. benthamiana leaves formed IBs of a variety of shapes and sizes. Small IBs were dynamic, undergoing fusion/dissociation events. Co-expression of actin-binding polypeptides with FL-P6 altered IB size distribution and inhibited movement. This suggests that IB movement is required for fusion and growth. A P6 deletion mutant was discovered that formed a single large IB per cell, which suggests it exhibited altered fusion/dissociation dynamics. Myosin-inhibiting drugs did not affect small IB movement, while those inhibiting actin polymerization did. Large IBs colocalized with components of the aggresome pathway, while small ones generally did not. This suggests a possible involvement of the aggresome pathway in large IB formation.

Proteasome inhibitor b-AP15 induces enhanced proteotoxicity by inhibiting cytoprotective aggresome formation

Proteasome inhibitors have been shown to induce cell death in cancer cells by triggering an acute proteotoxic stress response characterized by accumulation of poly-ubiquitinated proteins, ER stress and the production of reactive oxygen species. The aggresome pathway has been described as an escape mechanism from proteotoxicity by sequestering toxic cellular aggregates. Here we show that b-AP15, a small-molecule inhibitor of proteasomal deubiquitinase activity, induces poly-ubiquitin accumulation in absence of aggresome formation. b-AP15 was found to affect organelle transport in treated cells, raising the possibility that microtubule-transport of toxic protein aggregates is inhibited, leading to enhanced cytotoxicity. In contrast to the antiproliferative effects of the clinically used proteasome inhibitor bortezomib, the effects of b-AP15 are not further enhanced by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Our results suggest an inhibitory effect of b-AP15 on the transport of misfolded proteins, resulting in a lack of aggresome formation, and a strong proteotoxic stress response.

Abstract 1859: Inhibition of proteasome deubiquitinase activity prevents cytoprotective aggresome formation in cancer cells

Abstract Proteasome inhibitors kill cancer cells by inducing an acute proteotoxic stress response characterized by the cytosolic accumulation of poly-ubiquitinated proteins, ER stress and the production of reactive oxygen species (ROS). The aggresome pathway has been described as an escape mechanism from proteasome inhibitor-induced cytotoxicity that functions by sequestering the accumulated protein. We show that b-AP15, an established small molecule inhibitor of proteasome deubiquitinase activity, induces the accumulation of cytosolic poly-ubiquitin in the absence of aggresome formation. In contrast to the clinically used proteasome inhibitor bortezomib, the accumulated poly-ubiquitin complexes induced by b-AP15 remain localized in the cytoplasm and fail to form typical aggresome structures. Inhibition of aggresome formation is dependent on ubiquitin but independent of tubulin dynamics, and may result in the accumulation of a range of substrates of the intracellular transport system on microtubules. Finally, b-AP15 induces hyper-ubiquitination of the aggresome chaperone HDAC6, as well as altering HDAC6 association with the proteasome. This suggests a potential mechanism for the present findings, which will require further investigation. Our data suggests that b-AP15 inhibition of aggresome formation may circumvent a potential resistance pathway following cytotoxicity due to proteasome inhibition via the blocking of microtubule transport. Citation Format: Ellin-Kristina Hillert, Slavica Brjnic, Magdalena Mazurkiewicz, Rolf Larsson, Mårten Fryknäs, Lisa Swanton, Stephen High, Stig Linder, Padraig B. D'Arcy. Inhibition of proteasome deubiquitinase activity prevents cytoprotective aggresome formation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1859.

Both p62/SQSTM1-HDAC6-dependent autophagy and the aggresome pathway mediate CDK1 degradation in human breast cancer

Cyclin-dependent kinase 1 (CDK1) is the central mammalian regulator of cell proliferation and a promising therapeutic target for breast cancer. In fact, CDK1 inhibition downregulates survival and induces apoptosis. Due to its essential role, CDK1 expression and activity are strictly controlled at various levels. We previously described that CDK1 stability is also regulated and that SCF(βTrCP) ubiquitinates CDK1, which is degraded via the lysosomal pathway. In addition, in breast tumors from patients, we found a negative correlation between CDK1 accumulation and βTrCP levels, and a positive correlation with the degree of tumor malignancy. This prompted us to study the molecular mechanism involved in CDK1 clearance. In this report, we determine that both chemotherapeutic agents and proteolytic stress induce CDK1 degradation in human breast cancer MCF7 cells through p62/HDAC6-mediated selective autophagy. On the one hand, CDK1 binds to p62/SQSTM1-LC3 and, on the other hand, it interacts with HDAC6. Both complexes are dependent on the presence of an intact βTrCP-binding motif on CDK1. Furthermore, we also show that CDK1 is recruited to aggresomes in response to proteasome inhibition for an extended period. We propose CDK1 clearance as a potential predictive biomarker of antitumor treatment efficacy.

The Role of Panobinostat Plus Bortezomib and Dexamethasone in Treating Relapsed or Relapsed and Refractory Multiple Myeloma: A European Perspective.

Panobinostat is an oral pan-histone deacetylase inhibitor developed by Novartis. Panobinostat acts via epigenetic modification and inhibition of the aggresome pathway. In August 2015, the European Commission authorized panobinostat for use in combination with bortezomib and dexamethasone for the treatment of relapsed or relapsed and refractory multiple myeloma (MM) in patients who have received ≥2 prior regimens including bortezomib and an immunomodulatory drug. In January 2016, the National Institute for Health and Care Excellence recommended panobinostat for use in the same combination and patient population. The authorization and recommendation were based on results from the pivotal phase 3 PANORAMA 1 (NCT01023308) clinical trial, which demonstrated an improvement in median progression-free survival of 7.8 months for the three-drug combination compared with placebo plus bortezomib and dexamethasone in this patient population. This review will discuss the current treatment landscape for relapsed/refractory MM, the mechanism of action of panobinostat, clinical data supporting the European authorization, concerns about safety and strategies for mitigating toxicity, and how panobinostat fits into the current MM landscape in Europe. Funding: Editorial support, funded by Novartis Pharmaceuticals.

Translational Focus on Targeting HDAC6 with Ricolinostat Confirms Potent Activity in Preclinical Models of Lymphoma and a Favorable Toxicity Profile in Patients with Relapsed or Refractory Lymphoma

Pan-class HDAC inhibitors are active agents for select subtypes of lymphoma but are often associated with toxicities such as fatigue, diarrhea/constipation, cardiac arrhythmias and myelosuppression. HDAC6 plays a critical role in protein homeostasis by managing misfolded proteins and inducing the unfolded protein response (UPR). Ricolinostat is an isoform selective HDAC6 inhibitor. We evaluated the preclinical effects of ricolinostat and translated the findings into a multicenter Phase 1b study in patients with lymphoma (NCT 02091063). Cytotoxicity was evaluated in 16 lymphoma cell lines (8 DBCL, 4 MCL, 4 TCL) by Cell TiterGlo and Annexin V. Ricolinostat has activity with IC50 values ranging from 0.9-4.7 uM range, which is in the range observed in clinical PK studies. Ricolinostat displayed marked synergy with bortezomib, PI3K inhibitors, disatinib, alisertib, and ibrutinib with synergy coeficients of 0.24, 0.29, 0.64, 0.07, 0.16 respectively (values In an effort to translate these findings, a multi-center Phase Ib open-label, single agent study designed to study the safety of 2 dose schedules of ricolinostat in patients with relapsed or refractory lymphoid malignancies was initiated. Patients were treated with 160 mg of oral ricolinostat continuously on a 28 day cycle. Patients were accrued sequentially to 2 dose cohorts: 3 patients were enrolled on single daily dosing (QD) schedule, and following safety assessment, patients were enrolled to twice daily dosing (BID). Samples were collected for PK and PD analysis. As of July 25th 2015, 11 patients were enrolled on the phase Ib study and were evaluated for safety. The median age was 36 yrs (24-86) and 7 (64%) were male. Sixty-four percent had 5 prior treatments (range: 1-15). Histologies included HL (N=3) and NHL (N=8) including DLBCL=3, MZL=2, FL=1, PTCL=1, PTLD=1. Three patients were enrolled in QD dosing and 8 patients in the BID schedule. There were no DLTs at either of the dosing schedules, and the maximum administrable dose was determined on the BID dosing schedule. The median number of cycles completed was 2 and ranged from 1 through 12+ (currently ongoing). The only grade 3 toxicities reported were anemia (27%), hyponatremia (9%), hypercalcemia (9%) and hypotension (9%), none thought to be attributed to the study drug. Other treatment emergent events included nausea (45%), diarrhea (36%), fatigue (36%) and vomiting (27%) which were all grade 1 and 2. Three patients achieved stable disease, all of whom were treated on the BID schedule (3 of 8, 37.5%), no complete or partial responses have been observed to date. One patient with refractory HL with 14 prior lines of therapy achieved stable disease (SD) and had symptomatic improvement corresponding to an increase of hemoglobin after 9 cycles, as well as resolution of B symptoms. Another patient with PTCL had immediate resolution of B symptoms such as fevers and pruritus after starting QD dosing. This is the first reported experience with an isoform selective HDAC inhibitor to be studied for the treatment of patients with relapsed or refractory lymphoid malignancies. Preclinical studies support the notion that ricolinostat mediates its effects through the UPR and potently synergizes with complementary agents. Ricolinostat is well tolerated with no DLTs. Thirty-seven percent of patients achieved SD on the BID schedule. PK and PD data will be evaluated for acetylation of alpha-tubulin and modulation of the UPR in an effort to confirm preclinical findings. Efficacy will be evaluated in a phase II study. We believe that ricolinostat in rational combinations will lead to effective treatment for patients with relapsed lymphoid malignancies. Disclosures Amengual:Acetylon Pharmaceuticals, INC: Consultancy, Research Funding. Off Label Use: Ricolinostat is being studied for the treatment of lymphoma. Neylon:Seattle Genetics: Speakers Bureau; Celgene: Speakers Bureau; Genentech: Speakers Bureau; Gilead: Speakers Bureau. Deng:TG Therapeutics: Research Funding; Gilead: Research Funding; Amgen/Onyx: Research Funding. Sawas:Seattle Genetics: Research Funding; Gilead Sciences: Honoraria. Shah:Pharmacyclics: Speakers Bureau; NCCN: Consultancy; SWOG: Consultancy; Rosetta Genomics: Research Funding; Acetylon Pharmaceuticals, INC: Membership on an entity9s Board of Directors or advisory committees; Plexus Communications: Honoraria; Spectrum: Speakers Bureau; Bayer: Honoraria; Celgene: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; DeBartolo Institute for Personlaized Medicine: Research Funding; Seattle Genetics: Research Funding; Janssen: Speakers Bureau. Wheeler:Acetylon Pharmaceuticals, INC: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. O9Connor:Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Takeda Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Bristol-Myers Squibb Company: Consultancy; Novartis: Consultancy, Honoraria; Seattle Genetics: Consultancy; Bayer: Consultancy, Honoraria; Acetylon Pharmaceuticals, INC: Consultancy; Mundipharma: Consultancy, Honoraria, Research Funding.

A Phase 2 Study of Panobinostat (PAN) in Combination with Bortezomib (BTZ) in Patients with Relapsed/Refractory Peripheral T-Cell Lymphoma (PTCL) or NK/T-Cell Lymphoma (NKL)

BackgroundRelapsed/refractory PTCL and NKL after conventional chemotherapy carry a poor prognosis and there is currently no proven salvage treatment available. Numerous preclinical studies have demonstrated synergistic interactions between proteasome and histone deacetylase (HDAC) inhibitors. PAN inhibits the aggresome pathway of protein degradation, which is upregulated when the proteasome pathway is inhibited by BTZ. Primary end point of this phase II multi-center open-label clinical study (NCT00901147) is the objective response rate (ORR) according to the Revised Response Criteria (Cheson 2007) among eligible patients (pts) treated with this novel combination of BTZ and PAN. Secondary end points include the evaluation of the progression-free survival (PFS) and the assessment of the safety and tolerability of the combination. We report the final clinical results of our study exploring this novel combination.MethodsPts with histologically confirmed PTCL or NKL who failed or were refractory to 1 or more prior systemic therapy, and had measurable disease and ECOG performance status 0–2 were eligible. Pts were accrued according to a 2-stage Gehan design. Pts receive thrice weekly oral PAN (20 mg) and twice weekly BTZ (IV 1.3 mg/m2), both for 2 of 3 weeks for up to 8 cycles. CT scanning and/or FDG-PET were performed after every two cycles.Results:Among 25 pts enrolled, histologies included: angioimmunoblastic T-cell lymphoma (AITL) n=8, PTCL (unspecified) n=11, Anaplastic large cell lymphoma, ALK+ and ALK- n=1 and 2 respectively, NKL, nasal type n=2 and subcutaneous panniculitis-like T-cell lymphoma n=1. The median age was 59 (35-79) years, and 64% were male. Outcomes are available on 23 patients as 2 patients withdrew consent before any response assessment could be made. The ORR (CR+PR) was 43% (10/23) with 22% (5/23) attaining a CR. Median time to response was 6 weeks. Five pts (22%) had stable disease while 8 pts developed progressive disease (35%) while on study. Pts received a median of 2 prior therapies (range 1-4); 28% had prior autologous stem cell transplantation (SCT). Common treatment-related grade 3/4 adverse events included thrombocytopenia (68%), neutropenia (36%), diarrhoea (28%) and asthenia/fatigue (16%). Peripheral neuropathy of any grade was observed in 40%. 5 pts successfully underwent subsequent allogeneic SCT. Updated survival analysis will be presented.ConclusionsThe study regimen is generally well tolerated and shows encouraging activity across different T/NK-cell lymphomas. The novel combination could successfully serve as a bridge to allogeneic SCT for many transplant-eligible patients who have failed conventional chemotherapy. These results form the basis for further validation studies on proteasome and HDAC inhibition in PTCL or NKL. Ongoing correlative studies are designed to determine if the study regimen is more active in diseases with up-regulation of NF-kappa B activity or transcription factors/ co-regulators known to be modified by acetylation. DisclosuresGoh:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Jannsen Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Gilead Sciences: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity’s Board of Directors or advisory committees. Kim:Novartis, Celgene, Takeda: Research Funding. Tan:JANSEN: Honoraria, Research Funding; NOVARTIS: Research Funding.

Factors regulating the subcellular localization of Activators of G‐protein Signaling 3: The role of serine/threonine residues in the G‐protein regulatory domain

Activators of G‐protein Signaling (AGS) proteins contain one to four G‐protein regulatory motifs (GPR) that serve as docking sites for Gαi/o/t. The GPR‐Gα signaling module is regulated by guanine nucleotide exchange factors and provides unexpected signaling diversity for the “G‐switch”. AGS3, which contains seven tetratricopeptide repeats (TPR) and four GPR motifs, is involved in a wide range of biological functions including asymmetric cell division, autophagy and addictive behavior. Interaction of AGS3 with Gαi or FRMPD1 stabilizes AGS3 at the cell cortex, whereas disruption of the TPR motif stabilizes the protein in the aggresome pathway. We asked if AGS3 trafficking was regulated by phosphorylation. As a first approach to this issue, we determined the subcellular distribution of a mutant GFP‐tagged AGS3 (AGS3‐GFP‐PM1) in which 24 S/T residues in the GPR domain of the protein were changed to alanine to eliminate S/T phosphorylation in this region. GFP tagged AGS3 WT, PM1 and TPR mutants were expressed in HEK‐293 cells and the subcellular distribution determined by immunofluorescence microscopy. In contrast to wild type AGS3, both the PM1 and TPR mutants distributed to preaggresomal structures in the cytosol. These data suggest that phosphorylation of AGS3 may play a regulatory role in its trafficking within the cell. Dysregulation of the subcellular location of AGS3 may contribute to pathologies involving altered protein processing through the aggresomal and autophagic pathways.

The novel role of Sav1 in aggresome formation

Intracellular deposition of misfolded protein aggregates into ubiquitin‐rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Recent genetic screens of fly mutants and molecular analysis have revealed that the Hippo/Salvador/Warts pathway controls both cell proliferation and cell death. Specifically, adaptor protein Salvador is the key component of this pathway. Using proteomics screening, we identified the molecular chaperone Hsp70 as a novel protein associated with Sav1 (mammalian homologue of Salvador). Sav1 targets to the aggresome and its sequestration to the aggresome is enhanced by the proteasome inhibitor MG132. Furthermore, Sav1 co‐localizes with Hsp70 and CHIP (carboxy terminus of Hsp70‐interacting protein) that has been shown to mediate substrate ubiquitination and degradation by the proteasome. This study suggests that Sav1 has a previously unexpected critical role in the aggresome pathway.

Protein Aggregates Are Recruited to Aggresome by Histone Deacetylase 6 via Unanchored Ubiquitin C Termini

The aggresome pathway is activated when proteasomal clearance of misfolded proteins is hindered. Misfolded polyubiquitinated protein aggregates are recruited and transported to the aggresome via the microtubule network by a protein complex consisting of histone deacetylase 6 (HDAC6) and the dynein motor complex. The current model suggests that HDAC6 recognizes protein aggregates by binding directly to polyubiquitinated proteins. Here, we show that there are substantial amounts of unanchored ubiquitin in protein aggregates with solvent-accessible C termini. The ubiquitin-binding domain (ZnF-UBP) of HDAC6 binds exclusively to the unanchored C-terminal diglycine motif of ubiquitin instead of conjugated polyubiquitin. The unanchored ubiquitin C termini in the aggregates are generated in situ by aggregate-associated deubiquitinase ataxin-3. These results provide structural and mechanistic bases for the role of HDAC6 in aggresome formation and further suggest a novel ubiquitin-mediated signaling pathway, where the exposure of ubiquitin C termini within protein aggregates enables HDAC6 recognition and transport to the aggresome.

P62/SQSTM1 in autophagic clearance of a non-ubiquitylated substrate

Proteolytic systems and the aggresome pathway contribute to preventing accumulation of cytotoxic aggregation-prone proteins. Although polyubiquitylation is usually required for degradation or aggresome formation, several substrates are processed independently of ubiquitin through a poorly understood mechanism. Here, we found that p62/SQSTM1, a multifunctional adaptor protein, was involved in the selective autophagic clearance of a non-ubiquitylated substrate, namely an aggregation-prone isoform of STAT5A (STAT5A_ΔE18). By using a cell line that stably expressed STAT5A_ΔE18, we investigated the properties of its aggregation and degradation. We found that STAT5A_ΔE18 formed non-ubiquitylated aggresomes and/or aggregates by impairment of proteasome functioning or autophagy. Transport of these aggregates to the perinuclear region was inhibited by trichostatin A or tubacin, inhibitors of histone deacetylase (HDAC), indicating that the non-ubiquitylated aggregates of STAT5A_ΔE18 were sequestered into aggresomes in an HDAC6-dependent manner. Moreover, p62 was bound to STAT5A_ΔE18 through its PB1 domain, and the oligomerization of p62 was required for this interaction. In p62-knockdown experiments, we found that p62 was required for autophagic clearance of STAT5A_ΔE18 but not for its aggregate formation, suggesting that the binding of p62 to non-ubiquitylated substrates might trigger their autophagic clearance.

Efficient Induction of Nuclear Aggresomes by Specific Single Missense Mutations in the DNA-binding Domain of a Viral AP-1 Homolog

Nuclear aggresomes induced by proteins containing an expanded polyglutamine (polyQ) tract are pathologic hallmarks of certain neurodegenerative diseases. Some GFP fusion proteins lacking a polyQ tract may also induce nuclear aggresomes in cultured cells. Here we identify single missense mutations within the basic DNA recognition region of Bam HI Z E B virus replication activator (ZEBRA), an Epstein-Barr virus (EBV)-encoded basic zipper protein without a polyQ tract, that efficiently induced the formation of nuclear aggresomes. Wild-type (WT) ZEBRA was diffusely distributed within the nucleus. Four non-DNA-binding mutants, Z(R179E), Z(R183E), Z(R190E), and Z(K178D) localized to the periphery of large intranuclear spheres, to discrete nuclear aggregates, and to the cytoplasm. Other non-DNA-binding mutants, Z(N182K), Z(N182E), and Z(S186E), did not exhibit this phenotype. The interior of the spheres contained promyelocytic leukemia and HSP70 proteins. ZEBRA mutants directly induced the nuclear aggresome pathway in cells with and without EBV. Specific cellular proteins (SC35 and HDAC6) and viral proteins (WT ZEBRA, Rta, and BMLF1) but not other cellular or viral proteins were recruited to nuclear aggresomes. Co-transfection of WT ZEBRA with aggresome-inducing mutants Z(R183E) and Z(R179E) inhibited late lytic viral protein expression and lytic viral DNA amplification. This is the first reported instance in which nuclear aggresomes are induced by single missense mutations in a viral or cellular protein. We discuss conformational changes in the mutant viral AP-1 proteins that may lead to formation of nuclear aggresomes.

Movement of Activator of G‐Protein Signaling 3 within the Aggresome Pathway

AGS3, a receptor independent activator of G‐protein signaling, is involved in unexpected functional diversity for G‐protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G‐protein regulatory (GPR) motifs, each of which bind and stabilizes the GDP bound conformation of Giα. The positioning of AGS3 within the cell and the intra‐molecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G‐protein signaling. We report that AGS3 enters into the aggresome pathway and that this positioning of the protein is differentially regulated by the AGS3 binding partners Giα and mInsc. Giα rescues AGS3 from the aggresome, whereas mInsc augments the aggresomal distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain and it is accelerated by disruption of the TPR organizational structure or introduction of a non‐synonymous single nucleotide polymorphism. These data present AGS3, G‐proteins and mInsc as candidate proteins involved in regulating cellular stress associated with protein processing pathologies.

Distribution of activator of G-protein signaling 3 within the aggresomal pathway: role of specific residues in the tetratricopeptide repeat domain and differential regulation by the AGS3 binding partners Gi(alpha) and mammalian inscuteable.

AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gialpha-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gialpha and mammalian Inscuteable (mInsc). Gialpha rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.

A Phase I Clinical Trial Testing the Combination of Bortezomib and Tipifarnib in Relapsed/Refractory Multiple Myeloma.

Abstract 3851Poster Board III-787 BackgroundPreclinical work from our group and others has demonstrated that the combination of a farnesyl transferase inhibitor (FTI) and the proteasome inhibitor bortezomib results in enhanced plasma cell apoptosis and is associated with AKT activation (David, Blood 2005). Sequence of bortezomib followed by a FTI synergistically inhibited cell growth compared to concurrent treatment. More recently, further preclinical data suggests that the mechanism responsible for this profound synergy is due to inhibition of HDAC6 with a resultant inhibition of both the proteasome and aggresome pathway (David ASH 2007). Based upon these observations, with the MMRC, we initiated a phase I trial combining the FTI tipifarnib with bortezomib to clinically evaluate the efficacy of this combination. MethodsPatients with relapsed or refractory myeloma were treated with bortezomib at 1.0 mg/m2 or 1.3mg/m2 given on days 1,4,8, and 11 in conjunction with escalating doses of tipifarnib (100-400mg PO BID) given on days 2-15 every 21 days, respectively. Dose escalation was accomplished using an adaptive phase I design (Escalation With Overdose Control (EWOC)). Eligibility criteria included a serum creatinine of <2.5, normal liver function, ANC>500, and platelets >25. ResultsTwenty-six patients have been enrolled to date: bortezomib 1.0 mg/m2 with tipifarnib 100 mg (n=6), 200mg (n=5), 300mg (n=7), 400mg (n=2) and bortezomib 1.3mg/m2 with tipifarnib 300mg (n=6), 400mg to be enrolled. Median age is 63 (range 42-77). 19/26 patients had received prior high dose therapy. The average number of prior therapies was 4, and, of the 26 patients, 10 were refractory to bortezomib (relapsed on therapy or within 6 months) 11 were bortezomib naïve, and 5 were previously exposed to bortezomib but not refractory. Among these patients with advanced myeloma and refractory disease, 50% had stabilization of disease or better. Maximum number of cycles received was 10. Of note, among the patients achieving clinical benefit, 1 had a stable M-protein, but experienced an 80% reduction in circulating plasma cells while on therapy, and another has had a 75% reduction in the free light chain assay. The most common drug-related toxicities were GI (17.0%) with nausea/vomiting occurring most frequently. Hematologic toxicities were difficult to ascertain as patients had advanced myeloma and many entered with platelet counts of 25-50. Additional grade 3 toxicities included renal insufficiency (related to PD), pneumonia and altered mental status which were all considered due to disease progression. There were no Grade 3- 5 non-hematologic drug related toxicities. There were no cardiac events or DVT, and 1 patient experienced grade 2 peripheral neuropathy who did not have pre- existing PN at baseline. ConclusionsThe combination of bortezomib and tipifarnib is supported by preclinical rationale and has produced stable disease or better among a group of patients with refractory and advanced myeloma. To date the optimal doses of both tipifarnib and bortezomib have yet to be defined, and additional patients will be enrolled to define the MTD by escalation of tipifarnib to 400 with 1.3mg/m2 of bortezomib. Correlative studies evaluating the effect of the combination on HDAC6 and plasma cell apoptosis will be presented. Disclosures:Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Kaufman:Millennium : Consultancy; Celgene: Consultancy, Research Funding; Genzyme: Consultancy; Merck: Research Funding.

The Combination of Romidepsin and Bortezomib Results in Synergistic Induction of Apoptosis in Human B-Lymphoma Cell Lines.

Abstract 1689Poster Board I-715 IntroductionThe use of the proteasome inhibitor bortezomib has demonstrated activity in multiple myeloma and lymphomas. The HDAC inhibitor romidepsin is being evaluated in CTCL and PTCL, though its activity in B-cell lymphomas is less clear. We hypothesized that the combination of bortezomib and romidepsin would result in synergistic apoptosis in different B-cell NHL cell lines based upon the observed activity of this combination in more mature B-cell malignancies such as myeloma. Experimental DesignDaudi, HT, Ramos and SUDHL-4 cell lines were exposed to different concentrations of bortezomib and romidepsin, separately, concurrently, and sequentially. Cell viability was assessed using MTT-assay, induced apoptosis was evaluated using Annexin V and PI staining from 24-48 hours. Apoptosis was also evaluated using western blot analysis of caspases and PARP cleavage. LC3 and HDAC6 level expressions were performed to determine if the effect of the combination was a result of the aggresome or autophagy pathway. Cell cycle studies were also performed to study if there were any changes after treating cells with the combination. ResultsThe combination of bortezomib and romidepsin resulted in synergistic B-cell apoptosis as measured by MTT-assay with combination indices of < 0.5. This was associated with increased caspases and PARP cleavage as early as 24 hours after exposure. Order of addition experiments demonstrated definite sequence specificity. When romidepsin was added first, and 6 hours later followed by bortezomib, apoptosis was enhanced, compared to both agents being given concurrently or when bortezomib was administered first. Cell cycle analysis studies demonstrated that pretreatment of cells with romidepsin for 6 hours followed by the addition of bortezomib arrested the cells in G2M phase. HDAC6 expression was significantly reduced following combination therapy, and LC3-I was cleaved to LC3-II in treated cells suggesting that the combination affected aggresome formation and autophagy. ConclusionThe combination of romidepsin and bortezomib at low nanomolar concentrations suggests that this may be an important clinical combination to test in patients with relapsed or refractory B-cell malignancies. Sequence of administration data is currently being tested to determine if the effect is a result of autophagy inhibition as is seen in myeloma cell lines. Additional mechanistic studies will be presented with the goals of identifying predictors of response that can then be validated in prospective clinical trials. DisclosuresLechowicz:Gloucester: Consultancy. Kaufman:Millennium: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Gloucester: Research Funding; Novartis: Consultancy; BMS: Consultancy; Millennium: Consultancy, Research Funding; Celgene: Consultancy. Flowers:Millennium: Research Funding.

A phase I MMRC clinical trial testing the combination of bortezomib and tipifarnib in relapsed/refractory multiple myeloma

8597 Background: Preclinical work from our group has demonstrated that the combination of a farnesyl transferase inhibitor and bortezomib results in enhanced plasma cell apoptosis, and more recently, we have suggested that the mechanism for this synergy is due to inhibition of HDAC6, and inhibition of the proteasome and aggresome pathway. Methods: Patients with relapsed or refractory myeloma were treated with bortezomib at 1.0 or 1.3 mg/m2 on a standard schedule in conjunction with escalating doses of tipifarnib (100–400mg/BID) given on days 2–15 every 21 days. Dose escalation was accomplished using an adaptive phase I design (Escalation With Overdose Control). Results: 22 patients have been enrolled, of which 18 are evaluable, into respective tipifarnib dose levels 100 mg (n=6), 200mg (n=5) and 300mg (n=5) and 400mg (n=2). Median age for the enrolled patients is 59 and median time from myeloma diagnosis was 4.7 years. The average number of prior therapies was 4.5. Among these patients with advanced myeloma and refractory disease, stabilization of disease or better was seen among 8/10 patients with 2 of the 8 achieving an MR. Among the patients achieving clinical benefit, 1 patient had a stable M-protein, but experienced an 80% reduction in circulating plasma cells while on therapy, and another has had a 75% reduction in the free light chain assay. The most common drug related side effect was was Gr2 diarrhea (23.5%). Hematologic toxicities were difficult to ascertain as patients had advanced myeloma and many were entered onto study with platelet counts between 25 and 50. Additional grade 3 toxicities included renal insufficiency (related to PD), pneumonia and altered mental status which were all considered not related to study drug but to progression of disease. There were no Grade 3-5 drug related toxicities. One patient experienced grade 2 peripheral neuropathy who did not have pre existing PN at baseline. Conclusions: The combination of bortezomib and tipifarnib is supported by preclinical rationale and has produced stable disease or better among 10 of 18 patients with refractory myeloma. Optimal dose of tipifarnib and bortezomib have yet to be defined, and additional patients are being enrolled with 1.3mg/m2 of bortezomib, and escalating doses of tipifarnib. [Table: see text]

A Critical Role for CHIP in the Aggresome Pathway

Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed "physiologic aggresome," is exemplified by aggresome formation of inducible nitric oxide synthase (iNOS), an important host defense protein. CHIP (carboxy terminus of Hsp70-interacting protein) is a highly conserved protein that has been shown to mediate substrate ubiquitination and degradation by the proteasome. In this study, we show that CHIP has a previously unexpected critical role in the aggresome pathway. CHIP interacts with iNOS and promotes its ubiquitination and degradation by the proteasome as well as its sequestration to the aggresome. CHIP-mediated iNOS targeting to the proteasome sequentially precedes CHIP-mediated iNOS sequestration to the aggresome. CHIP is required for iNOS preaggresome structures to form a mature aggresome. Furthermore, CHIP is required for targeting the mutant form of cystic fibrosis transconductance regulator (CFTRDeltaF508) to the aggresome. Importantly, the ubiquitin ligase function of CHIP is required in targeting preaggresomal structures to the aggresome by promoting an iNOS interaction with histone deacetylase 6, which serves as an adaptor between ubiquitinated proteins and the dynein motor. This study reveals a critical role for CHIP in the aggresome pathway.

A Phase I MMRC Clinical Trial Testing the Combination of Bortezomib and Tipifarnib in Relapsed/Refractory Multiple Myeloma

Background: Preclinical work from our group and others has demonstrated that the combination of a farnesyl transferase inhibitor (FTI) and the proteasome inhibitor bortezomib results in enhanced plasma cell apoptosis and is associated with AKT activation (David, Blood 2005). More recently, further preclinical data suggests that the mechanism responsible for this profound synergy is due to inhibition of HDAC6 with a resultant inhibition of both the proteasome and aggresome pathway (David ASH 2007). Based upon these observations, with the MMRC we initiated a phase I trial combining the FTI tipifarnib with bortezomib to clinically evaluate the efficacy of this combination.Methods: Patients with relapsed or refractory myeloma were treated with bortezomib at 1.0 mg/m2 given on days 1,4,8, and 11 in conjunction with escalating doses of tipifarnib (100–400mg/BID) given on days 2–15 every 21 days. Dose escalation was accomplished using an adaptive phase I design (Escalation With Overdose Control (EWOC)). Eligibility criteria included a serum creatinine of <2.5, normal liver function, ANC>500, and platelets >25. If dose escalation is able to proceed to 400mg of tipifarnib with 1.0 mg/m2, the tipifarnib dose escalation will restart with bortezomib given at 1.3 mg/m2.Results: Sixteen patients have been enrolled to date into respective tipifarnib dose levels 100 mg(n=6),200mg (n=5) and 300mg (n=5). Median age for the enrolled patients is 59 (range 43–76) and median time from myeloma diagnosis was 4.7 years. 15/16 patients had received prior high dose therapy. The average number of prior therapies was 4.5, and of the16 patients, 8 were refractory to prior bortezomib (relapsed on therapy or within 6 months) 4 were bortezomib naïve, and 4 were previously exposed to bortezomib but not known to be refractory. Among these patients with advanced myeloma and refractory disease, stabilization of disease or better was seen among 7/16 patients with 2 of the 7 achieving an MR. Of note, among the patients achieving clinical benefit, 1 patient had a stable M-protein, but experienced an 80% reduction in circulating plasma cells while on therapy, and another has had a 75% reduction in the free light chain assay. The most common drug related side effects were was Gr2 diarrhea (23.5%). Hematologic toxicities were difficult to ascertain as patients had advanced myeloma and many were entered onto study with platelet counts between 25 and 50. Additional grade 3 toxicities included renal insufficiency (related to progression), pneumonia and altered mental status which were all considered unrelated to study drug, but were associated with progression of disease. There were no Grade 3 –5 drug related toxicities. There were no cardiac events or DVT, and 1 patient experienced grade 2 peripheral neuropathy who did not have pre existing PN at baseline.Conclusions: The combination of bortezomib and tipifarnib is supported by preclinical rationale and has produced stable disease or better among a group of patients with refractory and advanced myeloma. To date the optimal dose of both tipifarnib and bortezomib have yet to be defined, and additional patients will be enrolled to define the MTD for tipifarnib with 1.0mg/m2 of bortezomib, followed by escalation of tipifarnib with 1.3mg/m2 of bortezomib. Correlative studies evaluating the effect of the combination on HDAC6 and plasma cell apoptosis will be presented.