Expression Of Apoptosis Inhibitor Research Articles

The expanded inhibitor of apoptosis gene family in oysters possesses novel domain architectures and may play diverse roles in apoptosis following immune challenge

BackgroundApoptosis plays important roles in a variety of functions, including immunity and response to environmental stress. The Inhibitor of Apoptosis (IAP) gene family of apoptosis regulators is expanded in molluscs, including eastern, Crassostrea virginica, and Pacific, Crassostrea gigas, oysters. The functional importance of IAP expansion in apoptosis and immunity in oysters remains unknown.ResultsPhylogenetic analysis of IAP genes in 10 molluscs identified lineage specific gene expansion in bivalve species. Greater IAP gene family expansion was observed in C. virginica than C. gigas (69 vs. 40), resulting mainly from tandem duplications. Functional domain analysis of oyster IAP proteins revealed 3 novel Baculoviral IAP Repeat (BIR) domain types and 14 domain architecture types across gene clusters, 4 of which are not present in model organisms. Phylogenetic analysis of bivalve IAPs suggests a complex history of domain loss and gain. Most IAP genes in oysters (76% of C. virginica and 82% of C. gigas), representing all domain architecture types, were expressed in response to immune challenge (Ostreid Herpesvirus OsHV-1, bacterial probionts Phaeobacter inhibens and Bacillus pumilus, several Vibrio spp., pathogenic Aliiroseovarius crassostreae, and protozoan parasite Perkinsus marinus). Patterns of IAP and apoptosis-related differential gene expression differed between the two oyster species, where C. virginica, in general, differentially expressed a unique set of IAP genes in each challenge, while C. gigas differentially expressed an overlapping set of IAP genes across challenges. Apoptosis gene expression patterns clustered mainly by resistance/susceptibility of the oyster host to immune challenge. Weighted Gene Correlation Network Analysis (WGCNA) revealed unique combinations of transcripts for 1 to 12 IAP domain architecture types, including novel types, were significantly co-expressed in response to immune challenge with transcripts in apoptosis-related pathways.ConclusionsUnprecedented diversity characterized by novel BIR domains and protein domain architectures was observed in oyster IAPs. Complex patterns of gene expression of novel and conserved IAPs in response to a variety of ecologically-relevant immune challenges, combined with evidence of direct co-expression of IAP genes with apoptosis-related transcripts, suggests IAP expansion facilitates complex and nuanced regulation of apoptosis and other immune responses in oysters.

Selection of the sex‐linked inhibitor of apoptosis in mountain pine beetle (Dendroctonus ponderosae) driven by enhanced expression during early overwintering

The mountain pine beetle (Dendroctonus ponderosae) is an insect native to western North America; however, its geographical range has recently expanded north in BC and east into Alberta. To understand the population structure in the areas of expansion, 16 gene‐linked microsatellites were screened and compared to neutral microsatellites using outlier analyses of F st and F ct values. One sex‐linked gene, inhibitor of apoptosis (IAP), showed a strong signature of positive selection for neo‐X alleles and was analyzed for evidence of adaptive variation. Alleles of IAP were sequenced, and differences between the neo‐X and neo‐Y alleles were consistent with neutral evolution suggesting that the neo‐Y allele may not be under functional constraints. Neo‐Y alleles were amplified from gDNA, but not effectively from cDNA, suggesting that there was little IAP expression from neo‐Y alleles. There were no differences in overall IAP expression between males and females with the common northern neo‐X allele suggesting that the neo‐X allele in males compensates for the reduced expression of neo‐Y alleles. However, males lacking the most common northern neo‐X allele thought to be selected for in northern populations had reduced overall IAP expression in early October—at a time when beetles are preparing for overwintering. This suggests that the most common allele may have more rapid upregulation. The reduced function of neo‐Y alleles of IAP suggested by both sequence differences and lower levels of expression may foster a highly selective environment for neo‐X alleles such as the common northern allele with more efficient upregulation.

Inhibitors of apoptosis: clinical implications in cancer

Inhibitor of apoptosis (IAP) family comprises a group of endogenous proteins that function as main regulators of caspase activity and cell death. They are considered the main culprits in evasion of apoptosis, which is a fundamental hallmark of carcinogenesis. Overexpression of IAP proteins has been documented in various solid and hematological malignancies, rendering them resistant to standard chemotherapeutics and radiation therapy and conferring poor prognosis. This observation has urged their exploitation as therapeutic targets in cancer with promising pre-clinical outcomes. This review describes the structural and functional features of IAP proteins to elucidate the mechanism of their anti-apoptotic activity. We also provide an update on patterns of IAP expression in different tumors, their impact on treatment response and prognosis, as well as the emerging investigational drugs targeting them. This aims at shedding the light on the advances in IAP targeting achieved to date, and encourage further development of clinically applicable therapeutic approaches.

Racial differences in the expression of inhibitors of apoptosis (IAP) proteins in extracellular vesicles (EV) from prostate cancer patients.

African-American men with prostate cancer typically develop more aggressive tumors than men from other racial/ethnic groups, resulting in a disproportionately high mortality from this malignancy. This study evaluated differences in the expression of inhibitors of apoptosis proteins (IAPs), a known family of oncoproteins, in blood-derived exosomal vesicles (EV) between African-American and European-American men with prostate cancer. The ExoQuick™ method was used to isolate EV from both plasma and sera of African-American (n = 41) and European-American (n = 31) men with prostate cancer, as well as from controls with no cancer diagnosis (n = 10). EV preparations were quantified by acetylcholinesterase activity assays, and assessed for their IAP content by Western blotting and densitometric analysis. Circulating levels of the IAP Survivin were evaluated by ELISA. We detected a significant increase in the levels of circulating Survivin in prostate cancer patients compared to controls (P<0.01), with the highest levels in African-American patients (P<0.01). African-American patients with prostate cancer also contained significantly higher amounts of EVs in their plasma (P<0.01) and sera (P<0.05) than European-American patients. In addition, EVs from African-American patients with prostate cancer contained significantly higher amounts of the IAPs Survivin (P<0.05), XIAP (P<0.001), and cIAP-2 (P<0.01) than EVs from European-American patients. There was no significant correlation between expression of IAPs and clinicopathological parameters in the two patient groups. Increased expression of IAPs in EVs from African-American patients with prostate cancer may influence tumor aggressiveness and contribute to the mortality disparity observed in this patient population. EVs could serve as reservoirs of novel biomarkers and therapeutic targets that may have clinical utility in reducing prostate cancer health disparities.

The identification of molecular target of (20S) ginsenoside Rh2 for its anti-cancer activity

The 20S ginsenoside Rh2 (G-Rh2) effectively inhibits cancer cell growth and survival in both animal models and cell lines. However, its molecular targets and mechanism of action remain largely unknown. By screening for molecules that interact with (20S)G-Rh2 in a phage display assay, we have identified Annexin A2 as a potential target that mediates its anti-cancer activity. Isothermal titration calorimetry and a cellular thermal shift assay demonstrated that (20S)G-Rh2 directly bound to either recombinant or intracellular Annexin A2. This binding inhibited the interaction between Annexin A2 and the NF-кB p50 subunit, which attenuated the nuclear translocations of NF-кB p50 subunit and reduced the transactivation activity of NF-кB. Correspond to this result, (20S)G-Rh2 treatment significantly down-regulated the expression of IAPs (inhibitors of apoptosis), the well-established NF-кB targets that promote cell survival. Moreover, (20S)G-Rh2 synergized with Annexin A2 inactivation to promote apoptosis. Taken together, this study for the first time suggests a cellular target and a molecular pathway by which (20S)G-Rh2 inhibits cancer cell growth. As over-expression of Annexin A2 was evident in human hepatoma, (20S)G-Rh2 might be a promising natural compound for targeted liver cancer therapy.

Morphological characteristics of uterine fibroids among women of reproductive age

The article deals with modern views on etiopathogenesis of uterine fibroids. Increased knowledge of the markers of proliferation and their study will help to predict the course of the disease and thus to develop the optimum tactics of the further treatment of the early stages. Much attention is paid to the study of markers that characterize apoptosis. By immunohistochemistry in tissue fibroids 15 patients studied protein expression Ki-67 and Bcl-2. Analyzed values of the correlation expression of the proteins according to the clinical and morphological features of the tumor. It showed a reduction in the proliferation marker Ki-67, and increased expression of apoptotic Bcl-2 inhibitor.Background. Presented by changes in the nature of proliferation and apoptotic activity in myoma nodes among women of reproductive age who underwent conservative myomectomy.The aim of the research was to study the performance of protein antibody Ki-67 proliferation and apoptosis inhibitor of Bcl-2 in women with uterine myoma.Material and methods. Surveyed 15 women with uterine fibroids who underwent conservative myomectomy. Inclusion criteria were: the reproductive age, uterine fibroids the size of which does not exceed the 12-week pregnancy. Exclusion criteria were the size of fibroids greater than 12 weeks of pregnancy, acute inflammatory diseases of the pelvic organs, severe somatic diseases, cancer of the reproductive organs. It was carried out histological and immunohistochemical study of remote myoma node. We examined by immunohistochemistry proliferation protein monoclonal antibody Ki-67 (Clone MIB-1, 1 : 150, Dako), inhibitor of apoptosis Bcl-2 (NCL-bcl-2-486). (Novocastra). Evaluation of the expression of markers studied conducted qualitative and quantitative methods in computer image analysis system “Morphology 5.0” (Video Test, Russia). Statistical processing of the results was performed using statistical analysis packages (STATGRAPHICS v. 6.0).Results. Age of women surveyed averaged 33,4 ± 4,1 years. Histological study of benign tumors of the uterus body showed the presence of degenerative changes, necrosis and inflammatory infiltration in every fourth case of the material.Conclusions. Immunogistohimichesoe study of uterine leiomyomas body revealed a significant increase in the expression of apoptosis inhibitor of Bcl-2 and reducing the proliferation marker Ki-receptor expression in all 67 tested samples, which is consistent with a histological study of the absence of mitotic activity in leiomyomas.

ViscumTT induces apoptosis and alters IAP expression in osteosarcoma in vitro and has synergistic action when combined with different chemotherapeutic drugs

BackgroundOsteosarcoma is the most common bone tumor and is associated with a poor prognosis. Conventional therapies, surgery and chemotherapy, are still the standard but soon reach their limits. New therapeutic approaches are therefore needed. Conventional aqueous mistletoe extracts from the European mistletoe (Viscum album L.) are used in complementary cancer treatment. These commercial extracts are water-based and do not include water-insoluble compounds such as triterpenic acids. However, both hydrophilic and hydrophobic triterpenic acids possess anti-cancer properties. In this study, a whole mistletoe extract viscumTT re-created by combining an aqueous extract (viscum) and a triterpene extract (TT) was tested for its anti-cancer potential in osteosarcoma.MethodsTwo osteosarcoma cell lines were treated with three different mistletoe extracts viscum, TT and viscumTT to compare their apoptotic potential. For this purpose, annexin/PI staining and caspase-3, −8 and −9 activity were investigated by flow cytometry. To determine the mechanism of action, alterations in expression of inhibitors of apoptosis (IAPs) were detected by western blot. Apoptosis induction by co-treatment of viscum, TT and viscumTT with doxorubicin, etoposide and ifosfamide was examined by flow cytometry.ResultsIn vitro as well as ex vivo, the whole mistletoe extract viscumTT led to strong inhibition of proliferation and synergistic apoptosis induction in osteosarcoma cells. In the investigations of mechanism of action, inhibitors of apoptosis such as XIAP, BIRC5 and CLSPN showed a clear down-regulation after viscumTT treatment. In addition, co-treatment with doxorubicin, etoposide and ifosfamide further enhanced apoptosis induction, also synergistically.ConclusionViscumTT treatment results in synergistic apoptosis induction in osteosarcoma cells in vitro and ex vivo. Additionally, conventional standard chemotherapeutic drugs such as doxorubicin, etoposide and ifosfamide were able to dramatically enhance apoptosis induction. These results promise a high potential of viscumTT as an additional adjuvant therapy approach for osteosarcoma.

A Phase 1b Study of Birinapant in Combination with 5-Azacitadine in Patients with Myelodysplastic Syndrome Who Are Naïve, Refractory or Have Relapsed to 5-Azacitadine

Background: Although 5-azacitidine (5-AZA) is the standard of care for patients with higher-risk patients with Myelodysplastic Syndrome (MDS) almost all patients will fail 5-AZA therapy with the majority of patients progressing within 2 years. Two-year survival for MDS patients refractory to 5-AZA is only 15%. There is a need for more effective therapies for these patients with higher-risk MDS. The Inhibitors of Apoptosis (IAP) Proteins are a family of molecules that suppress apoptotic cell death. Expression of IAPs is dysregulated in MDS, supporting potential as therapeutic targets. SMAC (second mitochondrial activator of caspases) is an IAP antagonist, resulting in caspase activation, inhibition of the pro-survival transcription factor NF-kB and increased apoptosis leading to tumor cell death. Birinapant is a potent bivalent SMAC mimetic in clinical development for the treatment of several cancers. Pre-clinical data support its investigation in myeloid malignancies, specifically MDS. Methods: This Phase 1b study was designed to determine the MTD and recommended Phase 2 dose and schedule of birinapant in combination with 5-AZA in patients with higher-risk MDS who are naïve to or have relapsed or are refractory to 5-AZA. Secondary objectives included determining the clinical activity of the combination of birinapant plus 5-AZA, and assessing the pharmacokinetics (PK) and exploratory biomarkers. Results: Twenty-one patients were enrolled, of whom 15 had high/very high risk MDS, with median IPSS score of 3 (range, 1.5-8). Median age (range) was 73 years (48-84 years). Eight patients were therapy-naïve. All patients received 5-AZA, administered either intravenous (IV; 13 patients) or subcutaneous (SC; 8 of whom 4 later switched to IV), at 75 mg/m2 for 7 days of a 28-day cycle in combination with birinapant. Birinapant was administered IV twice weekly (Days 1 and 4) at 13 mg/m2 for either 3 of 4 weeks (15 patients; Cohort 1 and Expansion Cohort) or 4 of 4 weeks (6 patients; Cohort 2) in 28-day cycles. No cycle 1 dose-limiting toxicities were observed. Adverse events ≥Grade 3 observed in two or more patients were thrombocytopenia (9 patients); pneumonia (including fungal pneumonia; 8); anemia (8); leukopenia (7); febrile neutropenia (7) neutropenia (5); fatigue (4); respiratory failure (3); cellulitis or soft tissue necrosis (3); and nausea, vomiting, or hypoxia (2 patients each). SAEs seen in more than one patient included pneumonia (6 patients, including fungal pneumonia); febrile neutropenia (4); sepsis (3) and cellulitis or soft tissue necrosis (3). Grade 3 abdominal soft tissue necrosis occurred in the area of subcutaneous injections of 5-AZA, and two events of Grade 3 cellulitis were judged related to treatment. These local injection site reactions were more severe than expected with SC 5-AZA, and were not observed with subsequent IV administration. These events were not considered DLTs. Treatment-related suppression of NF-k B activity was evident in circulating blast cells from patients in Cohort 1 suggesting pharmacodynamic activity of birinapant at this dose and schedule. Based on this, the Phase 2 dosing regimen has been recommended as birinapant administered at 13 mg/m2 IV twice weekly for 3 weeks of a 28-day cycle in combination with 5-AZA at 75 mg/m2 IV daily for 7 days of a 28-day cycle. Although not a primary endpoint, evidence of clinical activity, as demonstrated by significant reduction in marrow blast percentages, was seen, including 3 patients considered relapsed or refractory to prior therapy. Nine patients of 20 evaluable (45%) demonstrated either a ≥50% decrease in marrow blast count, or a blast count of ≤5% at the end of Cycle 1 or 2. Two additional patients (10%) demonstrated hematological improvement in one or more peripheral cell lineages without transfusions. In addition, 2 patients, one of whom had previously received 5-AZA, could be bridged to stem cell transplant subsequent to response with birinapant plus 5-AZA. Several patients remain in active follow-up. Conclusion: This Phase 1b trial confirms the acceptable safety profile of the combination of birinapant administered with IV 5-AZA. Evidence of clinical activity in both 5-AZA naïve and refractory patients provide the rationale for an ongoing global randomized, blinded Phase 2 study comparing birinapant in combination with 5-AZA vs. 5-AZA alone in patients with higher-risk MDS in the front-line setting. Disclosures Wang: Immunogen: Research Funding. Rakkar:TetraLogic Pharmaceuticals: Research Funding. Minderman:TetraLogic Pharmaceuticals: Research Funding. Burns:TetraLogic Pharmaceuticals: Employment. Tibes:TetraLogic Pharmaceuticals: Research Funding.

A Phase 2 Study of Birinapant in Combination with 5-Azacitadine in Patients with Myelodysplastic Syndrome Who Are Naïve to 5-Azacitadine: A Preliminary Analysis of Phase 2a

Background: The Inhibitors of Apoptosis (IAP) proteins are a family of molecules that suppress apoptotic cell death. Expression of IAPs is dysregulated in Myelodysplastic Syndrome (MDS) and is a potential path for therapeutic intervention. SMAC (second mitochondrial-derived activator of caspases) is an IAP antagonist, resulting in caspase activation, inhibition of NF-k B and increased apoptosis. Birinapant is a potent bivalent SMAC mimetic under clinical investigation for several cancer types. Pre-clinical data support its investigation in myeloid malignancies, including MDS, and a previous phase 1b study demonstrated promising clinical activity in patients with higher-risk MDS who were naïve or had relapsed or were refractory to prior therapy. In this trial birinapant is combined with 5-azacitidine (5-AZA) in high-risk MDS patients who have not yet been treated with a hypomethylating agent.Methods: This phase 2 ongoing study of birinapant plus 5-AZA in patients with MDS or CMMoL recently completed enrollment to the unblinded portion, in which 9 patients were dosed with birinapant 13 mg/m2 IV twice weekly for 3 weeks of a 28-day cycle plus 5-AZA 75 mg/m2 by IV infusion daily for 7 days of a 28-day cycle. The primary objective of this study is to determine the objective response rate by International Working Group criteria of birinapant plus 5-AZA in patients with high-risk MDS or CMMoL who are naïve to hypomethylating agents. Secondary objectives include assessing the tolerability of this regimen, and determining the pharmacokinetics and exploratory pharmacodynamics of the combination.Results: Of the 9 patients enrolled, 6 patients had high or very high risk MDS; no patients had CMMoL. 8 patients were age ≥60 years. All patients were naïve to 5-AZA. Of the 9 patients enrolled, 6 completed up to four cycles of chemotherapy and were considered evaluable for response; three discontinued therapy prior to Cycle 4 (patient or investigator decision, or occurrence of adverse events (AEs)). Of the 6 evaluable patients, 3 had a complete response (CR), one had a marrow CR, and one had a partial response (PR). A sixth patient had a best response of stable disease at Cycle 4. Of these six patients evaluable, one (PR) was able to proceed to stem cell transplantation. The median duration of response (range) in these patients has not yet been assessed. Adverse events were consistent with the disease under investigation or the known profile of birinapant or 5-AZA. The most common ≥Grade 3 AEs seen in ≥2 patients were neutropenia and thrombocytopenia (5 patients each); fatigue, anemia, increased lipase and syncope (2 patients each). One patient developed a Grade 1 VIth cranial nerve palsy. No serious adverse events were reported in more than one patient, and one fatal event (sepsis) was reported. No patients developed infusion site reaction. Data showing inhibition of NF-k B, a downstream pro-survival molecule activated by IAPs, in circulating blast cells, demonstrated evidence of a potential biomarker in this population.Conclusion: This ongoing phase 2 study provide data for an acceptable safety profile of the combination of birinapant administered with IV 5-AZA. Preliminary evidence of promising clinical activity in this non-randomized, open-label phase was observed. These results provide rationale for the continuation to the next phase of this global randomized, blinded phase 2 study comparing birinapant in combination with 5-AZA vs. 5-AZA alone in patients with high-risk MDS or CMMoL in the front-line setting. DisclosuresRitchie:Celgene: Speakers Bureau; Incyte: Speakers Bureau. Skolnik:TetraLogic Pharmaceuticals: Employment, Equity Ownership.

Targeting the X-Linked Inhibitor of Apoptosis Protein (XIAP) Can Promote Tumor Cell Death, and Increase the Cytotoxic Effects of Chemotherapy Agents in in Vitro and In Vivo Models of Rituximab-Resistant Lymphoma

In diffuse large B-cell lymphoma (DLBCL) the addition of rituximab to front line multi-agent therapy has changed the biology and clinical outcome of patients with relapsed/refractory disease. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines, which also display significant resistance to a wide range of chemotherapy agents. These therapy resistant cell lines (TRCL)s have multiple deregulations the apoptotic response pathway, including a loss of Bak and Bax. We previously demonstrated that small molecule mimetics of the second mitochondrial activator of caspases (SMAC), which antagonizes the inhibitor of apoptosis (IAP) family of proteins, could boost response to chemotherapy agents and extend survival in TRCL xenograft animal models. To investigate how SMAC mimetics overcome chemotherapy resistance in TRCL (RL-4RH and Raji-4RH) models we used RNA interference to transiently knockdown expression of individual IAPs (cIAP1, cIAP2, livin, survivin, and XIAP), and then exposed those cells to a panel of chemotherapy agents. Apoptosis induction was analyzed by flow cytometry staining for annexin V and Sytox blue (a DNA stain). While SMAC mimetics can decrease levels of the cellular inhibitor of apoptosis proteins (cIAP1/cIAP2), siRNA knockdown of cIAP1/cIAP2 did not increase chemotherapy-induced apoptosis. On the other hand, siRNA knockdown of XIAP in TRCLs substantially increased rates of apoptosis compared to cells transfected with non-targeting siRNA (5% in controls vs. 22% in XIAP knockdown RL-4RH cells). An additional 24% of XIAP knockdown RL-4RH cells showed signs of necrosis compared to 0.5% in control RL-4RH. Results were confirmed by western blot (PARP cleavage). In addition, siRNA XIAP knockdown re-sensitized TRCLs (Raji-4RH and RL-4H) to the cytotoxic effects of multiple chemotherapy agents (gemcitabine, etoposide, and vincristine). To investigate if XIAP knockdown could improve chemotherapy response in vivo we created a TRCL (Raji-4RH) that stably expresses XIAP targeting shRNA along with luciferase to enable in vivo imaging. SCID mice were inoculated with either the Raji-4RH XIAP knockdown or Raji-4RH non-targeting shRNA control cell line (10x106 cells per animal given i.v.). Tumors were allowed to engraft for 7 days prior to treatment administration. Animal were either given no treatment, or rituximab in combination with ifosfamide, carboplatin, etoposide (RICE) and mesna. Following treatment animals were imaged on a weekly basis to track tumor progression. At the time of the submission disease progression was present in animals in the observation groups, and in animals inoculated with non-targeting control shRNA Raji-4RH treated with RICE. InterestingHowever, animals inoculated with XIAP knockdown Raji-4RH and treated with RICE therapy remain tumor free with no detectable disease. In summary, XIAP acts as a key cell survival regulator in rituximab-chemotherapy resistant lymphoma models. Downregulation of XIAP can enhance chemotherapy activity and promote tumor cell death. Additionally, XIAP knockdown appears to enhance the activity of RICE, a commonly used regiment in the relapsed/refractory setting for DLBCL patients. Our results support the hypothesis that selective XIAP inhibitors, currently in development, may be highly active against relapsed/refractory B-cell lymphoma. DisclosuresCzuczman:Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board; MorphoSys: Consultancy; Celgene: Employment.

PI3K-independent mTOR activation promotes lapatinib resistance and IAP expression that can be effectively reversed by mTOR and Hsp90 inhibition

Although HER2 targeted therapies have substantially improved outcomes in HER2 overexpressing (HER2+) breast cancer, resistance to these therapies remains a clinical challenge. To better understand the mechanisms of resistance to lapatinib, a HER2 and EGFR dual kinase inhibitor, we treated HER2+ breast cancer cells with lapatinib for an extended period to generate a lapatinib-resistant (LapR) cell line model and examined cancer-promoting signaling activation in LapR cells. We found that LapR cells possess enhanced mTOR activation, which was independent of PI3K and other known mTOR activators. Lapatinib resistance could be reversed by mTOR kinase inhibition. Intriguingly, LapR cells had constitutive cytosolic cytochrome C, indicating that LapR cells suppress lapatinib-induced apoptosis downstream of cytochrome C release from mitochondria into the cytosol rather than by preventing its release into the cytosol. Consistent with this notion, LapR cells possessed increased levels of 2 of the inhibitors of apoptosis (IAPs), survivin and c-IAP-2, which are reported to block caspase activation downstream of cytosolic cytochrome C release. Further, treatment with the mTOR kinase inhibitor AZD8055 or the Hsp90 inhibitor 17-AAG reversed expression of IAPs and overcame lapatinib resistance in LapR cells. Together, these data suggest that suppression of apoptosis downstream of cytosolic cytochrome C release, possibly through increased expression of IAPs or other caspase-suppressing proteins, may promote lapatinib resistance. Further, PI3K is thought to be the main driver of lapatinib resistance, but our findings indicate that PI3K inhibitors may be ineffective in some lapatinib-resistant HER2+ breast cancers with PI3K-independent activation of mTOR kinase, which may instead benefit from mTOR or Hsp90 inhibitors.

A Phase 1b/2a Study of Birinapant in Combination with 5-Azacitadine in Patients with Myelodysplastic Syndrome Who Are Naïve, Refractory to or Have Relapsed on 5-Azacitadine: a Preliminary Analysis

▪5-azacitidine (5-AZA) is standard of care in the first-line setting for higher-risk patients with Myelodysplastic Syndrome (MDS). However, 40-50% of patients are refractory to 5-AZA and most responders ultimately demonstrate disease progression within 2 years, often progressing to AML. For patients refractory to 5-AZA, 2-year survival probability is low (15%). Thus, there is a need for more effective initial and second-line therapies for patients with higher-risk MDS. The Inhibitors of Apoptosis (IAP) Proteins are a family of molecules that suppress apoptotic cell death. Expression of IAPs is dysregulated in MDS, suggesting a potential path for therapeutic intervention. SMAC (second mitochondrial activator of caspases) is an IAP antagonist, resulting in caspase activation, inhibition of NF-kB and increased apoptosis. Birinapant is a potent bivalent SMAC mimetic with improved tolerability and therapeutic index compared to other SMAC mimetics. This Phase Ib/2a study was designed primarily to determine the MTD and recommended Phase 2 dose and schedule of birinapant in combination with 5-AZA in patients with higher-risk MDS who are naïve or have relapsed or are refractory to 5-AZA.To date, 11 patients have been enrolled in the Phase 1b portion of the study, including 7 with IPSS classification high-risk MDS (5 male, 2 female; median age 75, range 64-82) and 4 with intermediate-risk MDS (2 male, 2 female; median age 72.5, range 65-84). All patients received standard 5-AZA, administered via either intravenous (IV) or subcutaneous (SC) routes, at 75 mg/m2 for 7 days of a 28-day cycle in combination with birinapant. Birinapant was administered IV twice weekly (Days 1 and 4) for either 3 of 4 weeks or 4 of 4 weeks per 28-day cycle at a dose of 13 mg/m2.No cycle 1 dose-limiting toxicities have been observed. Adverse events ≥ Grade 2 observed in two or more patients and judged to be related to treatment were rash/pruritis (Grade 2, 2 patients), thrombocytopenia (Grade 4, 2 patients) and neutropenia (Grade 4, 1 patient; Grade 3, 1 patient). Two SAEs, Grade 3 abdominal soft tissue necrosis with abdominal pain in the area of subcutaneous injections of 5-AZA, and Grade 3 cellulitis were judged to be related to treatment. These local injection site reactions/cellulitis were more severe than usually expected with SC 5-AZA, suggesting an on target pharmacodynamic (TNF mediated) localized synergistic effect in skin, but were not observed with IV administration. Both occurred after cycle 1 and were not considered DLTs. Data showing inhibition of NF-kB, a downstream pro-survival molecule activated by IAPs, in circulating blast cells from patients treated with birinapant at a dose of 13 mg/m2 twice weekly for 3 weeks out of 4, suggests that birinapant is pharmacologically active at this dosing schedule. Based on these and other pharmacodynamic data, despite not determining the formal MTD, the Phase 2 dosing regimen has been established as birinapant administered at 13 mg/m2 IV twice weekly for 3 weeks of a 28-day cycle in combination with 5-AZA at 75 mg/m2 by IV infusion for 7 days of a 28-day cycle.Although not a primary endpoint, evidence of clinical activity in 5-AZA resistant/refractory patients was observed in 2 of 11 patients to date, with an additional good clinical response in a 5-AZA naive high-risk MDS/CMML patient. One patient with prior 5-AZA-refractory MDS (cytogenetics: hypodiploid and +11q) showed bone marrow (BM) blast count reduction from 25% to 2% after 1 cycle of 5-AZA/birinapant treatment. Another patient with MDS (cytogenetics: +X; +19; +21) refractory to single-agent 5-AZA showed BM blast count reduction from 21% to 7% after 2 cycles. A third patient with treatment-naïve MDS/CMML-2 (cytogenetics: 46, XY, inv(6)(p11.2q15), t(6;21)(q21;q22) ) who had received prior hydroxyurea demonstrated BM blast count reduction from 17% to 2% after 3 cycles and underwent hematopoietic stem cell transplant. Several patients remain on study and are in active follow-up.This Phase 1b trial provide data for an acceptable safety profile of the first combination of a SMAC mimetic (birinapant) administered with IV 5-AZA. Early signs of clinical activity including in 5-AZA naïve and refractory patients were observed. These results provide rationale for a randomized Phase 2 study comparing birinapant in combination with 5-AZA against 5-AZA alone in the first-line setting for patients with higher-risk MDS. DisclosuresBorthakur:Tetralogic Pharmaceuticals: Research Funding. Foran:TetraLogic Pharmaceuticals: Research Funding. Wang:Tetralogic Pharmaceuticals: Research Funding. Rakkar:Tetralogic Pharmaceuticals: Research Funding. Hager:Tetralogic Pharmaceuticals: Research Funding. Frey:Tetralogic Pharmaceuticals: Research Funding. Andreeff:TetraLogic Pharmaceuticals: Research Funding. Carter:Tetralogic Pharmaceuticals: Research Funding. Minderman:Tetralogic Pharmaceuticals: Research Funding. Russell:Tetralogic Pharmaceuticals: Employment. Tibes:Tetralogic Pharmaceuticals: Research Funding.

A novel antagonist to the inhibitors of apoptosis (IAPs) potentiates cell death in EGFR-overexpressing non-small-cell lung cancer cells.

In the effort to develop an efficient chemotherapy drug for the treatment of non-small-cell lung cancer (NSCLC), we analyzed the anti-tumorigenic effects of a novel small molecule targeting the inhibitor of apoptosis (IAPs), HM90822B, on NSCLC cells. HM90822B efficiently decreased IAP expression, especially that of XIAP and survivin, in several NSCLC cells. Interestingly, cells overexpressing epidermal growth factor receptor (EGFR) due to the mutations were more sensitive to HM90822B, undergoing cell cycle arrest and apoptosis when treated. In xenograft experiments, inoculated EGFR-overexpressing NSCLC cells showed tumor regression when treated with the inhibitor, demonstrating the chemotherapeutic potential of this agent. Mechanistically, decreased levels of EGFR, Akt and phospho-MAPKs were observed in inhibitor-treated PC-9 cells on phosphorylation array and western blotting analysis, indicating that the reagent inhibited cell growth by preventing critical cell survival signaling pathways. In addition, gene-specific knockdown studies against XIAP and/or EGFR further uncovered the involvement of Akt and MAPK pathways in HM90822B-mediated downregulation of NSCLC cell growth. Together, these results support that HM90822B is a promising candidate to be developed as lung tumor chemotherapeutics by targeting oncogenic activities of IAP together with inhibiting cell survival signaling pathways.

Abstract 335: Aspirin, di-aspirin and their toxicity to colorectal cancer

Abstract Introduction The lifetime risk for developing colorectal cancer (CRC) is approximately 7%. In the USA over 140,000 people a year develop CRC, with approximately 51,000 deaths/year. Epidemiological studies indicate that long-term aspirin usage reduces the incidence of CRC, and may be protective against other non-CRC related adenocarcinoma, including esophageal cancer. We have investigated the mechanism by which aspirin exhibits toxicity to CRC by synthesising novel analogues of aspirin in an effort to identify functionally significant moieties. We previously suggested that “di-aspirin” (bis-carboxyphenol succinate, BCS) and analogues thereof retain relatively specific toxicity to colorectal cancer cells cultured in vitro. The cellular functions of the Inhibitors of Apoptosis (IAPs) correlate with suppression of apoptotic cell death, tumor progression and metastases. Aspirin triggered apoptosis correlates with its capacity to decrease expression of Survivin, a member of the IAP family. Survivin exhibits dual functions; as an inhibitor of apoptosis and regulator of mitosis. We hypothesised that studying the alterations in the expression of IAPs would give an insight into the cytotoxic nature of the compounds. Experimental procedure A cell viability assay (MTT) was employed to investigate the cytotoxicity of the analogues in various CRC (SW480, HCT116 and LoVo) and non-CRC cell lines (A549, MCF7 and EA.hy926). Expression levels of IAPs (c-IAP1, cIAP2, XIAP, Livin and Survivin) were examined in the SW480 cell line by immunoblotting. Survivin expression was further examined by immunofluorescence analysis. Changes in Survivin expression in the CRC and non-CRC cell lines was also determined by immunoblotting. Summary of data Herein, we show that the “di-aspirin” analogues are relatively more toxic to colorectal cancer cells compared to non-colorectal cancer cell lines. In addition we suggest that these analogues can potently reduce the expression of Survivin in comparison to other IAPs. Conclusion We conclude that the analogues of aspirin used in the study promote cell death in CRC and non-CRC cell lines. We propose that the compounds induce apoptosis by decreasing the expression of Survivin. We also propose that the anti-proliferative activity of “di-aspirins” and their analogues have potential benefit in tackling human cancer. Citation Format: Chandra S. Kankipati, Christopher J. Perry, Iain D. Nicholl. Aspirin, di-aspirin and their toxicity to colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 335. doi:10.1158/1538-7445.AM2014-335

X-chromosome-linked inhibitor of apoptosis as a key factor for chemoresistance in clear cell carcinoma of the ovary.

Background:X-chromosome-linked inhibitor of apoptosis (XIAP) is one of the anti-apoptotic proteins leading to chemoresistance in several cancers. The aim of this study is to evaluate the impact of XIAP expression upon ovarian clear cell carcinoma (CCC) that has a platinum-resistant phenotype.Methods:Tissue microarrays made from 90 CCC patients were analysed for immunohistochemical expression levels of XIAP, c-Met, p-Akt and Bcl-XL. In addition, CCC cell lines were evaluated whether XIAP silencing could modulate sensitivity to platinum agent in vitro.Results:High XIAP expression was observed in 30 (33%) of 90 CCC cases, and was associated with c-Met (<0.01) and Bcl-XL (<0.01) expression. Cases with high XIAP expression had lower response rate to primary platinum-based chemotherapy (10% vs 65%, P=0.02). In stages II–IV tumours, high XIAP expression was related with worse progression-free survival (PFS, P=0.02). Furthermore, high XIAP expression was identified as an independent worse prognostic factor for PFS and overall survival. Finally, downregulation of XIAP using XIAP-specific small interfering RNA increased sensitivity to cisplatin in human cancer cells derived from CCC.Conclusions:X-chromosome-linked inhibitor of apoptosis expression was correlated with chemoresistance of primary chemotherapy, and identified as a prognostic marker for CCC. X-chromosome-linked inhibitor of apoptosis could be a candidate for new therapeutic target in CCC.

Evodiamine synergizes with doxorubicin in the treatment of chemoresistant human breast cancer without inhibiting P-glycoprotein.

Drug resistance is one of the main hurdles for the successful treatment of breast cancer. The synchronous targeting of apoptosis resistance and survival signal transduction pathways may be a promising approach to overcome drug resistance. In this study, we determined that evodiamine (EVO), a major constituent of the Chinese herbal medicine Evodiae Fructus, could induce apoptosis of doxorubicin (DOX)-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells in a caspase-dependent manner, as confirmed by significant increases of cleaved poly(ADP-ribose) polymerase (PARP), caspase-7/9, and caspase activities. Notably, the reversed phenomenon of apoptosis resistance by EVO might be attributed to its ability to inhibit the Ras/MEK/ERK pathway and the expression of inhibitors of apoptosis (IAPs). Furthermore, our results indicated that EVO enhanced the apoptotic action of DOX by inhibiting the Ras/MEK/ERK cascade and the expression of IAPs without inhibiting the expression and activity of P-glycoprotein (P-gp). Taken together, our data indicate that EVO, a natural product, may be useful applied alone or in combination with DOX for the treatment of resistant breast cancer.

IAP proteins as targets for drug development in oncology

The inhibitors of apoptosis (IAPs) constitute a family of proteins involved in the regulation of various cellular processes, including cell death, immune and inflammatory responses, cell proliferation, cell differentiation, and cell motility. There is accumulating evidence supporting IAP-targeting in tumors: IAPs regulate various cellular processes that contribute to tumor development, such as cell death, cell proliferation, and cell migration; their expression is increased in a number of human tumor samples, and IAP overexpression has been correlated with tumor growth, and poor prognosis or low response to treatment; and IAP expression can be rapidly induced in response to chemotherapy or radiotherapy because of the presence of an internal ribosome entry site (IRES)-dependent mechanism of translation initiation, which could contribute to resistance to antitumor therapy. The development of IAP antagonists is an important challenge and was subject to intense research over the past decade. Six molecules are currently in clinical trials. This review focuses on the role of IAPs in tumors and the development of IAP-targeting molecules for anticancer therapy.

Abstract SY06-01: Targeting IAP proteins for the treatment of cancer.

Abstract Evasion of cell death is a characteristic feature of human cancers and represents a key cause of resistance to current treatment approaches. Therefore, reactivation of cell death programs in cancer cells is a promising strategy to overcome treatment resistance, one of the major unsolved problems in clinical oncology. Inhibitor of apoptosis (IAP) protein family members block cell death in response to diverse stimuli and are expressed at elevated levels in the majority of human malignancies, which makes them attractive targets for developing a novel class of cancer therapeutics. We have designed small-molecule IAP antagonists (smac mimetics) that bind with high affinities to select baculovirus IAP repeat (BIR) domains of IAPs resulting in a dramatic induction of c IAP auto-ubiquitination activity and rapid proteasomal degradation. IAP antagonists inhibit tumor growth in vivo as single agents and in combination with a number of anti-tumor agents. Using several different proteomic approaches we have discovered novel binding partners for IAP proteins and determined the spatial and temporal pattern of endogenous IAP-mediated ubiquitination during proliferative, apoptotic and necrotic signaling. The differential expression levels and posttranslational modifications of identified proteins will be used to build diagnostic profiles for the treatment with IAP antagonists. These compounds can be used in the treatment of malignancies in which IAP expression contributes to tumor progression and resistance to conventional chemotherapeutic agents. Citation Format: Domagoj Vucic. Targeting IAP proteins for the treatment of cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY06-01. doi:10.1158/1538-7445.AM2013-SY06-01

Cell death and survival mechanisms are concomitantly active in the hippocampus of patients with mesial temporal sclerosis

Mesial temporal lobe epilepsy (MTLE) is often characterized pathologically by severe neuronal loss in the hippocampus. In this study we investigated concomitant appearance of the pro-apoptotic and anti-apoptotic mechanisms in injured neurons in epileptic human hippocampi. Postsurgical hippocampal specimens of randomly selected 25 patients with MTLE were studied with standard immunohistochemical techniques to detect the below markers of cell death pathways: truncated Bid – tBid, mitochondrial translocation of Bax (markers of pro-apoptotic Bcl-2 protein activation) and nuclear translocation of AIF (caspase-independent pro-apoptotic pathway). For cell survival pathways, we investigated the expression of c-IAP1, c-IAP2 and Hsp70 (heat shock protein). Immunopositive cells were counted in different regions of the hippocampus. We also verified IAP (inhibitor of apoptosis) expression with Western blotting. The results were statistically compared with hippocampi from non-epileptic autopsy controls. In patient hippocampi, Bax and tBid immunoreactivity were significantly increased and Bax staining was consistent with mitochondrial translocation. AIF was not translocated to the nucleus. c-IAP1 and c-IAP2 were barely detectable in control hippocampi, whereas their expression was dramatically increased in the patients in all hippocampal subfields. Interestingly, these neurons were also positively co-labeled for tBid and translocated Bax. Hsp70 immunreactivity was significantly increased in all surviving neurons in patient hippocampi whereas degenerating neurons failed to express Hsp70. Our findings are consistent with both pro-apoptotic and anti-apoptotic mechanisms being active within the same hippocampal neurons of patients with MTLE, illustrating an ongoing struggle between cell death and survival mechanisms in neurons under stress.

Expression of the IAP protein family acts cooperatively to predict prognosis in human bladder cancer patients

The inhibitors of apoptosis (IAPs) are a group of anti-apoptotic factors in the apoptotic pathway that render cancer cells insensitive to apoptotic stimulation. Recently, several members of the IAP family have been investigated in the context of bladder cancer, and some of these have been associated with specific clinical and pathological tumor features, and with prognosis. These data suggested that the expression of an individual nuclear IAP has an important relationship with the progression of bladder cancer. To date, there are no studies concerning the overall tendencies of IAPs and their comparative therapeutic values in bladder cancer. In this study, we investigated the overall expression trends of the five tumor-related proteins, Survivin, cIAP1, cIAP2, XIAP and Livin, in normal bladder tissues and bladder cancer tissues. We classified and compared the gene expression data of these IAPs with the corresponding clinical and pathological tumor features, and with prognosis, in the development and progression of bladder cancer. The differences in IAP expression levels between archival bladder specimens from 36 normal controls and 105 patients who underwent surgery at our facility were examined using western blot analysis. The localization and expression level of each protein in low- and high-grade bladder cancer tissues were examined through immunohistochemistry. The cytoplasmic expression levels of each protein were scored as 0 (negative), +1 (weak), +2 (medium) or +3 (strong). The nuclear expression levels of cIAP1 and Survivin were scored as 0 (0%), +1 (1–25%), +2 (26–50%) or +3 (>50%). The results demonstrated that the expression of IAPs acted cooperatively to predict prognosis in human bladder cancer patients.