Olga Tayber

Human CARD4 Protein Is a Novel CED-4/Apaf-1 Cell Death Family Member That Activates NF-κB

The nematode CED-4 protein and its human homolog Apaf-1 play a central role in apoptosis by functioning as direct activators of death-inducing caspases. A novel human CED-4/Apaf-1 family member called CARD4 was identified that has a domain structure strikingly similar to the cytoplasmic, receptor-like proteins that mediate disease resistance in plants. CARD4 interacted with the serine-threonine kinase RICK and potently induced NF-κB activity through TRAF-6 and NIK signaling molecules. In addition, coexpression of CARD4 augmented caspase-9-induced apoptosis. Thus, CARD4 coordinates downstream NF-κB and apoptotic signaling pathways and may be a component of the host innate immune response.

Identification and Characterization of the Mouse Obesity Gene tubby: A Member of a Novel Gene Family

The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.

Novel mutations and a mutational hotspot in the MODY3 gene

Maturity-onset diabetes of the young 3 (MODY3) is a type of NIDDM caused by mutations in the transcription factor hepatocyte nuclear factor-1a (HNF-1α) located on chromosome 12q. We have identified four novel HNF-1α missense mutations in M0DY3 families. In four additional and unrelated families, we observed an identical insertion mutation that had occurred in a polycytidine tract in exon 4. Among those families, one exhibited a de novo mutation at this location. We propose that instability of this sequence represents a general mutational mechanism in M0DY3. We observed no HNF-1α mutations among 86 unrelated late-onset diabetic patients with relative insulin deficiency. Hence mutations in this gene appear to be most strongly associated with early-onset diabetes.

Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies.

Purpose: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). Experimental Design: Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease. Results: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. Conclusions: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. Clin Cancer Res; 17(23); 7313–23. ©2011 AACR.

Novel DNA Damage Checkpoints Mediating Cell Death Induced by the NEDD8-Activating Enzyme Inhibitor MLN4924

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

Sequence analysis of β-subunit genes of the 20S proteasome in patients with relapsed multiple myeloma treated with bortezomib or dexamethasone

Variations within proteasome β (PSMB) genes, which encode the β subunits of the 20S proteasome, may affect proteasome function, assembly, and/or binding of proteasome inhibitors. To investigate the potential association between PSMB gene variants and treatment-emergent resistance to bortezomib and/or long-term outcomes, in the present study, PSMB gene sequence variation was characterized in tumor DNA samples from patients who participated in the phase 3 Assessment of Proteasome Inhibition for Extending Remissions (APEX) study of bortezomib versus high-dose dexamethasone for treatment of relapsed multiple myeloma. Twelve new PSMB variants were identified. No associations were found between PSMB single nucleotide polymorphism genotype frequency and clinical response to bortezomib or dexamethasone treatment or between PSMB single nucleotide polymorphism allelic frequency and pooled overall survival or time to progression. Although specific PSMB5 variants have been identified previously in preclinical models of bortezomib resistance, these variants were not detected in patient tumor samples collected after clinical relapse from bortezomib, which suggests that alternative mechanisms underlie bortezomib insensitivity.

Analysis of two pharmacodynamic biomarkers using acoustic micro magnetic particles on the ViBE bioanalyzer.

Pharmacodynamic responses to drug treatment are often used to confirm drug-on-target biological responses. Methods ranging from mass spectrometry to immunohistochemistry exist for such analyses. By far, the most extensively used methodologies employ antigen-specific antibodies for detection (at a minimum) and, in some cases, target quantitation as well. Using a novel frequency-modulating technology from BioScale called acoustic micro magnetic particle (AMMP) detection, two pathway biomarkers were chosen for pharmacodynamic analysis and compared with either AlphaScreen or LI-COR Western blot assays. For these studies, pharmacodynamic biomarkers for both proteasome and phosphoinositol 3-kinase inhibition were used. Our results show clearly that the BioScale technology is a robust and rapid method for measuring recombinant standards or endogenously derived proteins from both tissue culture and mouse xenograft tumor lysates. Moreover, the sensitivity obtained with the BioScale platform compares favorably with LI-COR Western blot and AlphaScreen technologies. Furthermore, the use of the ViBE Bioanalyzer eliminates the labor-intensive effort of Western blot analysis and is devoid of the optical and other endogenous interfering substances derived from lysates of xenograft tumors typically observed with AlphaScreen.

Determination of complementary antibody pairs using protein A capture with the AlphaScreen assay format.

The utility of antibody reagents for the detection of specific cellular targets for both research and diagnostic applications is widespread and continually expanding. Often it is useful to develop specific antibodies as reagent pairs that distinguish different epitopes of the target such that sandwich enzyme-linked immunosorbent assay can be used for selective and specific detection. However, the identification of pairing antibodies is often cumbersome and labor-intensive even with the use of designed peptide-specific epitopes as antigens. We have developed a robust and high-throughput method for identifying pairing complementary antibodies derived either from commercial sources or during a rabbit hybridoma monoclonal screening and selection process using protein A capture with the AlphaScreen bead-based assay format. We demonstrate the value and effectiveness of this assay with three protein targets: Akt2, ATF3, and NAEβ (the β-subunit of the neddylation activation enzyme).

Abstract 3104: Genome-wide screen for modulators of cell death induced by the NEDD8-activating enzyme inhibitor MLN4924

The NEDD8-activating enzyme (NAE) is an E1 involved in the activation of a large family of ubiquitin E3 ligases termed the cullin-RING ligases (CRLs) through conjugation of the cullin proteins with the ubiquitin-like modifier NEDD8. Polyubiquitination of CRL substrate proteins targets them for degradation by the proteasome. In this way, NAE regulates the stability of proteins required for cancer cell growth and survival. MLN4924 is an investigational small molecule that is a potent and selective inhibitor of NAE in Phase I clinical trials. In order to investigate the primary genetic determinants that confer sensitivity of cells to NAE inhibition, we have performed a genome-wide synthetic lethal RNAi screen using MLN4924 in the A375 melanoma cell line. We have also investigated the biological consequences of NAE inhibition by studying the regulation of protein and transcript levels in MLN4924-treated A375 cells using large-scale quantitative proteomics and gene expression profiling, respectively. The RNAi screen has identified 123 genes whose down-regulation modulates MLN4924-induced cell death, and approximately one-third of these interfere with components of the NEDD8 pathway itself, the cell cycle and apoptotic machinery, and DNA damage-response pathways. Of these genes, 99 were subsequently assessed using high throughput FACS analysis for their contribution to the major phenotype induced by MLN4924. The results emphasize replication, p53, BRCA1/BRCA2, and transcription-coupled repair as being particularly important for MLN4924-induced cell death. In addition, the interactions of 80 genes with roles in cell cycle and DNA damage repair were explored with proteasome inhibitors and 12 DNA damaging agents, demonstrating that MLN4924 induces DNA damage by a distinct mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3104. doi:1538-7445.AM2012-3104

Abstract B5: Sequence analysis of HSP90-encoding genes in breast tumor tissue identifies a novel 9 bp deletion in HSP90AB1

The 90 kDa heat-shock protein (HSP90) functions as a ubiquitous cellular chaperone in cells, and is required for the function and stability of many client proteins such as ERBB2, EGFR, BCR-ABL, MET, androgen and estrogen receptors, BRAF and HIF-1α. 1 There are four primary genes that encode for HSP90 in humans: HSP90AA1 and HSP90AB1 (cytosolic), HSP90B1 (endoplasmic reticulum) and TRAP1 (mitochondrial). Increased HSP90 expression has been associated with poor prognosis in cancer, and inhibiting HSP90 can lead to degradation of many oncogenic client proteins via the proteasome pathway. 1 HSP90 has been the subject of intense drug discovery activities, with several therapeutics under clinical investigation. 2 However, the data on single nucleotide polymorphisms (SNPs) and potential somatic mutations within the HSP90-encoding genes in tumor tissue is limited. Target allelic variation can alter drug binding and therapeutic response, as demonstrated by Iressa™9s reported specific efficacy in EGFR-mutant non-small cell lung cancer patients. 3 Therefore, it is critical to understand target allelic variance as part of a drug development program. To facilitate drug development efforts around HSP90, we investigated allelic variation within the coding regions of HSP90AA1 (second isoform) and HSP90AB1 genes in 49 formalin-fixed paraffin- embedded human breast tumor samples. Sample SNP data were compared to control data from the NCBI Entrez SNP database. We observed four synonymous changes within HSP90AA1, and two synonymous changes within HSP90AB1. Intriguingly, we also observed a heterozygous 9-base deletion in exon 1 of HSP90AB1 within one breast tumor patient sample. This deletion results in an alanine>valine substitution at amino acid 22 and a loss of amino acid 23-25 (glutamine, leucine, methionine). The deleted region includes one turn of a 3-turn alpha helix, located within the N-terminal domain structure of the HSP90AB1 protein. Full details of HSP90 allelic variance within breast tumor tissue, and a discussion of the potential functional significance of the deletion, will be provided. References: 1. Pearl LH, Prodromou C, Workman P. The HSP90 molecular chaperone: an open and shut case for treatment. Biochem J. 2008;410:439-53. 2. Hahn JS. The HSP90 chaperone machinery: From structure to drug development. BMB Reports. 2009;42(10):623-30. 3. Sequist LV, Bell DW, Lynch TJ, Haber DA. Molecular predictors of response to epidermal growth factor receptor antagonists in non- small-cell lung cancer. J Clin Oncol. 2007;25(5):587-95. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B5.