Leutz Buon

Interactions of the Hdm2/p53 and Proteasome Pathways May Enhance the Antitumor Activity of Bortezomib

Purpose: p53 is inactivated in many human malignancies through missense mutations or overexpression of the human homologue of Mdm2 (Hdm2), an E3 ubiquitin ligase that ubiquitinates p53, thereby promoting its proteasomal degradation. The cis-imidazoline nutlin-3 can disrupt the p53-Hdm2 interaction and activate p53, inducing apoptosis in vitro in many malignancies, including multiple myeloma (MM). Experimental Design: We hypothesized that suppression of Hdm2-mediated p53 ubiquitination may augment sequelae of p53 accumulation caused by proteasomal inhibition. We compared the response of MM cells versus several epithelial cancer models to the proteasome inhibitor bortezomib in combination with nutlin-3. Results: The combination of sublethal concentrations of bortezomib plus nutlin-3 induced additive cytotoxicity against bortezomib-sensitive MM cell lines. Importantly, however, in breast, prostate, colon, and thyroid (papillary, follicular, anaplastic, and medullary) carcinoma cell lines, this combination triggered synergistic cytotoxicity, and increased expression of p53, p21, Hdm2, Bax, Noxa, PUMA, and cleavage of caspase-3 and poly ADP ribose polymerase. Coculture with bone marrow stromal cells attenuated MM cell sensitivity to nutlin-3 monotherapy and was associated with evidence of suppression of p53 activity in MM cells, whereas combined bortezomib-nutlin-3 treatment maintained cytotoxicity even in the presence of bone marrow stromal cells. Conclusions: This differential response of MM versus epithelial carcinomas to combination of nutlin-3 with bortezomib sheds new light on the role of p53 in bortezomib-induced apoptosis. Concurrent Hdm2 inhibition with bortezomib may extend the spectrum of bortezomib applications to malignancies with currently limited sensitivity to single-agent bortezomib or, in the future, to MM patients with decreased clinical responsiveness to bortezomib-based therapy. (Clin Cancer Res 2009;15(23):7153–60)

Molecular and Cellular Effects of NEDD8-Activating Enzyme Inhibition in Myeloma

The NEDD8-activating enzyme is upstream of the 20S proteasome in the ubiquitin/proteasome pathway and catalyzes the first step in the neddylation pathway. NEDD8 modification of cullins is required for ubiquitination of cullin-ring ligases that regulate degradation of a distinct subset of proteins. The more targeted impact of NEDD8-activating enzyme on protein degradation prompted us to study MLN4924, an investigational NEDD8-activating enzyme inhibitor, in preclinical multiple myeloma models. In vitro treatment with MLN4924 led to dose-dependent decrease of viability (EC50 = 25–150 nmol/L) in a panel of human multiple myeloma cell lines. MLN4924 was similarly active against a bortezomib-resistant ANBL-6 subline and its bortezomib-sensitive parental cells. MLN4924 had submicromolar activity (EC50 values <500 nmol/L) against primary CD138+ multiple myeloma patient cells and exhibited at least additive effect when combined with dexamethasone, doxorubicin, and bortezomib against MM.1S cells. The bortezomib-induced compensatory upregulation of transcripts for ubiquitin/proteasome was not observed with MLN4924 treatment, suggesting distinct functional roles of NEDD8-activating enzyme versus 20S proteasome. MLN4924 was well tolerated at doses up to 60 mg/kg 2× daily and significantly reduced tumor burden in both a subcutaneous and an orthotopic mouse model of multiple myeloma. These studies provide the framework for the clinical investigation of MLN4924 in multiple myeloma. Mol Cancer Ther; 11(4); 942–51. ©2012 AACR.

Genomic evolution in Barrett's adenocarcinoma cells: Critical roles of elevated hsRAD51, homologous recombination and Alu sequences in the genome

A prominent feature of most cancers including Barretts adenocarcinoma (BAC) is genetic instability, which is associated with development and progression of disease. In this study, we investigated the role of recombinase (hsRAD51), a key component of homologous recombination (HR)/repair, in evolving genomic changes and growth of BAC cells. We show that the expression of RAD51 is elevated in BAC cell lines and tissue specimens, relative to normal cells. HR activity is also elevated and significantly correlates with RAD51 expression in BAC cells. The suppression of RAD51 expression, by short hairpin RNA (shRNA) specifically targeting this gene, significantly prevented BAC cells from acquiring genomic changes to either copy number or heterozygosity (P<0.02) in several independent experiments employing single-nucleotide polymorphism arrays. The reduction in copy-number changes, following shRNA treatment, was confirmed by Comparative Genome Hybridization analyses of the same DNA samples. Moreover, the chromosomal distributions of mutations correlated strongly with frequencies and locations of Alu interspersed repetitive elements on individual chromosomes. We conclude that the hsRAD51 protein level is systematically elevated in BAC, contributes significantly to genomic evolution during serial propagation of these cells and correlates with disease progression. Alu sequences may serve as substrates for elevated HR during cell proliferation in vitro, as they have been reported to do during the evolution of species, and thus may provide additional targets for prevention or treatment of this disease.

Targeting homologous recombination and telomerase in Barrett's adenocarcinoma: Impact on telomere maintenance, genomic instability and tumor growth

Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in Barrett’s esophageal adenocarcinoma (BAC). The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for β-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those that resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contributes to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase renders telomeres more vulnerable to degradation and significantly increases/expedites their attrition, leading to apoptosis. We therefore demonstrate that a therapy targeting HR and telomerase has the potential to prevent both tumor growth and genomic evolution in BAC.

Molecular and cellular effects of multi-targeted cyclin-dependent kinase inhibition in myeloma: biological and clinical implications

Cell cycle regulators, such as cyclin‐dependent kinases (CDKs), are appealing targets for multiple myeloma (MM) therapy given the increased proliferative rates of tumour cells in advanced versus early stages of MM. We hypothesized that a multi‐targeted CDK inhibitor with a different spectrum of activity compared to existing CDK inhibitors could trigger distinct molecular sequelae with therapeutic implications for MM. We therefore studied the small molecule heterocyclic compound NVP‐LCQ195/AT9311 (LCQ195), which inhibits CDK1, CDK2 and CDK5, as well as CDK3 and CDK9. LCQ195 induced cell cycle arrest and eventual apoptotic cell death of MM cells, even at sub‐μmol/l concentrations, spared non‐malignant cells, and overcame the protection conferred to MM cells by stroma or cytokines of the bone marrow milieu. In MM cells, LCQ195 triggered decreased amplitude of transcriptional signatures associated with oncogenesis, drug resistance and stem cell renewal, including signatures of activation of key transcription factors for MM cells e.g. myc, HIF‐1α, IRF4. Bortezomib‐treated MM patients whose tumours had high baseline expression of genes suppressed by LCQ195 had significantly shorter progression‐free and overall survival than those with low levels of these transcripts in their MM cells. These observations provide insight into the biological relevance of multi‐targeted CDK inhibition in MM.

Impact of RAD51C-mediated homologous recombination on genomic integrity in Barrett’s adenocarcinoma cells

BACKGROUND In normal cells, RAD51-mediated homologous recombination (HR) is a precise DNA repair mechanism which plays a key role in the maintenance of genomic integrity and stability. However, elevated (dysregulated) RAD51 is implicated in genomic instability and is a potential target for treatment of certain cancers, including Barrett’s adenocarcinoma (BAC). In this study, we investigated genomic impact and translational significance of moderate vs. strong suppression of RAD51 in BAC cells. METHODS BAC cells (FLO-1 and OE33) were transduced with non-targeting control (CS) or RAD51-specific shRNAs, mediating a moderate (40–50%) suppression or strong (80-near 100%) suppression of the gene. DNA breaks, spontaneous or following exposure to DNA damaging agent, were examined by comet assay and 53BP1 staining. Gene expression was monitored by microarrays (Affymetrix). Homologous recombination (HR) and single strand annealing (SSA) activities were measured using plasmid based assays. RESULTS We show that although moderate suppression consistenly inhibits/reduces HR activity, the strong suppression is associated with increase in HR activity (by ~15 – ≥ 50% in various experiments), suggesting activation of RAD51-independent pathway. Contrary to moderate suppression, a strong suppression of RAD51 is associated with a significant induced DNA breaks as well as altered expression of genes involved in detection/processing of DNA breaks and apoptosis. Stronger RAD51 suppression was also associated with mutagenic single strand annealing mediated HR. Suppression of RAD51C inhibited RAD51-independent (SSA-mediated) HR in BAC cells. CONCLUSION Elevated (dysregulated) RAD51 in BAC is implicated in both the repair of DNA breaks as well as ongoing genomic rearrangements. Moderate suppression of this gene reduces HR activity, whereas strong or near complete suppression of this gene activates RAD51C-dependent HR involving a mechanism known as single strand annealing (SSA). SSA-mediated HR, which is a mutagenic HR pathway, further disrupts genomic integrity by increasing DNA breaks in BAC cells.

Molecular and cellular effects of multi-targeted CDK inhibition in myeloma: Biological and clinical implications

Cell cycle regulators, such as cyclin‐dependent kinases (CDKs), are appealing targets for multiple myeloma (MM) therapy given the increased proliferative rates of tumour cells in advanced versus early stages of MM. We hypothesized that a multi‐targeted CDK inhibitor with a different spectrum of activity compared to existing CDK inhibitors could trigger distinct molecular sequelae with therapeutic implications for MM. We therefore studied the small molecule heterocyclic compound NVP‐LCQ195/AT9311 (LCQ195), which inhibits CDK1, CDK2 and CDK5, as well as CDK3 and CDK9. LCQ195 induced cell cycle arrest and eventual apoptotic cell death of MM cells, even at sub‐μmol/l concentrations, spared non‐malignant cells, and overcame the protection conferred to MM cells by stroma or cytokines of the bone marrow milieu. In MM cells, LCQ195 triggered decreased amplitude of transcriptional signatures associated with oncogenesis, drug resistance and stem cell renewal, including signatures of activation of key transcription factors for MM cells e.g. myc, HIF‐1α, IRF4. Bortezomib‐treated MM patients whose tumours had high baseline expression of genes suppressed by LCQ195 had significantly shorter progression‐free and overall survival than those with low levels of these transcripts in their MM cells. These observations provide insight into the biological relevance of multi‐targeted CDK inhibition in MM.

Molecular and cellular effects of multi-targeted cyclin-dependent kinase inhibition in myeloma: biological and clinical implications: Anti-myeloma Activity of a Multi-targeted CDK Inhibitor

Cell cycle regulators, such as cyclin‐dependent kinases (CDKs), are appealing targets for multiple myeloma (MM) therapy given the increased proliferative rates of tumour cells in advanced versus early stages of MM. We hypothesized that a multi‐targeted CDK inhibitor with a different spectrum of activity compared to existing CDK inhibitors could trigger distinct molecular sequelae with therapeutic implications for MM. We therefore studied the small molecule heterocyclic compound NVP‐LCQ195/AT9311 (LCQ195), which inhibits CDK1, CDK2 and CDK5, as well as CDK3 and CDK9. LCQ195 induced cell cycle arrest and eventual apoptotic cell death of MM cells, even at sub‐μmol/l concentrations, spared non‐malignant cells, and overcame the protection conferred to MM cells by stroma or cytokines of the bone marrow milieu. In MM cells, LCQ195 triggered decreased amplitude of transcriptional signatures associated with oncogenesis, drug resistance and stem cell renewal, including signatures of activation of key transcription factors for MM cells e.g. myc, HIF‐1α, IRF4. Bortezomib‐treated MM patients whose tumours had high baseline expression of genes suppressed by LCQ195 had significantly shorter progression‐free and overall survival than those with low levels of these transcripts in their MM cells. These observations provide insight into the biological relevance of multi‐targeted CDK inhibition in MM.

T1176 Role of Recombinase (RAD51) in Ongoing Genomic Instability and Proliferation in Barrett's Adenocarcinoma Cells

activity. Exposure to 15mmHg increased extracellular pressure stimulated serine phosphorylation of FAK (p-FAK) in Caco-2 and primary human colon cancer cells isolated from surgical specimens. This pressure-induced increase in serine p-FAK was blocked by Akt inhibitor and by siRNA silencing of Akt1 but not by silencing Akt2. Co-precipitation demonstrated that Akt associates directly with FAK. Akt-FAK association was increased by pressure and this increased association was blocked by inhibiting FAK or silencing Akt1 but not Akt2. Scanning the FAK sequence with Scansite software revealed three serine-containing consensus sequences for AKT phosphorylation in the FAK sequence. We therefore constructed a FAK non-phosphorylatable mutant with point mutations (S®A) at these three putative serine phosphorylation sites (S517/601/695) of FAK by Akt to investigate their relevance to pressurestimulated cell adhesion and tyrosine p-FAK. Indeed, overexpression of the triple mutant of FAK (S517/601/695®A) in Caco-2 cells, in contrast to wild type FAK, prevented the increase in p-FAK at Y397 and cancer cell adhesion induced by extracellular pressure. These results suggest that Akt regulates pressure-induced cancer cell adhesion by binding directly to and phosphorylating FAK at S509/601/695. This serine phosphorylation, in turn, permits the pressure-dependent tyrosine autophosphorylation of p-FAK at Y397, the conventional initiator of FAK activation. Although Akt is therefore required for FAK activation in response to pressure, further studies demonstrated that FAK also potentiates Akt activation. Blocking or silencing FAK by three different FAK-specific siRNA sequences prevented the increases in serine p-Akt (S473) and tyrosine p-FAK(Y397) induced by increased pressure. Thus, FAK and Akt bind directly and potentiate each others activation. This novel mechanism of FAKAkt interaction suggests that FAK and Akt1 may be important dual therapeutic targets for preventing cancer cell adhesion, and eventually cancer metastasis.