CKS1B Expression Research Articles

Integrated bulk and single-cell RNA sequencing identifies an aneuploidy-based gene signature to predict sensitivity of lung adenocarcinoma to traditional chemotherapy drugs and patients’ prognosis

Background Patients with lung adenocarcinoma (LUAD) often develop a poor prognosis. Currently, researches on prognostic and immunotherapeutic capacity of aneuploidy-related genes in LUAD are limited. Methods Genes related to aneuploidy were screened based on bulk RNA sequencing data from public databases using Spearman method. Next, univariate Cox and Lasso regression analyses were performed to establish an aneuploidy-related riskscore (ARS) model. Results derived from bioinformatics analysis were further validated using cellular experiments. In addition, typical LUAD cells were identified by subtype clustering, followed by SCENIC and intercellular communication analyses. Finally, ESTIMATE, ssGSEA and CIBERSORT algorithms were employed to analyze the potential relationship between ARS and tumor immune environment. Results A five-gene ARS signature was developed. These genes were abnormally high-expressed in LUAD cell lines, and in particular the high expression of CKS1B promoted the proliferative, migratory and invasive phenotypes of LUAD cell lines. Low ARS group had longer overall survival time, higher degrees of inflammatory infiltration, and could benefit more from receiving immunotherapy. Patients in low ASR group responded more actively to traditional chemotherapy drugs (Erlotinib and Roscovitine). The scRNA-seq analysis annotated 17 cell subpopulations into seven cell clusters. Core transcription factors (TFs) such as CREB3L1 and CEBPD were enriched in high ARS cell group, while TFs such as BCLAF1 and UQCRB were enriched in low ARS cell group. CellChat analysis revealed that high ARS cell groups communicated with immune cells via SPP1 (ITGA4-ITGB1) and MK (MDK-NCl) signaling pathways. Conclusion In this research, integrative analysis based on the ARS model provided a potential direction for improving the diagnosis and treatment of LUAD.

Identification of CKS1B as a prognostic indicator and a predictive marker for immunotherapy in pancreatic cancer.

As a regulatory subunit of cyclin kinase, CKS1B promotes cancer development and is associated with poor prognosis in multiple cancer patients. However, the intrinsic role of CKS1B in pancreatic cancer remains elusive. In our research, CKS1B expression in pancreatic tumor tissue was higher than that in normal tissue by TCGA, Oncomine and CPTAC databases analysis. Similar result was verified in our center tissues by qRT-PCR. CKS1B expression was closely relevant to histologic grading, prognosis, and TMB. GSEA showed that CKS1B mainly participated in the regulation of autophagy and T cell receptor signaling pathway. Furthermore, CIBERSORT analysis showed that there was a strong correlation between CKS1B expression and tumor immune cells infiltration. Drug sensitivity analysis showed that patients with high CKS1B expression appeared to be more sensitive to gemcitabine, 5-fluorouracil, and paclitaxel. We then investigated cell viability and migratory ability by CCK8 and transwell assay, respectively. Results indicated that CKS1B knockdown by short hairpin RNA significantly reduced pancreatic cancer cell viability and invasion via regulating PD-L1 expression. In conclusion, our research further demonstrates the role of CKS1B in pancreatic cancer and the signaling pathways involved. The association of CKS1B with immune infiltration and immune checkpoint may provide a new direction for immunotherapy of pancreatic cancer.

Super-Enhancer Driven Regulation of CKS1B in Multiple Myeloma: Implications in Mediating Response to BET Inhibitor and Celmod Agent Combination

Introduction: The Myeloma Genome Project (MGP) characterized the genomic landscape of patients with newly diagnosed multiple myeloma (NDMM) (Walker BA, et al. Blood 2018; 132[6]:587-597). Using a multi-omics unsupervised clustering approach, 12 molecularly-defined disease segments were identified (Ortiz M, et al. Blood 2018; 132[suppl 1]:3165). Here, we performed experimental validation of CDC28 Protein Kinase Regulatory Subunit 1B (CSK1B) that was identified as a putative target from the disease segment with poorest clinical outcome. CKS1B was selected for in-depth validation due to their role in cell cycle pathways associated with high-risk disease, biological mechanisms of chromosome 1q amplification and druggability.Methods: Association of CKS1B with outcomes was analyzed in NDMM patients, across relapses and with clinical outcome datasets from MGP and Mayo clinic. Inducible shRNAs of CKS1B and bromodomain containing protein 4 (BRD4, a member of the BET [bromodomain and extra terminal domain] family) were generated in MM cell lines. BRD4 and Aiolos ChIP-seq datasets were analyzed for binding on CKS1B gene. BRD4 inhibitors JQ1 and CC-90010 were utilized for inhibition studies in MM cell lines.Results: Higher expression of CKS1B was associated with significantly poorer PFS, OS, disease severity and relapse. Knock-down of CKS1B in MM cells led to a significant decrease in proliferation (P<0.001) and enhanced apoptosis in MM cell lines. BRD4-ChIP sequencing studies revealed that the expression of CKS1B was regulated by super-enhancer (SE) associated elements. As expected, two BRD4 inhibitors, JQ1 and CC-90010 and inducible BRD4 shRNAs downregulated the expression of CKS1B resulting in decreased proliferation, cell cycle arrest and apoptosis in MM cell lines. Furthermore, MM cell lines harboring chromosome 1q gain/amp showed higher sensitivity to BRD4 inhibition compared to cell lines with normal 1q copy number. Mechanistic studies revealed that BRD4inh and BRD4 shRNAs downregulated the expression of Aiolos and Ikaros in MM cell lines. Interestingly, Aiolos ChIP-sequencing studies demonstrated the binding of Aiolos at the transcriptional start sites of CKS1B with the transcriptional activation mark. The immunomodulatory agent (IMiD ®) pomalidomide (Pom) transcriptionally downregulated CKS1B in Pom-sensitive cells downstream of Aiolos, Ikaros degradation. Based on these mechanisms, IMiD agents, lenalidomide, Pom and the novel Cereblon E3 ligase modulating degrader (CELMoD ®) agent CC-92480 in combination with BRD4inh promoted a synergistic decrease in proliferation, cell cycle arrest and increase in apoptosis in both Pom-sensitive and -resistant cell lines. The combination of IMiD or novel CELMoD agent with BRD4inh also promoted deeper downregulation of CKS1B, Aiolos, Ikaros, c-Myc and survivin proteins with enhanced levels of apoptotic marker cleaved Caspase 3 as compared to single agents alone.Conclusions: In summary, we have identified CKS1B as a key target associated with poor outcome in MM patients. Translational studies suggest a profound downregulation of CKS1B and key pro-survival effector proteins following combination treatment with BRD4inh and IMiD agents/novel CELMoD agents resulting in synergistic anti-tumor effects. These data provide rationale for testing these agents in the clinic for high-risk and IMiD-relapsed patients.Figure: Changes in cell proliferation and protein levels of key signaling mediators were studied in K12PE cell line treated with increasing doses of Lenalidomide, Pomalidomide and CC-92480 in combination with JQ1. [Display omitted] DisclosuresAhsan: BMS: Current Employment, Current equity holder in publicly-traded company. Polonskaia: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hsu: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bahlis: Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Knockdown of LINC00657 inhibits the viability, migration and invasion of pancreatic cancer cells by regulating the miR-520h/CKS1B axis.

Long non-coding RNA LINC00657 has a critical role in multiple cancers. The aim of the present study was to investigate the regulatory effect of LINC00657 in pancreatic cancer (PC) and reveal its molecular mechanism of function. The expression levels of LINC00657 and microRNA (miR)-520h were detected by reverse transcription-quantitative PCR in PC tissues and cell lines. MTT, wound healing and Transwell assays were used to detect cell viability, migration and invasion, respectively. Dual-luciferase reporter assay was utilized to examine the relationship between LINC00657 and miR-520h and that between miR-520h and cyclin-dependent kinases regulatory subunit 1 (CKS1B). Western blotting was performed to detect CKS1B expression. The expression levels of LINC00657 and CKS1B were enhanced and miR-520h expression level was reduced in PC tissues and cell lines compared with adjacent normal tissues or HPDE6 cells. LINC00657 knockdown decreased the viability, migration and invasion of PC cells. Additionally, LINC00657 targeted miR-520h and negatively modulated miR-520h expression. Furthermore, miR-520h overexpression inhibited the viability, migration and invasion of PC cells. In addition, miR-520h targeted CKS1B and reversely regulated CKS1B expression. miR-520h inhibition and CKS1B overexpression alleviated the inhibition effect of LINC00657 knockdown on the viability, migration and invasion of PACA-2 PC cells. In conclusion, the results of the present study demonstrated that LINC00657 knockdown repressed the viability, migration and invasion of PC cells via targeting the miR-520h/CKS1B axis, which may offer a future target for PC therapy.

A Synthetic Dosage Lethal Genetic Interaction Between CKS1B and PLK1 Is Conserved in Yeast and Human Cancer Cells.

The CKS1B gene located on chromosome 1q21 is frequently amplified in breast, lung, and liver cancers. CKS1B codes for a conserved regulatory subunit of cyclin–CDK complexes that function at multiple stages of cell cycle progression. We used a high throughput screening protocol to mimic cancer-related overexpression in a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential only when CKS1 is overexpressed, a synthetic dosage lethal (SDL) interaction. Mutations in multiple genes affecting mitotic entry and mitotic exit are highly enriched in the set of SDL interactions. The interactions between Cks1 and the mitotic entry checkpoint genes require the inhibitory activity of Swe1 on the yeast cyclin-dependent kinase (CDK), Cdc28. In addition, the SDL interactions of overexpressed CKS1 with mutations in the mitotic exit network are suppressed by modulating expression of the CDK inhibitor Sic1. Mutation of the polo-like kinase Cdc5, which functions in both the mitotic entry and mitotic exit pathways, is lethal in combination with overexpressed CKS1. Therefore we investigated the effect of targeting the human Cdc5 ortholog, PLK1, in breast cancers with various expression levels of human CKS1B. Growth inhibition by PLK1 knockdown correlates with increased CKS1B expression in published tumor cell data sets, and this correlation was confirmed using shRNAs against PLK1 in tumor cell lines. In addition, we overexpressed CKS1B in multiple cell lines and found increased sensitivity to PLK1 knockdown and PLK1 drug inhibition. Finally, combined inhibition of WEE1 and PLK1 results in less apoptosis than predicted based on an additive model of the individual inhibitors, showing an epistatic interaction and confirming a prediction of the yeast data. Thus, identification of a yeast SDL interaction uncovers conserved genetic interactions that can affect human cancer cell viability.

Regulation of Transient Site-specific Copy Gain by MicroRNA

Intra-tumor copy number heterogeneity is commonly observed in cancer; however, the molecular mechanisms that contribute to heterogeneity remain poorly understood. Up-regulation of the histone demethylase KDM4A promotes transient site-specific copy gain (TSSG) in cells; therefore, uncovering how KDM4A levels are controlled is important for understanding the regulation of copy number heterogeneity. Here, we demonstrate that KDM4A is regulated by hsa-mir-23a-3p, hsa-mir-23b-3p, and hsa-mir-137. Altering expression of these microRNAs (miRNAs) regulates KDM4A-dependent TSSG. miRNA inhibition promoted copy gains and increased expression of the drug-resistant oncogene CKS1B, which was further substantiated in primary breast tumors. Consistent with increased CKS1B expression, miRNA inhibition reduced breast cancer cell sensitivity to cisplatin. Our data identify these miRNAs as regulators of TSSG and copy gains of a drug resistance gene.

NEDD8 Inhibition Overcomes CKS1B-Induced Drug Resistance by Upregulation of p21 in Multiple Myeloma.

CKS1B is significantly upregulated in multiple myeloma and associated with poor prognosis. The identification of novel therapies is essential for effective treatment of patients resistant to chemotherapy. The NEDD8 inhibitor MLN4924 selectively targets SCF(Skp2) activation and offers a more specific approach to protein degradation inhibition than total proteasomal inhibition. The goal of this study was to evaluate whether MLN4924 is effective in high CKS1B conditions and identify mechanisms regulating drug potency. Bortezomib and MLN4924 sensitivity was assessed through proliferation, viability, clonogenic potential, and senescence induction in cells overexpressing CKS1B. The mechanism for MLN4924 sensitivity was elucidated by immunoblot analysis of SCF(skp) substrates and confirmed by shRNA knockdown. The clinical relevance of the NEDD8 pathway was examined in gene expression profiles (GEP) derived from healthy people, patients with monoclonal gammopathy of undetermined significance (MGUS), and multiple myeloma. Cells overexpressing CKS1B were resistant to bortezomib but sensitive to MLN4924. Treatment of CKS1B-overexpressing cells with MLN4924 decreased proliferation, clonogenicity, and induced senescence. MLN4924, but not bortezomib, induced stabilization of p21 and knockdown of p21 resulted in loss of MLN4924 sensitivity. Patients with MGUS and multiple myeloma exhibited increased expression of NEDD8 pathway genes relative to normal plasma cells. Multiple myeloma patients with high NEDD8 expression were linked to bortezomib resistance in clinical trials, and had inferior outcomes. Our data demonstrate that cells with elevated CKS1B expression are resistant to bortezomib but sensitive to MLN4924 and offer a mechanism through the stabilization of p21. These findings provide rationale for targeting the NEDD8 pathway in multiple myeloma patients exhibiting elevated expression of CKS1B. Clin Cancer Res; 21(24); 5532-42. ©2015 AACR.

Targeting Neddylation to Overcome Drug Resistance in Multiple Myeloma

Background: The overexpression of CKS1B resulting from chromosomal amplification of the 1q21 region identifies a subset of multiple myeloma (MM) patients with poor clinical outcomes. Bortezomib (Btz) is an FDA-approved first-in-class proteasome inhibitor that has greatly improved clinical outcomes in MM. However, toxicities affiliated with pan-proteasomal inhibition and resistant populations remain a problem, highlighting the need for improved therapies. We have previously demonstrated that elevated expression of CKS1B results in the destabilization of p21 and contributes to insensitivity to bortezomib. MLN4924 is an inhibitor of the NEDD8-Activating Enzyme E1 (NAE1) and prevents the neddylation and subsequent activation of Cullin-1, a crucial component in the activation of SCF-driven ubiquitin-mediated degradation. We therefore explored if MLN4924 might serve as an alternative inhibitor to proteasomal degradation in MM.Materials and Methods: CKS1B was overexpressed or knocked down in myeloma cell lines using lentiviral vectors and shRNA. Cell proliferation and viability were assessed by cell counts and using PrestoBlue reagent. Clonogenicity was assessed by colony formation in soft agar. SA-b-galactosidase staining was used to assess senescence. Gene expression profiling was performed using the publicly available databases Total Therapy 2 (TT2) and APEX trials.Results: To examine the efficacy of Btz and MLN4924 in elevated CKS1B expression environments, we compared treatment with the MLN4924 to Btz in CKS1B OE cells in vitro. Cells with basal levels of CKS1B were sensitive to treatment with either Btz or MLN4924, with treatment leading to decreased proliferation and cellular viability. In the CKS1B OE background, we found cells were resistant to Btz repression of proliferation but sensitive to MLN4924. We also found that MLN4924 could more potently reduce colony formation in soft agar and more potently induce senescence in CKS1B OE cells compared to treatment with Btz. Immunoblot analyses confirmed a correlation between CKS1B expression and Cullin-1 neddylation. Further immunoblotting of known SCF-mediated ubiquitin targets in cells with and without CKS1B OE demonstrated a stabilization of p21 in all cells upon MLN4924 treatment that was not exhibited upon Btz treatment. To investigate the role of p21 in CKS1BOE cell sensitivity to MLN4924, we stably knocked down expression of p21. We found the knockdown of p21 partially abrogated the sensitivity of CKS1B OE cells to MLN4924, increasing cell viability, increasing colony formation in soft agar, and decreasing senescence induction.The importance of neddylation in the clinic was confirmed using GEP. We found that expression of the neddylation-related genes (i.e. NAE1, UBA3, and UBC12) is significantly upregulated (p<0.05) in MM patients relative to patients in MGUS or to healthy donors. Expression of NEDD8 was also significantly elevated in patients unresponsive to either Btz treatment or treatment with the combination of Btz and dexamethasone compared to responsive patients. The elevated expression of CKS1B highlights a population of patients with poor clinical outcomes. Considering the elevated expression of neddylation-related genes in MM progression, we performed Kaplan Meier survival analyses looking at patients segregated on two axes by CKS1B expression levels (high/low) and neddylation-related gene expression levels (either UBA3 or UBC12). We found patients with high/high expression of CKS1B and neddylation-related genes had significantly decreased survival relative to patients with mixed high/low expression from either axis. Together, these data suggest the importance of neddylation contributions to MM progression and clinical outcomes.Conclusions: These results shed light on the subtle differences in the blockade of proteasomal inhibition, illustrating the distinct effects of pan inhibition from inhibition of SCF-driven ubiquitin-mediated degradation. Mechanistically, we demonstrate at least a partial role for p21 in mediating cell sensitivity to MLN4924 in CKS1B overexpressing cells. Our findings highlight the important contributions of neddylation to MM disease progression and suggest the utility of targeting neddylation as a means of overcoming drug resistance in MM. DisclosuresNo relevant conflicts of interest to declare.

Too little O2 Too much gain

Too little O2 Too much gain

Abstract 693: A yeast synthetic lethal screen identifies a conserved interaction between PLK1 and CKS1b affecting cancer cell viability

Abstract The CKS1b gene is located on chromosome 1q21 and is frequently amplified in breast, lung and liver cancers. CKS1b codes for a conserved regulatory subunit of cyclin-CDK complexes that functions at multiple stages of cell cycle progression. We used a high throughput screening protocol to mimic cancer-related over-expression in Saccharomyces cerevisiae mutants to identify genes whose functions become essential only when CKS1 is over- expressed, a genetic interaction termed synthetic dosage lethality (SDL). Mutations in genes affecting the mitotic entry checkpoint and genes affecting mitotic exit are evident among the SDL interactions identified. The mitotic entry checkpoint controls the stability of the mitotic inhibitor Swe1 (Human WEE1), resulting in delayed mitosis when cell polarity is disrupted. Swe1 is a tyrosine kinase that inhibits the cyclin-dependent kinase (CDK) complex by phosphorylation of a conserved tyrosine-19 on Cdc28 (Human CDK1). We find that the SDL interaction between Cks1 and the mitotic entry checkpoint requires both Swe1 and the Cdc28 tyrosine-19 residue. The mitotic exit network is a signaling cascade that controls post-anaphase inhibition of CDK activity necessary for progression into G1. Since the polo-like kinase Cdc5 (Human PLK1) functions in both the mitotic entry and mitotic exit pathways, we investigated the effect of targeting PLK1 in breast cancers with varying expression of CKS1b. We analyzed published datasets and found that growth inhibition resulting from PLK1 knockdown was strongly correlated with increased CKS1b expression levels in breast cancer cell lines [Marcotte, R., Brown, K., Suarez, F., &amp; Sayad, A. (2012). Cancer Discovery, 2:172]. We independently confirmed sensitivity to PLK1 knockdown in 8 breast cancer cell lines in which 3 of 4 sensitive lines had high CKS1b expression and 3 of 4 insensitive lines had low CKS1b expression. We modified CKS1b expression in hMEC cells and showed that increased expression enhanced apoptosis upon PLK1 knockdown while decreased CKS1b expression abrogated sensitivity. Thus identification of a yeast synthetic dosage lethal (SDL) interactions identifies a conserved interactions that may be targeted to kill cancer cells. Citation Format: Robert J. Reid, Xing Du, Ivana Sunjevaric, Vinayak Rayannavar, John Dittmar, Matthew Maurer, Rodney Rothstein. A yeast synthetic lethal screen identifies a conserved interaction between PLK1 and CKS1b affecting cancer cell viability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 693. doi:10.1158/1538-7445.AM2015-693

Abstract 5088: A synthetic genetic interaction screen in yeast identifies Plk1 as a promising therapeutic target in cancer cells that overexpress Cks1b

Abstract Cancer cells often over-express specific genes as a result of translocation or gene amplification. We developed a high throughput screening protocol to mimic cancer-related over-expression in Saccharomyces cerevisiae deletion strain libraries to identify genes whose functions become essential only when the cancer-related query gene is over-expressed. The resulting synthetic dosage lethal (SDL) interactions uncover conserved genes that can be targeted to kill cancer cells but leave normal cells unaffected, providing a cancer-specific therapy. The CKS1b gene is located on chromosome 1q21, a region that is frequently amplified in breast, lung and liver cancers. Cks1b is a conserved regulatory subunit of cyclin-CDK complexes which functions during cell cycle progression. The SDL screen identified multiple pathways affecting mitotic progression that are essential when yeast CKS1 is over-expressed. One pathway controls a morphological checkpoint that leads to stabilization of the mitotic inhibitor Swe1 (Wee1 in mammals) resulting in delayed mitosis when cell polarity is disrupted. Swe1, a tyrosine kinase, inhibits the cyclin dependent kinase (CDK) complex by phosphorylation of a conserved tyrosine-19 on the yeast CDK, Cdc28. We find that the SDL interaction between CKS1 and the morphological checkpoint requires both Swe1 and the Cdc28 tyrosine-19 residue. Normal turnover of Swe1 occurs via SCF-mediated degradation after phosphorylation by the cyclin-CDK complex and the Polo-like kinase (Cdc5). Since, mutant alleles of CDC5 were identified in the SDL screen, we investigated the effect of targeting human PLK1, the mammalian Cdc5 ortholog, in breast cancers with varying expression of Cks1b. We first correlated RNAi knock down of PLK1 across 28 breast cancer cell lines [1] with Cks1b expression data taken from the Cancer Cell Line Encyclopedia [2]. Importantly, we find that growth inhibition by PLK1 knockdown correlates with increased Cks1b levels (r = -0.554, top 0.8th percentile). TCGA data also provides evidence for this SDL in breast cancer, since CKS1b over-expression and down-regulation of PLK1 expression rarely co-occur (mutual exclusion p-value 6.47x10-8). Together, these analyses support the hypothesis that PLK1 activity is essential when CKS1b levels are elevated. Finally, using shRNAs, we independently confirmed the PLK1 knockdown sensitivity in 8 breast cancer cell lines. Three of the 4 sensitive lines show increased CKS1b expression, while 3 of the 4 insensitive lines show low CKS1b expression, corroborating the correlation of CKS1b and PLK1 levels and lending support to conservation of the genetic interaction. [1] Marcotte, et al., Cancer Discovery, 2:172 (2012) [2] Barretina, et al., Nature, 483:603 (2012) Citation Format: Robert J. D. Reid, Xing Du, John C. DIttmar, Vinayak Rayannavar, Ivana Sunjevaric, Matthew Maurer, Rodney Rothstein. A synthetic genetic interaction screen in yeast identifies Plk1 as a promising therapeutic target in cancer cells that overexpress Cks1b. [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 5088. doi:10.1158/1538-7445.AM2014-5088

Quantitative analysis of CKS1B mRNA expression and copy number gain in patients with plasma cell disorders.

In this study, we have examined CKS1B gene expression and copy number in a total of 114- patients at diagnosis: 83 with multiple myeloma (MM) and 31 with monoclonal gammopathy of undetermined significance (MGUS). Results were correlated with cytogenetics, FISH and clinical characteristic. Significant CKS1B mRNA levels in MM compared to MGUS cases (p<0.048) were detected. In MM, the frequency of 1q21 (CKS1B) copy gain was significantly higher in cases with abnormal karyotype compared to patients with normal karyotype (p=0.021). Global analysis showed a positive correlation between CKS1B expression and 1q21 copy number (p<0.0001). No association between CKS1B mRNA expression and clinical parameters was found. However, a significantly higher level of β2 microglobulin in cases with 1q21 gains than those without (p=0.0094) was observed. Overall survival was shorter in cases with 1q21 gain compared to those with normal 1q21 region (p=0.0082). Our results suggest a role for CKS1B in the multiple step process of progression of MGUS to MM and show that CKS1B copy gain has a more significant prognostic value than its overexpression. This adverse impact on survival probably reflects the genetic instability associated to chromosome 1q alterations resulting in a more aggressive behavior of the disease.

Elevated Expression Of CKS1B Inhibits Senescence Thorough Enhanced Degradation Of p21 In Multiple Myeloma

BackgroundHigh expression of CKS1B gene as a result of amplification of region 1q21 identifies a subset of patients with poor prognosis in multiple myeloma (MM). We have previously shown that over-expression of CKS1B enhances degradation of p27 and activates both MEK/ERK and JAK/STAT3 signaling pathways, promotes MM cell drug-resistance. However, other mechanisms of drug resistance induced by high expression of CKS1B remain to be identified. Materials and methodsCKS1B was over-expressed or knocked-down by shRNA in OCI-MY5 and KMS28PE myeloma cell lines. Cell viability was detected by PrestoBlue assay; Annexin-V assay was employed to detect cell apoptosis. Colony formation was assessed in soft agar. The direct dye efflux to assess multidrug resistance was performed using eFluxx-ID Multidrug resistance assay. Senescence was detected by SA-β-galactosidase (β-Gal) staining. Expression of SCF-Skp2 substrates was evaluated by western blots. ResultsWe demonstrated that overexpression of CKS1B significant decreased sensitivity to bortezomib and increased the clonogenic capacity compared to cells transfected with empty vector in the presence of bortezomib (p < 0.05). A prominent mechanism of drug resistance involved upregulation of multidrug resistance associated proteins (MRPs). We investigated whether the level of MDR1, BCRP, and MRP1, mitotic checkpoint protein MAD2 and ABC transporter family members was increased in cells with elevated CKS1B. However, none of drug pump proteins was up-regulated or stabilized by over-expression of CKS1B in OCI-MY5 and KMS28PE. Accordingly, over-expression of CKS1B did not promote efflux of the hydrophilic eFluxx-ID™ gold fluorescent dye from MM cells any greater than control cells, suggesting alternative mechanisms. We therefore tested whether CKS1B can inhibit drug-induced senescence in MM cells after treatment with bortezomib by quantifying the percentage of cells expressing senescence-associated β-Gal. Interestingly, MM cells with elevated CKS1B were significantly less β-Gal positive following treatment with bortezomib compared to control cells (p < 0.05). To explore molecular mechanism underlying CKS1B induced anti-senescence, expression of SCF-Skp2 substrates involved in senescence pathway was examined as CKS1B is an essential cofactor for SCF-Skp2 complex. Over-expression of CKS1B significantly reduced expression of p21, p27, and p57 while knock-down of CKS1B stabilized these proteins. Notably, cells with high expression of CKS1B failed to accumulate p21 protein in response to bortezomib. ConclusionOur findings suggest that anti-senescence is a novel mechanism in CKS1B-induced drug resistance in MM and the turnover of p21 regulated by CKS1B could have important implications not only for bortezomib therapy but also to develop therapies aimed at enhancing p21 function independent of p53. Disclosures:No relevant conflicts of interest to declare.

Abstract 214: High expression of CKS1B antagonizes MLN4924, a nedd8-activating enzyme inhibitor, that induces myeloma cell growth inhibition through decreased neddylation of SCF complex

Abstract Background: MLN4924 binds to the NEDD8-activating enzyme (NAE), and promotes neddylation of SCF (Skp1-Cul1 / Cdc53-F-box protein) complex resulting in inhibition of proteasomal degradation of several substrates. CKS1B acts as an adaptor of SCF complexes involved in cell cycle progression and DNA damage repair in many cancers, including multiple myeloma (MM). In this study, we investigate the functional role of CKS1B expression on MLN4924 in myeloma and determine whether CKS1B can be used as a potential biomarker for sensitivity to MLN4924 treatment in MM. Methods: Three myeloma cell lines, KMS12, KMS28PE, and OCI-MY5 in which CKS1B was artificially over-expressed (CKS1B-OE) as well as their empty vector (EV) controls were treated with MLN4924 at different concentrations. The IC50 and growth inhibition of cells were analyzed via a fluorencent screeing assay. APC-labeled annexinV and PI were assessed by flow cytometry to detect apoptosis and cell cycle changes. The expression of Cullin-NEDD8 and a series of ubiquitined SCF substrates were analyzed by non-reducing western blot. Results: MLN4924 effectively inhibited cell growth and induced cell apoptosis in both CKS1B-OE and EV MM cells. However, MM cells transfected with EV were more sensitive than those transfected with CKS1B-OE as evidenced by the IC50 values (KMS28PE OE vs. EV 99: 65 nM; KMS12 OE vs. EV 125: 72 nM; and OCI-MY5 OE vs. EV 106:89 nM). The cell viabilities at 100 nM were 49.6% vs. 26.7%, 54.5% vs. 34.2%, and 53.6% vs. 36.8% in CKS1B-OE versus EV KMS28PE, KMS12, and OCI-MY5 cells (P&amp;lt;0.05). Western blots showed that over-expression of CKS1B significantly decreased expression of cullin1, nedd8, and the conjugated complex of cullin-nedd8 in MLN4924 treated cells (100 nM, 24h) compared with EV in all tested three cell lines. We also found that high expression of CKS1B was negatively correlated with SCF substrates including p21, CDT-1, p27 and CDT-1. MLN4924 induced a dose-dependent accumulation of these substrates in all three MM cell lines. Conclusion: High expression of CKS1B induces SCF substrate ubiquitination and degradation in MM cells. The NAE inhibitor MLN4924 induces cell apoptosis through decreased neddylation of SCF complex resulting in increase of p27, p21, CDT-1 and p130. CKS1B can be used as a marker of drug resistance to MLN4924, suggesting that a combination of MLN4924 with other drugs will be necessary to treat myeloma patients with high endogenous expression of CKS1B. 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 214. doi:1538-7445.AM2012-214

CKS1B nuclear expression is inversely correlated with p27Kip1 expression and is predictive of an adverse survival in patients with multiple myeloma.

CKS1B is a member of the highly conserved cyclin kinase subunit 1 (CKS1) family that interacts with cyclin-dependent kinases and plays an important role in cell cycle progression. We and others have shown that CKS1B amplification located on chromosome 1q21 is an adverse prognostic factor in multiple myeloma, but its relationship with CKS1B nuclear protein expression, is unclear. The aim of this study was to correlate nuclear CKS1B protein immunoreactivity, 1q21 amplification status, p27(Kip1) expression and survival in patients with newly-diagnosed multiple myeloma. Nuclear expression of CKS1B and p27(Kip1) was evaluated by immunohistochemistry in decalcified, paraffin-embedded bone marrow biopsies from 94 patients with newly diagnosed multiple myeloma. Clonal plasma cells of the bone marrow aspirates from the same cohort were examined for CKS1B gene status by interphase cytoplasmic fluorescence in situ hybridization. Fluorescence in situ hybridization detected the 1q21 amplification in 36 (38%) of the 94 patients and immunohistochemistry showed CKS1B protein expression in 37 (39%). Thirty-two (86%) of the 36 amplified (1q21) cases expressed CKS1B and 31 (84%) of the 37 CKS1B immunore-active cases had amplified 1q21. 1q21 amplification and CKS1B protein expression were strongly correlated (P<0.0001). CKS1B protein expression was inversely correlated with p27(Kip1) immunostaining (P<0.0001) and was associated with a shorter overall survival (median 44.5 versus 89.3 months, P<0.0001). Immunohistochemistry for CKS1B is a simple, rapid method that appears to predict 1q21 amplification and adverse outcome for risk stratification of patients with multiple myeloma.

CKS1B Over-Expression Significantly Predicts for a Lower Rate of Response to Primary Therapy with Thalidomide-Dexamethasone (Thali-Dex) for Newly Diagnosed Multiple Myeloma (MM) Patients.

In MM tandem duplication and jumping translocations of the q21 band of chromosome (chr.) 1 are acquired during progression of the disease and can lead to chr.1q amplification. In several studies, chr. 1 amplification has been linked to poor prognosis after both conventional and high-dose chemotherapy. Recently, a subset of genes mapping on 1q21 band has been identified, whose increased expression may be due to increased DNA copy number. Among these genes, CKS1B has been one of the most significantly upregulated. CKS1B regulates SCF skp2 mediated ubiquitination and proteolysis of the cyclin dependent kinase inhibitor p27Kip1, whose low expression has been reported to be an independent adverse prognostic factor in MM patients. Aim of the present study was to investigate the relationship between CKS1B expression and response to primary therapy with thali-dex in a large series of patients with newly diagnosed MM. Secondary endpoint was to explore the relationship between CKS1B expression and del(13), as assessed by FISH analysis, and t(4;14), as evaluated by an RT-PCR assay designed to detect the presence of IgH/MMSET fusion gene. A total of 132 patients were analyzed. The presence of t(4;14) and CKS1B expression were investigated in all patients, while del(13) was studied in 129/132 patients. CKS1B expression was evaluated by Real-time RT-PCR. CKS1B values were separated in four different quartiles, with expression levels increasing progressively from quartile 1 to 4. Response to therapy was evaluated according to the criteria proposed by Bladè et al. The Fisher test and the Mann-Whitney test were applied for statistical analysis. On an intent-to-treat basis, the overall probability to respond (≥ partial response, PR) to up-front thali-dex therapy was 71%, while 38 patients (29%) either did not respond (NR) or progressed. Median CKS1B expression value was significantly higher in NR in comparison with patients who attained at least a PR: 1.42 (range 0.15–52.35) vs. 0.89 (range 0–11.88), respectively (p=0.01). In particular, the proportion of NR patients in the CKS1B expression quartile 4 was significantly higher as opposed to the frequency of NR in the CKS1B expression quartiles 1 to 3 (45.5% vs. 23.2%, respectively; p= 0.02). CKS1B over expression did not correlate with the presence of t(4;14) or del(13). Only 6 patients harbouring t(4;14) fell into the CKS1B expression quartile 4, as opposed to 32 patients included into the CKS1B expression quartiles 1–3 (18.2% vs. 32.3%; p, not significant). Similarly, the frequency of del(13) was comparable in the CKS1B expression quartile 4 and in the CKS1B expression quartiles 1–3 (34.4% vs. 44.3%; p, not significant). Likewise, only 2 patients carrying both t(4;14) and del(13) fell into the CKS1B expression quartile 4, as opposed to 15 patients who fell into the CKS1B expression quartiles 1–3 (6.5% vs. 15.3%; p, not significant). In conclusion, in patients with newly diagnosed MM, CKS1B over expression at baseline predicts for a significantly lower probability of response to primary remission induction therapy with thali-dex. Poor response to thali-dex conferred by CKS1B over expression is independent from the presence of t(4;14) and/or del(13). Supported by Università di Bologna, Progetti di Ricerca ex-60% (M.C.); Ministero dell'Università e Ricerca Scientifica (MIUR), progetto FIRB, RBAU012E9A_001 (M.C.); and Fondazione Carisbo.

Metaphase Cytogenetics Adds to Gene Expression Profiling in the Identification of High Risk Multiple Myeloma.

The International Staging System (ISS), based on B2-microglobulin and albumin levels at the time of diagnosis, has now generally been adopted as a new prognostic classification system for multiple myeloma (MM). While readily and widely applicable, ISS does not account for genetic disease features, such as metaphase (CA) and interphase fluorescence in situ hybridization (FISH) cytogenetic abnormalities, which when examined in the context of standard prognostic variables, confer higher hazards of relapse and disease-related death. Recently, gene expression profiling (GEP) uncovered the major prognostic significance for outcome of high expression of CKS1B, a gene mapping to an amplicon at chromosome 1q21. We have performed a comprehensive study of CA, FISH, GEP and ISS staging in 351 newly diagnosed MM patients, treated uniformly on Total Therapy 2. We have analyzed outcome based on a combination of high CKS1B by GEP and CA. GEP-based t(11;14) was prognostically favorable, irrespective of expression of CKS1B and, therefore, was removed from the group of patients with high CKS1B expression. After this adjustment, with the combination of both CA and high CKS1B (approximately 10% of all patients) conferred a very poor outcome with only 24% and 40% of such patients being event-free and/surviving at 3 years, compared with 72% and 84% for the others (p values : <.0001). Such patients fared poorly, irrespective of their ISS stage. Similar prognostic information could be gained by combining CA with FISH-defined amplification of 1q21 and t(11;14). Because of their major prognostic impact, all newly diagnosed patients should be tested for these genetic markers. Novel treatment modalities are justified in the small subgroup of such poor prognosis patients, since they derive only a minor benefit from advances in MM therapy. [Display omitted]

Inhibition of CKS1B Expression Affects Myeloma Cell Growth In Vivo.

CKS1B is the regulatory component of the SCF-Skp2 ubiquitin ligase that ubiquinates the cyclin-dependent kinase inhibitor p27kip1 targeting it for proteasomal degradation. Overexpression of CKS1B is associated with poor prognosis in malignancies such as oral squamous cell carcinomas, colorectal carcinoma, breast cancer, prostate cancer, and others. Recently, high expression levels of CKS1B were associated with an aggressive course of Multiple Myeloma (Shaughnessy J. Hematology . 2005; 10 Suppl 1:117). We investigated whether inhibition of CKS1B in myeloma cell lines with multiple copies of 1q21 and high expression of CKS1B affects in vivo tumor growth. Myeloma cell lines were transduced with lentivirus conditionally expressing (tet on) CKS1B shRNA and lentivirus constitutively expressing luciferase (for in vivo experiments), or with lentivirus constitutively expressing CKS1B shRNA (for in vitro work). Cells with inducible expression were injected into the human bones of SCID-hu mice, and, once tumors established, expression was induced with doxycycline in the drinking water. Changes in tumor sizes were followed by weekly luminescence imaging. Cells with constitutive shRNA expression were used to determine effects on cell cycle by propidium iodide flow cytometry. Effects of shRNA expression on cytoplasmic and nuclear CKS1B and p27kip1 levels were determined by western blot analysis. Induction of CKS1B shRNA expression in JJN3 and OCI-MY5 cells in SCID-hu mice inhibited tumor growth by 57% and 81% within one week, compared to non-targeting construct. As expected, viable cells recovered from these tumors had low levels of CKS1B and higher levels of p27kip1 proteins. Similar to observations in the MM cell lines JJN3 and OCI-MY5 (see Abstract by Zhan and colleagues), constitutive expression of shRNA in CAG and XG1 cells effectively reduced cytoplasmic and nuclear CKS1B protein levels. This was associated with increased levels of p27kip1 protein, marked (51%) increases of cells in the G1 phase of the cell cycle, and reduced S and G2 (33% and 52%, respectively), compatible with a G1 arrest, and a 76% increase in the number of sub G1 (apoptotic) cells. In contrast to these observations, cells constitutively over-expressing p27kip1 had no change in G1, 50% fewer cells in S, and a 150% increase in G2, indicating G1 and G2 arrests, with no change in the sub G1 fraction. Interestingly, cells over-expressing p27kip1 had lower levels of CKS1B proteins, compatible with the reported proteasomal degradation of the molecule during the later phases of the cell cycle (Hattori T et al, Genes Cells. 2003; 8:889). These observations suggest that the poor prognosis associated with 1q21 amplifications and high CKS1B expression in myeloma reflects an elevated proliferative state of the tumor cells that results in rapid regrowth of minimal residual disease.

CKS1B, Mapping to an Amplicon on Chromosome 1q21 and over Expressed in Aggressive Disease, Regulates Multiple Myeloma Growth and Survival through p27Kip1-Dependent and Independent Mechanisms.

While deregulated expression of a D-type cyclin is thought to represent an initiating event in myelomagenesis, the molecular mechanisms of disease progression are not understood. We recently reported that disease progression and high-risk disease are strongly correlated with increased copy number and expression of genes mapping to chromosome 1q. Here we report that elevated expression of CKS1B, a regulatory subunit of the SCF-Skp2 ubiquitin ligase that regulates p27Kip1 protein stability, mapping to an amplicon at 1q21 and over expressed in high-risk myeloma, is required for myeloma cell survival both in vitro and in-vivo. CKS1B expression, absent in plasma cells from healthy individuals and the benign plasma cell dyscrasia monoclonal gammopathy of undetermined significance, is inversely correlated with p27Kip1 protein levels in primary MM. Silencing of CKS1B in MM cell lines JJN3 and OCI-MY5 using shRNAs delivered by constitutive and inducible lentivirus vectors induced stabilization of p27Kip1, cell cycle arrest and apoptosis. Over expression of a non-degradable form of p27Kip1 in JJN3 and OCI-MY5 cells inhibited cell cycle progression, but did not induce apoptosis. We propose that CKS1B may play in important role in myeloma progression through p27Kip1-related and unrelated mechanisms.

A Gene Expression Based Centrosome Index Is a Powerful Prognostic Factor in Myeloma.

Previously, we have shown that centrosome amplification is common in multiple myeloma and is already present in monoclonal gammopathy of undetermined significance (MGUS), suggesting an early role in myelomagenesis. In addition, we derived a gene expression based centrosome index (CI) that correlated closely with centrosome amplification detected by immunofluorescence. We also showed in a heterogeneously treated and relatively small cohort of patients that the CI is an independent prognostic factor. In this study, we validated the prognostic significance of a gene expression based centrosome index (CI) in 2 large cohorts of homogenously treated myeloma patients entered into clinical trials. Cohort 1 consisted of newly diagnosed patients from the University of Arkansas for Medical Science (UAMS) entered treated with total therapy II. These patients have gene expression profiling performed on purified CD138+ plasma cells from pre-treatment bone marrow cells using the Affymetrix U133plus chip (data available from Gene Expression Omnibus). Cohort 2 consisted of relapsed or refractory patients entered into bortezomib trials. These patients have GEP performed on purified CD138+ PCs using the Affymetrix U133A and B chip as part of a pharmacogenomics study (data kindly provided by Millenium Pharmaceuticals). Expression values extracted using the MAS 5.0 software are log-transformed, median centered and analyzed using Genespring 7.0 (Agilent Technologies). Survival is calculated from date of entry into trial till death in both cohorts. In the UAMS dataset, a high CI is significantly associated with shorter survival and emerged as an independent prognostic factor with t(4;14) (detected by spike FGFR3/MMSET expression) when entered into a multivariate Cox proportional hazard analysis (other variables entered included CKS1B expression above median and elevated gene expression based proliferation index, both of which were significantly associated with shorter survival on univariate but not multivariate analysis). In the bortezomib cohort, a high CI is again significantly associated with shorter survival, and this effect is independent of response to bortezomib. Therefore, a high CI is a powerful independent prognostic factor in both newly diagnosed and relapsed patients even in patients treated by intensive therapy (total therapy II) and novel agents (bortezomib). We next compared the gene expression profiles of tumors with high and low CI, to define the molecular phenotype of these aggressive tumors and identify possible novel therapeutic targets. Tumors with high CI over-expressed genes coding for proteins associated with the centrosome (TUBG1, CETN2, TACC3, NEK2, PRKRA, STK6, AURKB and PLK4), cell cycle (CCNB1, CCNB2, CCND2, E2F2, CDC gene family, CDK5, CDK6, CDKN2C), proliferation (RAN, CKS1B, TOP2A, TTK, TYMS, MCM gene family, ASPM), involve in DNA repair / G2 cell cycle checkpoints (BRCA1, CHEK1, CHEK2, MAD2L1, BUB1, BUB1B, FANCD2, REV1L), kinetochore and microtubule attachment (AURKB, BIRC5, CENPA, CENPE, CENPH, ZWINT), genes coding for cancer testis antigens (GAGE and MAGE family) and implicated in other cancer (PTP4A3, EZH2). Consistent with this, the most significantly associated Gene Ontology biological process are cell cycle, mitosis, proliferation, DNA repair, microtubule related In particular, aurora kinases are significantly over-expressed in patients with high CI and may represent novel therapeutic targets in these patients with very poor prognosis.