Supplementary Figure Legends 1-4 from Cks1 Regulates cdk1 Expression: A Novel Role during Mitotic Entry in Breast Cancer Cells

Supplementary Table 1 from Cks1 Regulates cdk1 Expression: A Novel Role during Mitotic Entry in Breast Cancer Cells

DNA methyltransferase (DNMT) 1 is an enzyme that functions as a maintenance methyltransferase during DNA replication, and depletion of this enzyme from cells is considered to be a rational goal in DNA methylation-dependent disorders. Two DNMT1-depleting agents 5-aza-2'-deoxycytidine (aza-dCyd, decitabine) and 5-aza-cytidine (aza-Cyd, azacitidine) are currently used for the treatment of myelodysplastic syndromes and acute myeloid leukemia and have also been investigated for nononcology indications, such as sickle cell disease. However, these agents have several off-target activities leading to significant toxicities that limit dosing and duration of treatment. Development of more selective inhibitors of DNMT1 could therefore afford treatment of long durations at effective doses. We have discovered that 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) is as effective as aza-dCyd in depleting DNMT1 in mouse tumor models but with markedly low toxicity. In this review we describe the preclinical studies that led to the development of aza-T-dCyd as a superior DNMT1-depleting agent with respect to aza-dCyd and will describe its pharmacology, metabolism, and mechanism of action. In an effort to understand why aza-T-dCyd is a more selective DNMT1 depleting agent than aza-dCyd, we will also compare and contrast the activities of these two agents. SIGNIFICANCE STATEMENT: Aza-T-dCyd is a potent DNMT1-depleting agent. Although similar in structure to decitabine (aza-dCyd), its metabolism and mechanism of action is different than that of aza-dCyd, resulting in less off-target activity and less toxicity. The larger therapeutic index of aza-T-dCyd (DNMT1 depletion vs. toxicity) in mice suggests that it would be a better clinical candidate to selectively deplete DNMT1 from target cells and determine whether or not depletion of DNMT1 is an effective target for various diseases.

e21135 Background: Post progression interventions and their impact on survival, which can be different among lines of treatment (LOTs) can influence the association between progression-free survival (PFS) and overall survival (OS), thereby differentially affecting the strength of surrogacy of PFS to OS. To explore the potential benefit of PFS as a surrogate for OS we evaluated the correlation between PFS and OS among anti-PD-1/PD-L1 agents in NSCLC between sets of LOT1+ versus LOT2+ trials. Methods: Trial-level analyses were performed using the SMARTImmuno-Oncology (SMART-IO) repository of completed and ongoing trials evaluating IO treatments in NSCLC. Weighted (by trial size) mixed regression models were used to determine the correlation between mPFS and mOS from trial data of anti-PD-1/PD-L1 agents and the strength of association was estimated vis-à-vis LOT. Results: A majority of the NSCLC IO trials (n = 268) had single agent anti-PD-1 (22%) as the intervention regimen of which nivolumab trials (n = 31) were most common, followed by pembrolizumab (n = 27). Anti-PD-L1 agents accounted for 12% of the trials with atezolizumab being most common (n = 18). Among all trials with a comparator group (n = 49), docetaxel was the most commonly used (n = 7) control regimen. Overall PD1/L1 agents showed higher correlation in LOT2+ (r = 0.48) compared to LOT1+ (r = 0.38) trials. Also, mPFS correlated better with mOS in anti-PD-1 trials (range: 0.82-0.84) as opposed to trials where the intervention arm was an anti-PD-L1 agent, wherein no correlation was found (range: 0.08-0.1). Among PD-1/L1 agents, correlation was best for nivolumab (r = 0.84) followed by pembrolizumab (r = 0.82), whereas there was no correlation in atezolizumab (r = 0.10) and durvalumab (r = 0.08) trials. Pembrolizumab showed higher correlation in LOT2+ (r = 0.87) compared to LOT1+ (r = 0.79) trials. Conclusions: PFS acts as a reliable surrogate for OS in LOT2+ versus LOT1+. Whereas PFS is a strong surrogate for OS in anti PD-1 agent trials, it is currently unclear whether this also extends to anti PD-L1 agents. Future LOT2+ trials of anti PD-1 agents should further validate if PFS can be treated as a primary endpoint instead of OS.

Constitutive levels of Cks1 protein are very high in mammary carcinoma tissue and in breast tumor cell lines. However, despite being transcribed at relatively high levels, Cks1 protein is very low in normal mammary tissue. Also, basal Cks1 is barely detectable in primary human mammary epithelial cells (HMECs). Epoximicin, a proteasome inhibitor, induced detectable endogenous Cks1 in HMECs, and upregulated it above the basal level in MCF-7 breast cancer cells. Interestingly, transiently transfected Cks1 is remarkably unstable and accumulates only upon proteasomal blockade in multiple cell lines even when driven by the strong CMV promoter-enhancer. We examined the stability of site-directed Cks1 mutants in order to identify the structural determinants of its turnover in cancer cells. Since protein turnover is regulated by phosphorylation, and phosphoproteomic studies reveal phosphorylated tyrosines in Cks1, we replaced its five conserved tyrosines (Y) with phenylalanine (F), both individually and in combinations. We find that like wild-type, all transiently transfected mutant Cks1 vectors, even when driven by the CMV promoter-enhancer, expressed detectable protein only in cells treated with epoximicin. However, turnover of the Y8F, Y12F and Y19F Cks1 mutants was more rapid than that of wild-type, Y7F and Y57F. Since lysines are modified by ubiquitination or acetylation we also examined the consequences of lysine to arginine (K-R) substitutions on Cks1 proteasomal turnover. We found that the individual mutations K4R, K26R, K30R, and K34R slowed Cks1 turnover, while the K79R mutation or the combined mutation K75-76-78-79R increased turnover. Taken together, regulation of Cks1 protein stability is crucially dependent on specific tyrosine and lysine residues which are potential sites for post-translational modifications.

PURPOSE:Cks1, a conformationally heterogenous 9 kDa protein, is markedly overexpressed in cancer cells and contributes to tumor development. Cks1 is an essential component of the SCF-Skp2 ubiquitin ligase complex that targets the Cdk inhibitors p27(Kip1) and p21(Cip1). Cks1 is known to interact with the Hsp90-Cdc37 chaperone machinery, although whether this facilitates its conformational maturation and stability is not known. To test whether abrogating the chaperone function of Hsp90 could destabilize Cks1, we examined the effects of treating different cancer cell lines with the benzoquinone ansamycin 17-allylamino geldanamycin (17-AAG), a compound that selectively binds Hsp90 and potently inhibits its ATP-dependent chaperone activity.METHODS:The effect of Hsp90 inhibition using 17-AAG on Cks1 protein and associated cell cycle proteins including Skp2, p27(Kip1), p21(Cip1), and Cdk1 in cancer cells was determined by Western blotting. Ubiquitination analysis was carried out by transfecting cells with an HA-ubiquitin plasmid and specifically immunoprecipitating Cks1 to examine polyubiquitinated species. Flow cytometry was utilized to examine the effects of Hsp90 inhibition on cell cycle profiles.RESULTS:Here, we demonstrate for the first time that inhibition of Hsp90 utilizing 17-AAG destabilizes Cks1 in cancer cells by promoting its ubiquitination and proteasomal degradation. 17-AAG-induced Cks1 depletion was accompanied by concomitant decreases in Skp2 and Cdk1. 17-AAG treatment also induced G2/M accumulation in MCF-7 breast carcinoma cells, and G1 accumulation in the colon carcinoma lines HCT116 and SW620.CONCLUSIONS:We conclude that perturbing the Hsp90 pathway could provide a useful therapeutic strategy in tumors driven by Cks1 overexpression.

Abstract Targeting cancer epigenetic control of cell growth via DNA methylation has been successful in treating hematologic diseases, such as Decitabine (DAC) and Azacitidine for Myelodysplastic Syndrome including Acute Myelomonocytic Leukemia. This success has not extended to solid tissue tumors. The Division of Cancer Treatment and Diagnositcs of NCI has initiated pre-clinical development of TdCyd as an agent for treating solid tumors after promising early results in a lung adenocarcinoma xenograft model (NCI-H23). IP dosing at 5MKG (0.56 MTD) in nu/nu mice on a Q5D x 3 cycle schedule resulted in tumor stasis with no accompanying weight loss in the mice. A DAC-treated control arm treated at MTD resulted in tumor growth delay but not stasis, and a 10% weight loss was noted. Intratumoral levels of DNMT1 were reduced to undetectable levels in xenografts post administration of TdCyd by ELISA and Western Blot assays, but were unaffected by DAC treatment. Mass Spectrometry analysis demonstrated incorporation of both TdCyd and thiothymidine (TdThd) into H23 DNA. In vitro experiments on a selected panel of cancer cell lines confirmed the conversion of TdCyd to the triphosphate and re-expression of tumor suppressor proteins p15 and p16. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Robert J. Kinders, Melinda Hollingshead, Jaideep Thottassery, William B. Parker, Thomas D. Pfister, Lawrence W. Anderson, Joseph E. Tomaszewski, Jerry M. Collins, James H. Doroshow. Pre-clinical development of 4′-thio-2′-deoxycytidine (TdCyd) as a DNA-demethylating agent for use in treating solid tissue tumors. [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 2306. doi:10.1158/1538-7445.AM2014-2306

Currently approved DNA hypomethylating nucleosides elicit their effects in part by depleting DNA methyltransferase I (DNMT1). However, their low response rates and adverse effects continue to drive the discovery of newer DNMT1 depleting agents. Herein, we identified two novel 2′-deoxycytidine (dCyd) analogs, 4′-thio-2′-deoxycytidine (T-dCyd) and 5-aza-4′-thio-2′-deoxycytidine (aza-T-dCyd) that potently deplete DNMT1 in both in vitro and in vivo models of cancer and concomitantly inhibit tumor growth.

Background and purposeGlioblastoma multiforme (GBM) represents the most common and deadly primary brain malignancy, particularly due to temozolomide (TMZ) and radiation (RT) resistance. To better understand resistance mechanisms, we examined global kinase activity (kinomic profiling) in both treatment sensitive and resistant human GBM patient-derived xenografts (PDX or “xenolines”).Materials and methodsThirteen orthotopically-implanted xenolines were examined including 8 with known RT sensitivity/resistance, while 5 TMZ resistant xenolines were generated through serial TMZ treatment in vivo. Tumors were harvested, prepared as total protein lysates, and kinomically analyzed on a PamStation®12 high-throughput microarray platform with subsequent upstream kinase prediction and network modeling.ResultsKinomic profiles indicated elevated tyrosine kinase activity associated with the radiation resistance phenotype, including FAK and FGFR1. Furthermore, network modeling showed VEGFR1/2 and c-Raf hubs could be involved. Analysis of acquired TMZ resistance revealed more kinomic variability among TMZ resistant tumors. Two of the five tumors displayed significantly altered kinase activity in the TMZ resistant xenolines and network modeling indicated PKC, JAK1, PI3K, CDK2, and VEGFR as potential mediators of this resistance.ConclusionGBM xenolines provide a phenotypic model for GBM drug response and resistance that when paired with kinomic profiling identified targetable pathways to inherent (radiation) or acquired (TMZ) resistance.

Deregulation of the cell cycle results in loss of normal control mechanisms that prevent aberrant cell proliferation and cancer progression. Regulation of the cell cycle is a highly complex process with many layers of control. One of these mechanisms involves timely degradation of CDK inhibitors (CKIs) like p27Kip1 by the ubiquitin proteasomal system (UPS). Cks1 is a 9 kDa protein which is frequently overexpressed in different tumor subtypes, and has pleiotropic roles in cell cycle progression, many of which remain to be fully characterized. One well characterized molecular role of Cks1 is that of an essential adaptor that regulates p27Kip1 abundance by facilitating its interaction with the SCF-Skp2 E3 ligase which appends ubiquitin to p27Kip1 and targets it for degradation through the UPS. In addition, emerging research has uncovered p27Kip1-independent roles of Cks1 which have provided crucial insights into how it may be involved in cancer progression. We review here the structural features of Cks1 and their functional implications, and also some recently identified Cks1 roles and their involvement in breast and other cancers.

Abstract Epigenetic therapies using cytidine/deoxycytidine analogs are proving to be effective as indicated by the recent approvals of 5-azacytidine (5-azaCyd) and 5-aza-2’-deoxycytidine (5-azadCyd) in myelodysplastic syndromes (MDS) and certain leukemias. Studies suggest that the inhibition of DNA cytosine-5 methylation and the re-expression of silenced tumor suppressors contribute to the beneficial effects of these drugs. However inhibition of DNA synthesis and other toxicities of these compounds represent major drawbacks in the clinic. Recently another analog previously examined in our anticancer discovery program, 4’-thio-2’-deoxycytidine (T-dCyd), had been shown to be an inhibitor of methyl transfer by the M. HhaI DNA cytosine-5 methyltransferase. In the current studies we demonstrate that T-dCyd and its 5-aza analogue 4’-thio-5-aza-2’-deoxycytidine (5-aza-T-dCyd), can deplete human DNMT1 protein in NCI-H23 lung carcinoma and the myeloid leukemia lines THP-1 and KG1a. Consistent with this we also found that T-dCyd and 5-aza-T-dCyd were also effective in decreasing DNMT activities in cancer cells. Methylation-specific PCR (MSP) analysis also demonstrated that both T-dCyd and 5-aza-T-dCyd induced CpG demethylation and re-expression of the tumor suppressor p15 in KG1a cells. We have also found that T-dCyd is inserted into replicating DNA at nanomolar doses as readily if not better than the natural 2’-deoxycytidine (dCyd) and exhibited very little toxicity at these doses. T-dCyd is therefore readily activated to its triphosphate T-dCTP, which is a good substrate for DNA polymerase mediated incorporation, and DNA polymerases also readily extend the chain after incorporation. Furthermore we also observed that T-dCyd and 5-aza-T-dCyd are efficacious in in vivo tumor models. Collectively our data suggest that these compounds or their analogs could be developed as novel DNA methylation inhibitors with better properties for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2537. doi:10.1158/1538-7445.AM2011-2537

The outcomes in children with refractory/relapsed (R/R) acute lymphoblastic leukemia (ALL) are dismal. The efficacy and safety of intravenous clofarabine 40 mg/m(2) per day, cyclophosphamide 440 mg/m(2) per day, and etoposide 100 mg/m(2) per day for 5 consecutive days in pediatric patients with R/R ALL was evaluated in this phase 2 study. The primary endpoint was overall response rate (complete remission [CR] plus CR without platelet recovery [CRp]). Among the 25 patients (median age, 14 years; pre-B cell ALL, 84%; ≥ 2 prior regimens: 84%; refractory to previous regimen: 60%), the overall response rate was 44% (7 CR, 4 CRp) with a 67.3-week median duration or remission censored at last follow-up. Most patients proceeded to alternative therapy, and 10 patients (40%) received hematopoietic stem cell transplantation. Six patients (24%) died because of treatment-related adverse events associated with infection, hepatotoxicity, and/or multiorgan failure. The study protocol was amended to exclude patients with prior hematopoietic stem cell transplantation after 4 of the first 8 patients developed severe hepatotoxicity suggestive of veno-occlusive disease. No additional cases of veno-occlusive disease occurred. The regimen offered encouraging response rates and sustained remission in R/R patients. Future investigation should include exploration of patient selection, dosing, and supportive care. This trial was registered at www.clinicaltrials.gov as #NCT00315705.

Mesd is a specialized chaperone for low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6. In our previous studies, we found that Mesd binds to mature LRP6 on the cell surface and blocks the binding of Wnt antagonist Dickkopf-1 (Dkk1) to LRP6. Herein, we demonstrate that Mesd also binds to LRP5 with a high affinity and is a universal inhibitor of LRP5 and LRP6 ligands. Mesd not only blocks binding of Wnt antagonists Dkk1 and Sclerostin to LRP5 and LRP6 but also inhibits Wnt3A and Rspondin1-induced Wnt/beta-catenin signaling in LRP5- and LRP6-expressing cells. We also found that Mesd, Dkk1, and Sclerostin compete with one another for binding to LRP5 and LRP6 at the cell surface. More importantly, we demonstrated that Mesd is able to suppress LRP6 phosphorylation and Wnt/beta-catenin signaling in prostate cancer PC-3 cells and inhibits PC-3 cell proliferation. Our results indicate that recombinant Mesd protein is a useful tool for studying Wnt/beta-catenin signaling on the cell surface and has a potential therapeutic role in Wnt-dependent cancers.

Cks1 plays an essential role in SCFSkp2-mediated ubiquitination, and consequently turnover, of the cdk2 inhibitor and tumor supressor p27Kip1. High Cks1 expression is associated with aggressive breast tumors and correlates with low p27Kip1 levels in some cases, although it is also an independent prognostic marker for survival, and provides predictive information in addition to that provided by p27Kip1 alone. In this report we demonstrate that Cks1 protein and mRNA are elevated to very high levels in mammary tumors initiated by erbB2, c-myc and polyoma middle-T (PyMT) in transgenic mice, whereas Cks1 protein is hardly detectable in the normal mammary epithelium. Cks1 is also highly upregulated in rat mammary tumors initiated by methylnitrosourea (MNU). Despite high levels of Cks1 expression, p27Kip1 levels were not reduced, and were in fact slightly higher in mammary tumors initiated by erbB2, PyMT and MNU. In contrast mammary tumors from MMTV-c-myc mice did exhibit low p27Kip1 and higher levels of Skp2. Together, these data suggest that deregulated Cks1 expression might play roles in oncogene and carcinogen-initiated mammary tumorigenesis independent of p27Kip1 turnover in certain tumors. Stable overexpression of Cks1 in human breast carcinoma MCF-7 cells did not significantly reduce p27Kip1 expression, although it conferred resistance to Faslodex (ICI 182780)-mediated inhibition of colony outgrowth in these cells. In contrast, Cks1-depleted MCF-7 cells formed fewer colonies in estrogen-containing medium. Therefore, our studies also suggest that Cks1 levels regulate the responsiveness of ER+ breast cancers to estrogens and anti-estrogens.

Chapter 25 Clofarabine: From Design to Approval John A. Secrist III, Southern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305, USASearch for more papers by this authorJaideep V. Thottassery, Southern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305, USASearch for more papers by this authorWilliam B. Parker, Southern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305, USASearch for more papers by this author John A. Secrist III, Southern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305, USASearch for more papers by this authorJaideep V. Thottassery, Southern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305, USASearch for more papers by this authorWilliam B. Parker, Southern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305, USASearch for more papers by this author Book Editor(s):Prof. Dr. Piet Herdewijn, Rega Institute, Katholieke Universiteit, Minderbroedersstraat 10, 003000 Leuven, BelgiumSearch for more papers by this author First published: 13 August 2008 https://doi.org/10.1002/9783527623112.ch25Citations: 2 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary This chapter contains sections titled: Introduction Clofarabine: The Background The Beginnings The Next Generation of Compounds Mechanism of Action of Clofarabine Clofarabine to the Clinic Summary and Comments References Citing Literature Modified Nucleosides: in Biochemistry, Biotechnology and Medicine RelatedInformation

Clinical studies have shown that decreased tamoxifen effectiveness correlates with elevated levels of vascular endothelial growth factor (VEGF)-A(165) in biopsy samples of breast cancers. To investigate the mechanisms underlying tamoxifen resistance and metastasis, we engineered the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line to express VEGF to clinically relevant levels in a doxycycline-regulated manner. Induction of VEGF expression in orthotopically implanted xenografts that were initially tamoxifen responsive and noninvasive resulted in tamoxifen-resistant tumor growth and metastasis to the lungs. Lung metastases were also observed in a VEGF-dependent manner following tail vein injection of tumor cells. At both primary and metastatic sites, VEGF-overexpressing tumors exhibited extensive fibroblastic stromal content, a clinical feature called desmoplasia. VEGF-induced metastatic colonies were surrounded by densely packed stromal cells before detectable angiogenesis, suggesting that VEGF is involved in the initiation of desmoplasia. Because expression of VEGF receptors R1 and R2 was undetectable in these tumor cells, the observed VEGF effects on reduction of tamoxifen efficacy and metastatic colonization are most likely mediated by paracrine signaling that enhances tumor/stromal cell interactions and increases the level of desmoplasia. This study reveals new roles for VEGF in breast cancer progression and suggests that combination of antiestrogens and VEGF inhibitors may prolong tamoxifen sensitivity and prevent metastasis in patients with ER-positive tumors.

Abstract Cks1, a small protein whose expression is strongly associated with aggressive breast tumors, is a component of cyclin-cdk complexes, as well as the SCFSkp2 ubiquitin ligase. In these studies, we explored its roles in estrogen receptor–positive breast tumor cells. When exposed to the antiestrogen ICI 182780, these cells accumulate in G1 by reducing the expression of Cks1, and increasing the levels of p130/Rb2, a cdk2 inhibitor and SCFSkp2 target. Heregulin β1 or estradiol abrogate antiestrogen effects by increasing Cks1 expression, down-regulating p130/Rb2 and inducing S phase entry. Depletion of Cks1 in these cells by RNA interference concomitantly decreased Skp2 and up-regulated p130/Rb2 and another SCFSkp2 target, p27Kip1. Remarkably, however, Cks1-depleted cells not only exhibit slowed G1 progression, but also accumulate in G2-M due to blocked mitotic entry. Notably, we show that cdk1 expression, which is crucial for M phase entry, is drastically diminished by Cks1 depletion, and that restoration of cdk1 reduces G2-M accumulation in Cks1-depleted cells. cdk1 reduction in Cks1-depleted cells is a consequence of a marked decrease in its mRNA and not due to alteration in its proteolytic turnover. Both heregulin β1 and estradiol could neither restore cdk1 nor sustain cycling in Cks1-depleted cells, although classical estrogen receptor function remained unaltered. Cks1 depletion also decreased Skp2 in human mammary epithelial cells without altering cell cycle progression. Thus, the indispensability of Cks1 to the breast cancer cell cycle, versus its redundancy in normal cells, suggests that Cks1 abrogation could be an effective interventional strategy in breast cancer. [Cancer Res 2007;67(23):11393–401]