We present data suggesting a function of ␣ 2 -HS glycoproteins/fetuins in serum and in mineralization, namely interference with calcium salt precipitation. Fetuins occur in high serum concentration during fetal life. They accumulate in bones and teeth as a major fraction of noncollagenous bone proteins. The expression pattern in fetal mice confirms that fetuin is predominantly made in the liver and is accumulated in the mineralized matrix of bones. We arrived at a hypothesis on the molecular basis of fetuin function in bones using primary rat calvaria osteoblast cultures and salt precipitation assays. Our results indicate that fetuins inhibit apatite formation both in cell culture and in the test tube. This inhibitory effect is mediated by acidic amino acids clustering in cystatin-like domain D1. Fetuins account for roughly half of the capacity of serum to inhibit salt precipitation. We propose that fetuins inhibit phase separation in serum and modulate apatite formation during mineralization.

Kinesin-5 inhibitors (K5I) are promising antimitotic cancer drug candidates. They cause prolonged mitotic arrest and death of cancer cells, but their full range of phenotypic effects in different cell types has been unclear. Using timelapse microscopy of cancer and normal cell lines, we find that a novel K5I causes several different cancer and noncancer cell types to undergo prolonged arrest in monopolar mitosis. Subsequent events, however, differed greatly between cell types. Normal diploid cells mostly slipped from mitosis and arrested in tetraploid G 1 , with little cell death. Several cancer cell lines died either during mitotic arrest or following slippage. Contrary to prevailing views, mitotic slippage was not required for death, and the duration of mitotic arrest correlated poorly with the probability of death in most cell lines. We also assayed drug reversibility and long-term responses after transient drug exposure in MCF7 breast cancer cells. Although many cells divided after drug washout during mitosis, this treatment resulted in lower survival compared with washout after spontaneous slippage likely due to chromosome segregation errors in the cells that divided. Our analysis shows that K5Is cause cancer-selective cell killing, provides important kinetic information for understanding clinical responses, and elucidates mechanisms of drug sensitivity versus resistance at the level of phenotype. [Mol Cancer Ther 2008;7(11):3480 -9]

The role of Transforming growth factor beta (TGF-b) in carcinogenesis is complex. There are reports on both tumor inhibition and tumor promotion by TGF-b. To elucidate the complex role of TGF-b in epithelial carcinogenesis, we generated transgenic mice overexpressing a dominant negative type II TGF-b receptor in the basal cell compartment and in follicular cells of the skin. Despite the reduced responsiveness of transgenic keratinocytes to TGF-b, both proliferation and di erentiation were normal in non-irritated epidermis of these transgenic mice. Thus, interruption of signaling of all three isoforms of TGF-b in basal and follicular cells does not disturb tissue homeostasis. However, during tumor promotion transgenic mice showed an elevated level of proliferation in the epidermis. This hyperproliferation correlated with a very early onset of carcinoma development and a malignant conversion frequency of 30% from benign papillomas to carcinomas. By comparison, the conversion frequency in wild-type mice of this strain has previously been reported as 5.5%. Even without induction of hyperproliferation by tumor promoters, transgenic mice developed far more carcinomas as controls when treated with a carcinogen. This result indicates that there is a synergistic e ect between loss of TGF-b responsiveness and mutations caused by initiation with a carcinogen leading to an endogeneous tumor promotion in initiated cells only.

Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft tumors led to an increased number of persistent DSBs. Oral administration of M3814 to two xenograft models of human cancer, using a clinically established 6-week fractionated radiation schedule, strongly potentiated the antitumor activity of IR and led to complete tumor regression at nontoxic doses. Our results strongly support DNA-PK inhibition as a novel approach for the combination radiotherapy of cancer. M3814 is currently under investigation in combination with radiotherapy in clinical trials.

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Abstract.Combination therapies are widely accepted as a cornerstone for treatment of different cancer types. A tumor growth inhibition (TGI) model is developed for combinations of cetuximab and cisplatin obtained from xenograft mice. Unlike traditional TGI models, both natural cell growth and cell death are considered explicitly. The growth rate was estimated to 0.006 h −1 and the natural cell death to 0.0039 h −1 resulting in a tumor doubling time of 14 days. The tumor static concentrations (TSC) are predicted for each individual compound. When the compounds are given as single-agents, the required concentrations were computed to be 506 μg · mL −1 and 56 ng · mL −1 for cetuximab and cisplatin, respectively. A TSC curve is constructed for different combinations of the two drugs, which separates concentration combinations into regions of tumor shrinkage and tumor growth. The more concave the TSC curve is, the lower is the total exposure to test compounds necessary to achieve tumor regression. The TSC curve for cetuximab and cisplatin showed weak concavity. TSC values and TSC curves were estimated that predict tumor regression for 95% of the population by taking between-subject variability into account. The TSC concept is further discussed for different concentration-effect relationships and for combinations of three or more compounds.

Anti-EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR 1 solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibodydependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab-mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA-A2 and adoptively transferred human HLA-A2 1 PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti-EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti-EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8 1 T effector cells. This novel mechanism of ADCC conducted by CD8 1 T effector cells was restricted to IgG1 anti-EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8 1 T effector cell-mediated ADCC was enhanced in the presence of IL-15 and strongly improved after glycosylation of anti-EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.

The protein kinase ataxia telangiectasia mutated and Rad3-related ATR is one of the key mediators of the DNA damage response. ATR is recruited to regions of single-stranded DNA, which most commonly arise during replication stress (RS). RS occurs during S-phase when the cell’s DNA replication machinery encounters problems such as unresolved DNA lesions. In addition, treatment of cells with DNA-damaging agents can lead to RS as cells progress to S-phase without resolving damage incurred by such agents. Elevated levels of RS are evident in some cancer cells, even in the absence of a DNA-damaging agent resulting from expression of oncogenes that drive dysregulated replication, a hypoxic environment, or from defects in other repair pathways. RS in cancer cells can drive reliance on ATR for survival and, accordingly, ATR inhibitors may have benefit as monotherapy. M4344 was determined to be an adenosine triphosphate (ATP)-competitive, highly potent, and tight-binding inhibitor of ATR with a Ki of < 150 pM. Minimal inhibitory activity was observed against a large panel of unrelated protein kinases, with 308 of 312 kinases tested having a measured Ki corresponding to more than 100-fold selectivity. M4344 potently inhibits ATR-driven phosphorylated checkpoint kinase-1 (P-Chk1) phosphorylation with an IC50 of 8 nM. Profiling on a selected set of cancer cell lines showed synergy with several types of DNA damaging chemotherapeutics as well as PARP1/2 and CHK1 inhibitors. In monotherapy efficacy studies M4344 showed tumor stasis to regression in tumor models with alternative lengthening of telomeres (ALT). In combination with PARP inhibitors, tumor regression could be observed in triple-negative breast cancer xenograft models. A dose-escalation phase 1 study in patients with advanced solid tumors is currently ongoing. Citation Format: Frank T. Zenke, Astrid Zimmermann, Heike Dahmen, Brian Elenbaas, John Pollard, Philip Reaper, S Bagrodia, M E. Spilker, C Amendt, Andree Blaukat. Antitumor activity of M4344, a potent and selective ATR inhibitor, in monotherapy and combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 369.