George P. Studzinski

Highly active analogs of 1α,25-dihydroxyvitamin D3 that resist metabolism through C-24 oxidation and C-3 epimerization pathways

The secosteroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1alpha,25(OH)(2)-3-epi-D(3). A rational design for the synthesis of potent analogs of 1alpha,25(OH)(2)D(3) is developed based on the knowledge of the various metabolic pathways of 1alpha,25(OH)(2)D(3). Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D(3) analogs 1alpha,25(OH)(2)-16,23-E-diene-26,27-hexafluoro-19-nor-D(3) (Ro 25-9022), 1alpha,25(OH)(2)-16,23-Z-diene-26,27-hexafluoro-19-nor-D(3) (Ro 26-2198), and 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-D(3) (Ro 25-6760), as indicated by their antiproliferative activities.

Up-Regulation of Egr1 by 1,25-Dihydroxyvitamin D3 Contributes to Increased Expression of p35 Activator of Cyclin-Dependent Kinase 5 and Consequent Onset of the Terminal Phase of HL60 Cell Differentiation

Advances in differentiation therapy of cancer are likely to depend on improved understanding of molecular events that underlie cell differentiation. We reported recently that cyclin-dependent kinase (Cdk)5 and p35Nck5a (p35) are expressed in human leukemia HL60 cells induced to differentiate to monocytes by an exposure to 1,25-dihydroxyvitamin D3 (1,25D3), form a complex, and this complex has kinase activity (F. Chen and G. P. Studzinski, Blood 2001;97:3763). This laboratory has also provided evidence that the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway is active in the early (24–48 h) stages of HL60 cell differentiation induced by 1,25D3 but declines in the later, terminal phase of this form of differentiation (X. Wang and G. P. Studzinski, J Cell Biochem 2001;80:471). We examine now the hypothesis that Egr1 protein contributes to the up-regulation of p35 gene transcription and, thus, activated Cdk5/p35 kinase phosphorylates and inactivates mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1). Our data show that in 1,25D3-treated cells, p35 and Egr1 protein levels are elevated in a dose-dependent manner at the onset of the late stage of differentiation. We show also that 1,25D3 treatment of HL60 cells markedly increases the binding of Egr1 to an element in the p35 gene promoter, whereas transfection of an excess of this Egr1-binding oligonucleotide (“promoter decoy”) reduces p35 gene transcription and cell differentiation. Additionally, Cdk5/p35 phosphorylates MEK1 and inhibits its ability to phosphorylate its downstream target Erk2. These data suggest that in 1,25D3-treated HL60 cells, Egr1 up-regulates p35 gene transcription and that Cdk5/p35 kinase inactivates the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway by phosphorylation of MEK1, and this contributes to terminal differentiation of these cells.

Synergistic Antileukemic Activity of Carnosic Acid-Rich Rosemary Extract and the 19-nor Gemini Vitamin D Analogue in a Mouse Model of Systemic Acute Myeloid Leukemia

Objective: Differentiation therapy with the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D3), is a promising approach to treatment of acute myeloid leukemia (AML); however, 1,25D3 induces hypercalcemia at pharmacologically active doses. We investigated the in vitro and in vivoantileukemic efficacy of combined treatment with non-toxic doses of a low-calcemic 1,25D3 analogue, 1,25-dihydroxy-21(3-hydroxy-3-methyl-butyl)-19-nor-cholecalciferol (19-nor-Gemini; Ro27-5646), and rosemary plant agents in a mouse model of AML. Methods: Proliferation and differentiation of WEHI-3B D– (WEHI) murine myelomonocytic leukemia cellsin vitro were determined by standard assays. Reactive oxygen species, glutathione and protein expression levels were measured by flow cytometry, enzymatic assay and Western blotting, respectively. Systemic AML was developed by intravenous injection of WEHI cells in syngeneic Balb/c mice. Results: 19-nor-Gemini had a higher potency than its parent compounds, Gemini (Ro27-2310) and 1,25D3, in the induction of differentiation (EC50 = 0.059 ± 0.011, 0.275 ± 0.093 and 0.652 ± 0.085 nM, respectively) and growth arrest (IC50 = 0.072 ± 0.018, 0.165 ± 0.061 and 0.895 ± 0.144 nM, respectively) in WEHI cells in vitro, and lower in vivo toxicity. Combined treatment of leukemia-bearing mice with 19-nor-Gemini (injected intraperitoneally) and standardized rosemary extract (mixed with food) resulted in a synergistic increase in survival (from 42.2 ± 2.5 days in untreated mice to 66.5 ± 4.2 days, n = 3) and normalization of white blood cell and differential counts. This was consistent with strong cooperative antiproliferative and differentiation effects of low concentrations of 19-nor-Gemini or 1,25D3 combined with rosemary extract or its major polyphenolic component, carnosic acid, as well as with the antioxidant action of rosemary agents and vitamin D derivatives in WEHI cell cultures. Conclusion: Combined effectiveness of 1,25D3 analogues and rosemary agents against mouse AML warrants further exploration of this therapeutic approach in translational models of human leukemia.

DNA damage response: A barrier or a path to tumor progression?

Commentary to: DNA damage signaling is activated during cancer progression in human colorectal carcinoma Kazuhito Oka, Toshiki Tanaka, Tadahiko Enoki, Koichi Yoshimura, Mako Ohshima, Masayuki Kubo, Tomoyuki Murakami, Toshikazu Gondou, Yoshihide Minami, Yoshihiro Takemoto, Eijirou Harada, Takaaki Tsushimi, Tao-Sheng Li, Frank Traganos, Zbigniew Darzynkiewicz and Kimikazu Hamano

Differentiation and Cell Survival of Myeloid Leukemia Cells

The landscape of treatment for acute myeloid leukemia (AML) is currently a grim one. Apart from the AML subtype characterized by the 15:17 chromosome translocation known as acute promyelocytic leukemia (APL), which has shown lasting remissions when treated with all-trans retinoic acid (ATRA), especially when supplemented with the mildly toxic compound arsenic trioxide (ATO), mortality from the disease remains high. Thus, research into novel regimens of therapy is needed to supplement the current reliance on toxic compounds such as AraC and daunorubicin in the treatment of these diseases. Despite some spectacular clinical successes, ATRA-based differentiation therapy is not without its problems due to the induction of potentially life-threatening toxicities and the acquisition of therapeutic resistance in some patients. G. Brown and P. Hughes summarize the current state of knowledge in a comprehensive review entitled “Retinoid differentiation therapy for common types of acute myeloid leukemia” and suggest ways in which retinoid-based therapies can be improved by the inclusion of additional agents to increase the sensitivity of APL cells towards ATRA. This is followed in this issue by an example of cutting-edge research into the molecular basis for the efficacy of ATRA/ATO therapy for APL. B. Ozpolat et al. describe that at least part of the anti-APL effect of ATRA/ATO treatment can be explained by the inhibition of protein translation by these compounds. The report of these studies, entitled “PKCδ regulates translation initiation through PKR and eIF2α in response to retinoic acid in acute myeloid leukemia cells” also indicates that ATRA/ATO inhibit the PI3K/AKT/mTOR pathway, leading to an upregulation of the PKC delta/PKR axis. Both these articles add to our knowledge of the mechanism of ATRA in the treatment of myeloid leukemias and add to the debate on how this treatment may be improved. These new approaches may have importance outside the realms of leukemia treatments and may also lead to improved use of retinoids as therapies for other solid tumors. Chronic myeloid leukemia (CML) has considerably better prognosis than AML, and as with APL, treatment can be targeted to a hybrid gene, here Bcr-Abl, which results from a reciprocal translocation between chromosomes 9 and 22. Specific kinase inhibitors, such as Imatinib, have been identified and offer front-line treatment for CML. Unfortunately, resistance to kinase inhibitors frequently develops, and G. N. de Moraes et al. in a review entitled “The interface between BCR-ABL-dependent and -independent resistance signaling pathways in chronic myeloid leukemia” analyze the known causes for this resistance and offer several feasible molecular targets, which may overcome the development of resistance to kinase inhibitors in CML. The lessons from these two therapeutic successes are not easily transferred to other subtypes of myeloid leukemia, as the molecular lesions that can be attacked to eradicate the malignant cells appear to be “moving targets.” The mutations vary from AML case to case; secondary mutations appear and are often multiple, so current therapy largely depends on “brute-force” cell killing by administration of highly toxic agents, which have varying differential sensitivity for leukemic and normal cells. It seems, therefore, that alternate strategies are needed. The “by-pass the genetic lesion” suggested approach (G. P. Studzinski et al., PMID: 16046262) is based on the induction of cell differentiation by compounds such as vitamin D derivatives (VDDs). Here, instead of targeting a genetic lesion, the therapeutic agent induces the expression of transcription factors, which activate alternative but dormant routes to cell differentiation. Unfortunately, as pointed out by Harrison and J. A. Bershadskyi in a clinical perspective “Clinical experience using vitamin D and analogs in the treatment of myelodysplasia and acute myeloid leukemia: A review of the literature,” attempts to improve the therapy of AML with VDDs alone have not been successful so far. Further research on the mode of action of differentiation-inducing agents is, therefore, needed and the mechanisms involved should be explored in depth, to gain insights how to improve the differentiation regimens. One example of such exploration is provided in this issue by E. Gocek et al., who show multiple levels of regulation of the differentiation process in the report “Regulation of leukemic cell differentiation through the vitamin D receptor at the levels of intracellular signal transduction, gene transcription, protein trafficking and stability.” Another report on VDD action “Cell-type-specific effects of silibinin on vitamin D-induced differentiation of acute myeloid leukemia cells are associated with differential modulation of RXRα levels” by R. Wassermann et al. is more directly translational. This article addresses the potential pitfall of cell type specificity of biological responses. When attempts are made to enhance the effects of VDDs by combining them with an antioxidant silibinin, enhanced differentiation is seen in one AML subtype, but inhibition of differentiation is seen in another AML subtype. Advance knowledge of such antagonism is essential for future clinical trials. An exciting recent development in our understanding of the molecular basis of myeloid disorders is the realization that small noncoding RNAs play a role in these diseases. Y. Yuan et al. summarize a variety of such recent data in an article “MicroRNAs in acute myeloid leukemia and other blood disorders.” It seems that the information these and other studies described in this issue will provide a beginning of a long road that will eventually lead to much improved therapy, targeted or not, for myeloid leukemia. George P. Studzinski Geoffrey Brown Michael Danilenko Philip Hughes Ewa Marcinkowska

Abstract 1540: MAPK signaling profiles differ between 1,25D-sensitive HL60G and 1,25D-resistant 40AF human leukemia cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The resistance to 1,25-dihydroxyvitamin D3 (1,25D) could be an obstacle for potential clinical use of vitamin D in differentiation therapy of leukemia. 40AF cells are a subline of HL60 cells and provide a model for studies of 1,25D-resistance. It is known that 40AF cells grow more rapidly than the parental cells, and have a high percentage of cells in S phase. Also, the addition of the plant antioxidant carnosic acid and a kinase inhibitor SB202190 (the combination dubbed DCS) can partially overcome the resistance to 1,25D. To better understand the basis for this resistance we examined MAPK network profiles, previously shown to be important for HL60 cell differentiation, on mRNA level by RT2-PCR arrays. The results showed that 17 out of 84 genes studied had significantly altered mRNA levels (more than 2 fold) in 40AF cells compared to HL60G cells. Eighteen genes had lower basal level of expression in 40AF compared to HL60G cells, and after partial reversal of resistance by DCS treatment these genes showed increased expression; in contrast, 20 genes had higher basal levels in 40AF cells and their expression was decreased by DCS, suggesting that these genes may be involved in 1,25D resistance. Also, DCS treatment of 40AF cells changed the expression of the RAS-ERK pathway components more obviously than the JNK and the p38MAPK pathways. Mitotic cyclins and CDKs had a generally enhanced expression in 40AF cells compared to HL60G, and their expression decreased following DCS treatment, consistent with the slow down of the cell cycle by DCS, perhaps also because several CKIs, including p21Cip1, dramatically increased. Studies using Phospho-MAPK arrays showed that treatment of both HL60G and 40AF cells with 1,25D or DCS altered the levels of phosphorylated proteins of MAPK family members P-ERK2 in the ERK pathway, P-JNK2 in the JNK pathway, P-p38 alpha/delta/gamma in the p38 pathway, P-Akt1 in the PI3/Akt pathway. The gene expression studies will be validated by Westerns, and MAPK activity by kinase assays. In summary, MAPK signaling networks may play important roles in 1,25D resistance in AML cells, and modification of network expression can influence cell cycle progression and cell differentiation to partially reverse the resistance to 1,25D of non-responding leukemia cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1540.