J Cancer Res Clin Oncol (1992) 118:190-194

C~i~iieer~esearch Clinical 9 9 Springer-Verlag 1992

Effects of analogs of 1,25(OH)2 Vitamin D3 on the proliferation and differentiation of the human chronic myelogenous leukemia cell line, RWLeu-4 Jeffrey W. Clark 1, Marshall R. Posner 1, Jean M. Marsella 1, Anabel Santos 1, Milan Uskokovic 2, Charles Eil 3, and Stephen R. Lasky 3 Divisions of Hematology/Oncology and Endocrinology, Roger Williams General Hospital and Brown University School of Medicine, Providence, Rhode Island 02908, USA Hoffman-La Roche 3, Nutley, New Jersey 07110, USA Received 21 August 1991/Accepted 16 October 1991

Summary. We evaluated the proliferative and differentiative effects of analogs of 1,25(OH)2 vitamin D3 [1,25(OH)/D3] on a chronic myelogenous leukemia cell line, RWLeu-4, which is growth-inhibited and differentiates in response to 1,25(OH)2D 3 (EDs0=3-10nM). Side-chain-fluorinated analogs were more potent (EDso = 0.7-2 nM) while most of those with altered saturation of the D ring or side-chain carbon-carbon bonds were equally or less effective than 1,25(OH)/D 3. However, the two analogs with either two additional double bonds or an extra double and triple bond in the D ring had greater antiproliferatiive activity [1,25(OH)2-16,23diene D 3 (EDso = 2.7 nM) and 1,25(OH)z-16-ene-23-yne D 3 (EDso=0.7 nM)]. Since the latter of these has been reported to be less potent at mobilizing calcium than 1,25(OH)eD3, it (or a similar compound) may be a candidate for clinical use as an antineoplastic agent. Key words: Vitamin D 3 - Analogs - Chronic myelogenous leukemia cells - Differentiation - Proliferation

Introduction 1,25(OH)2 vitamin D 3 [1,25(OH)2D3] is a seco-sterol hormone with well-known actions in calcium and phosphate homeostasis. Most of its actions are thought to be mediated through the binding and activation of high-afAcknowledgements. This work was supported by grants from Rhode Island Foundation (to S.R.L. and C.E,) and National Institutes of Health (P30-CA13943 to the Cancer Research Center at Roger Williams General Hospital, RO1-CA50558 to S.R.L. and J.W.C. and RO1-CA50054 to M.R.P.). Abbreviations: 1,25(OH)zD3, lc~,25-(OH)2 vitamin D3; CML, chronic myelogenous leukemia; NBT, nitroblue tetrazolium; EDso, 50% effective dose; ICs0, 50% inhibitory concentration Offprint requests to: J. W. Clark, Department of Hematology/Oncology, Roger Williams General Hospital, 825 Chalkstone Ave., Providence, RI 02908, USA

finity nuclear receptors. Once bound with the hormone, activated receptors are capable of modulating tissue-specific gene expression by binding to the regulatory regions of the DNA in target genes. Extremely sensitive radioligand receptor-binding assays have now shown that the vitamin D receptors are not only present in the classical 1,25(OH)2D 3 target tissues of the bone, intestinal mucosa, and kidney, but are also found in the endocrine glands, the brain, the thymus and hematopoietic cells (Reichel et al. 1989; Minghetti et al. 1988; Rigby et al. 1987). The presence of high-affinity vitamin D 3 receptors in so many cell types suggests that the biological functions of 1,25(OH)2D3 may include the regulation of gene expression, cell growth, proliferation, and differentiation in many diverse systems. While 1,25(OH)2D3 stimulates the proliferation of at least certain normal myeloid cells, it can also inhibit the growth of myeloid leukemia cells and induce the differentiation of the promyelocytic leukemia cell line HL-60 into monocytic/macrophage-like cells (Koeffler et al. 1985; McCarthy et al. 1983; Mangelsdorf et al. 1984). Thus, this hormone can act as a modulator of lymphoid and myeloid cells as do other steroid and peptide hormones. While it has been clearly demonstrated that 1,25(OH)2D3 causes the cessation of.proliferation and the induction of differentiation of leukemic cells into more mature cells, the hypercalcemic effects of 1,25(OH)2D 3 in vivo severely limit the use of this agent in the treatment of leukemia. Reports from several groups have suggested that changing the aliphatic sidechains or the D ring on 1,25(OH)zD 3 may alter the potency of its calcium-mobilizing actions as compared with its effects on proliferation or differentiation (Abe et al. 1989; Binderup and Bramm 1988; Norman et al. 1990; Perlman et al. 1990; Zhou ct al. 1989). More recently, a number of 1,25(OH)2D 3 analogs have been shown to be significantly more potent at inducing differentiation and inhibiting proliferation of HL-60 cells than 1,25(OH)2D 3 while they are significantly less potent in bone calcium mobilization or intestinal calcium absorption (Zhou et al.

191

1991). Therefore, it may be possible to synthesize therapeutic 1,25(OH)2D3 hormone analogs that will inhibit the growth of and differentiated neoplastic cells without causing hypercalcemia and its attendant side-effects. The work presented here describes the effects of 1,25(OH)aD 3 and some of its analogs on the proliferation and differentiation of a 1,25(OH)zD3-responsive chronic myelogenous leukemia (CML) cell line, RWLeu-4. RWLeu-4 cells carry the Philadelphia chromosome (PH1), which is characteristic of CML, express constitutive p210 bcr-~bttyrosine kinase activity and thus provide a good in vitro model for evaluating the differentiation of CML cells (Lasky et al. 1990). 1,25(OH)aD 3 inhibits their proliferation and induces them to differentiate along a monocyte/macrophage-like pathway into cells with a more mature phenotype.

Materials and methods Reagents. Stock solutions of 1,25(OH)2D 3 and analogs of 1,25(OH)zD 3 were prepared in absolute ethanol, sparged with nitrogen gas and protected from direct light. [5-methyl-3H]Thymidine was purchased from ICN. All other chemicals were of the highest commercially available purity. Cells and cell culture. RWLeu4 was cultured in RPMI-1640 medium supplemented with penicillin, streptomycin, glutamine and 10% fetal calf serum in a 5% CO2 atmosphere at 37~ C. Cell number and viability were determined by fight-microscopic examination of trypan blue dye exclusion. Morphological characteristics of the cells were determined by examination of Wright-Geimsa-stained cells. Morphological changes consistent with a mature monocytic/macrophage-like phenotype of these cells include clumping, adherence to plastic, development of lobed nuclei, and a ruffled cytoplasmic membrane with pseudopod-like extensions (Lasky et al. 1990). The ability of the cells treated with 1,25(OH)2D 3 or various analogs to undergo oxidative metabolism, a characteristic of differentiation into more mature monocyte/macrophage-like cells, was determined by nitroblue tetrazolium (NBT) reduction. For this assay, 2 • 104 cells were resuspended in 100 I~1RPMI-1640 medium with 10% Fetal calf serum to which was added 50 Ixl phosphate-buffered saline (10 mM potassium phosphate/146 mM sodium chloride, pH 7.2) containing NBT (2 mg/ml) (Research Organics, Cleveland, Ohio) and 12-O-tetradecanoylphorbol 13-acetate (1.2 Ixg/ml). The cells were incubated in 5% CO2 at 37~ C for 30 min and evaluated by light microscopy for formation of dark blue crystals. Effects of 1,25(OH)2D 3 and analogs on cellular DNA synthesis. Triplicate cultures of 1 • 105 RWLeu-4 cells/ml were seeded into 96well microtiter plates in a final volume of 200 Isl RPMI-1640 containing diluent alone or serial threefold dilutions of 1,25(OH)2D 3 or analogs of 1,25(OH)2D3 . Concentrations ranged between 0.1 nM and 500 nM. After 72 h of treatment, 0.5 pCi [5-rnethyl-3H]thy midine was added to each well. After a 4-h pulse the cells were harvested onto glass-fiber filters using a Cambridge PHD cell harvester and the amount of label incorporated into the macromolecular fraction was determined by liquid scintillationcounting. Results are expressed as the percentage of control cell incorporation. Average control incorporation was approximately 90 000 cpm/well. Immunofluorescent detection of cell-surface antigens. In addition to morphological changes and ability to reduce NBT, the extent of monocytic differentiation was analyzed by changes in cell-surface antigens consistent with a more mature monocytic/macrophage cell using monoclonal antibodies reactive with CD4, CD1 lb (Mol), and CD14 (Mo2). An antibody reactive with the T-cell marker CD8 served as a negative control. Indirect immunofluorescent detection

of cell-surface antigens was performed on an EPICS C cell sorter (Coulter, Hialeah, Fla.) as previously described (Lasky et al. 1990; Posner et al. 1987). The significance of changes in the expression of cell-surface,antigens was analyzed using the Wilcoxon rank-sums test on data from three independent experiments.

Results

Table 1 lists the analogs that were tested for the inhibition of [3H]thymidine incorporation of RWLeu-4. As can be seen from the formulas listed in column one, there are three main classes of analogs of 1,25(OH)zD3: those with fluorine or deuterium atoms added to their side-chains (analogs 1-5); those with single-, double- or triple-bond alterations in their D rings or side-chains (analogs 6-9); and those with either two double-bond or a double- and triple-bond alteration (analogs 11 and 12). Analog 10 is a modification of 1,25-dihydroxyvitamin D 2 . Compounds are assigned numbers in the second column, which are used in the figure legends and Discussion. The third column represents the average IC5o [the concentration (molarity) of the compound that produced a 50% reduction in [3H]thymidine incorporation by the cells after 72 h of treatment] from at least three separate experiments. The final column represents the relative inhibitory index based on the result of the division of the IC50 of 1,25(OH)2D a by the IC5o of each analog. Figure 1 shows the dose/response graphs of analogs with fluorine or deuterium atoms in their side-chains. As can be seen, fluorination of the side-chain increased the inhibitory properties of the analogs, while deuteration of the same position had no effect (comparing compounds 1 and 5). There is a statistically significant difference in inhibition between all of the compounds except compounds in Fig. 1 and 1,25(OH)2D 3 at the 1 nM, 3 nM, and 10 nM doses. Figure 2 compares analogs that have been modified by the addition of a single, double or triple bond in the D ring (position 16) or in the side-chain (positions 22 and Table 1. The structure of the various vitamin D3 (VD3) analogs that were tested for their antiproliferative and differentiative effects on the RWLeu-4 CML cell line

Analog

Number ICso a

RI b

1,25(OH)zD3 1,25(OH)2-26,27-hexafluoro-Da 1,25(OH)z-22-ene-26,27-hexafluoro-D a 1,25S(OH)2-26-trifluoro-D a 1,25(OH)z-24-difluoro-D 3 1,25(OH)2-26,27-hexadeutero-Da 1,25(OH)z-16-ene-D 3 1,25(OH)2-22-ene-D 3 1,25(OH)2-23-yne-D 3 1,25S,26(OH)3-22-ene-D 3 1,25(OH)2-24epi-D z 1,25(OH)2-16-ene-23-yne-D3 1,25(OH)/-16,23-diene-Da

VD 3 1 2 3 4 5 6 7 8 9 10 11 12

1.0 14.3 10.0 5.9 5.0 1.0 1.0 1.0 1.0 0.5 1.0 14.3 3.7

10.0 0.7 1.0 1.7 2.0 10.0 10.0 10.0 10.0 50.0 10.0 0.7 ! 2.7

" The IC5o is the dose of the compound in nM that decreases [3H]thymidine incorporation by 50% as compared to untreated cells after 72 h of incubation. Each ICso represents the mean of three separate experiments b The relative inhibitory index (RI) is the ratio of the IC50 of vitamin Da to that of the compound

192 FLUORINATED

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Fig. 1. Inhibition of [3H]thymidine incorporation in RWLeu-4 ceils treated with various fluorinated or deuterated analogs [1,25(OH)2Da as a control]. Each dose point is the mean (+ SE) of three separate experiments with triplicate wells counted for each experiment. [], 1,25(OH)2Da; B, 1; D, 2; 9 3; o, 4;~,5 Numbers refer to compounds in Table 1

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Fig. 3. Inhibition of [3H]thymidine incorporation in RWLeu-4 cells treated with analogs of 1,25(OH)2D3 modified either with two double bonds (compound 12) or both a double and a triple bond (compound 11) [1,25(OH)2D3 as a control]. Each dose point is the mean (__.SE) of three separate experiments with triplicate wells counted for each experiment. [], 1,25(OH)zD3; o, 11; , , 12 Numbers refer to compounds in Table 1 Table 2. Changes in cell-surface markers on RWLeu-4 cells and per-

1,25(OH)2-D3 6 7

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centage of cells positive for nitroblue tetrazolium (NBT) reduction after 72 h of treatment with 1,25(OH)zD3 or various analogs (as numbered in Table 1). Control cells were treated with diluent alone9 Values represent the percentage of total cells positive for the indicated cell-surface marker

I N E F F E C T I V E SIDE C H A I N MODIFICATIONS 90

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Fig. 2. Inhibition of [3H]thymidine incorporation in RWLeu-4 ceils treated with non-fluorinated analogs containing single side-chain modifications of 1,25(OH)2D 3 or ergocalciferol (compound 10) [1,25(OH)2D3 as a control]. Each dose point is the mean (_SE) of three separate experiments with triplicate wells counted for each experimentt, n, 1,25(OH)2Da; B, 6 ; , , 7; n, 8;O, 9; o , 10

23). These analogs showed no significant change in their ICs0 values compared to 1,25(OH)2D3. The results also indicated that the 24 epi form of ergocalciferol (vitamin D2) (compound 10) was essentially as effective as cholecalciferol at inhibiting proliferation of RWLeu-4. Figur~ 3 illustrates the dose response of two analogs that have been modified by the addition of two double bonds or a double bond and a triple bond in their D ring and sidechain. These analogs were effective inhibitors of RWLeu4 cells, comparable in potency to the fluorinated analogs. Table 2 shows the differentiative abilities of various 1,25(OH)2D 3 analogs as determined by changes in cell-

surface antigen expression and NBT reduction. The RWLeu-4 cell line expresses moderate levels of C D l l b (Mol) and CD4 but negligible levels of CD14 (Mo2) in the unstimulated state. 1,25(OH)2D 3 induces monocytic differentiation in RWLeu-4 cells with significantly increased C D l l b and CDI4 expression, and reduced CD4 expression. As shown in Table 2, 1,25(OH)2D 3 and the various analogs tested all significantly induced the differentiation of RWLeu-4 cells. All analogs were used at 50 nM concentrations for the differentiating experiments and since this is significantly above the EDso values for these compounds, subtle differences in effects due to different EDso values would not be seen. In all cases, differentiation as measured by increases in C D l l b and CD14 cell-surface antigens and NBT reduction closely correlated with morphological differentiation as determined by light microscopy. Discussion

We have shown that analogs of 1,25(OH)2D 3 inhibited the proliferation of RWLeu-4 cells and that some D-ring

193 and side-chain modifications significantly affected this response. The addition of fluorine atoms to the aliphatic side-chains increased the antiproliferative potency of the analogs more than fivefold. The antiproliferative activities of many of the fluorinated analogs of 1,25(OH)2D 3 have been reported to correlate with their abilities to mobilize calcium uptake in chick duodenum as well as in human bone-derived cells (Norman et al. 1990; Kiriyama et al. 1991). This suggests that even though these compounds are more potent at inducing differentiation and inhibiting proliferation than 1,25(OH)2D3 on a molar basis, there is not a sufficient enough differential between their maturation-inducing and calcium-mobilizing effects for them to be clinically useful as antineoplastic agents. However, fluorinated compounds that also have both a double bond in the 16 and triple bond in the 23 position appear to have potent antiproliferative effects while being significantly less calcemic than 1,25(OH)zD3 (Zhou et al. 1991; see discussion below). Substituting deuterium atoms in the same positions did not increase the potency of the analog when compared to the antiproliferative action of 1,25(OH)zD 3. Although the 24-epi-ergocalciferol (compound 10) was essentially equal in efficacy to 1,25(OH)zD 3 at inhibiting RWLeu-4 (Fig. 2), it is significantly less calcemic and it has been shown to induce effectively the differentiation of the HL-60 human promyelocytic leukemia line also at concentrations comparable to those of 1,25(OH)2D 3 (Sato et al. 1991). Therefore, this or similar compounds might be potentially useful antitumor agents and further study of other analogs of ergocalciferol, which might be more potent at inducing differentiation, is warranted. Analogs with two or more double or triple bonds in their side-chains or D ring would appear to be the most promising antineoplastic compounds for clinical studies. Our data indicated that the addition of two double bonds at positions 16 (in the D ring) and 23 (in the side-chain) enhanced the anti-proliferative activity of 1,25(OH)2D 3 over threefold, whereas the addition of a double bond at carbon 16 and a triple bond at carbon 23 enhanced this activity more than tenfold. Addition of a single double or triple bond at either of these positions alone did not enhance the anti-proliferative activity of the analogs over that of 1,25(OH)zD3 (compounds 6 and 8 in Table 1). Most interestingly, as mentioned above, certain fluorinated compounds that also have the double bond at the 16 position and a triple bond at the 23 position are both more potent at inhibiting the proliferation of leukemic cells while being less calcemic (Zhou et al. 1991). Analogs with both of these features would appear to be promising for further study as potential differentiating agents, It has been recently shown that one of the analogs we tested [1,25(OH)z-16-ene-23-yne-D3, compound 11 in Table 1] is very effective at inhibiting the prqliferation of a number of leukemia cell lines while being significantly less potent in calcium mobilization than 1,25(OH)2D3 (Norman et al. 1990; Zhou et al. 1989). This analog has a decreased affinity for the 1,25(OH)2D 3 receptor and decreased binding to vitamin D3 serum binding protein, both of which may contribute to the fact that it is much less calcemic than 1,25(OH)2D3 (Dusso et al. 1991;

Norman et al. 1990). However, the exact mechanisms for the difference in its antiproliferative as opposed to calcemic effects remain to be elucidated and possible differences in the metabolism of the analog as compared to that of 1,25(OH)zD 3 may also be an important factor (Zhou et al. 1991). This analog is also able to inhibit leukemia growth in mice without untoward toxicity (Zhou et al. 1990). These findings, taken along with the data reported here, suggest that it may be possible at least partially to separate the calcemic effects of 1,25(OH)/D3 from its maturation-inducing effects by modifying its structure. The development of potentially new clinically useful differentiating agents for C M L is important for a number of reasons. This is a disease for which the only known curative therapy is bone marrow transplantation and this is not applicable for all patients, especially those who are older (McGlave 1990). Although the mechanisms by which interferons work against C M L are not certain, it is possible that some of the effect is mediated by differentiating effects on these cells (Appelbaum 1990). Therefore, agents that can induce differentiation of C M L cells have the potential for improving survival of these patients. It may be possible to use vitamin D 3 analogs such as number 11 at subtoxic doses in combination with other potential maturation-inducing agents such as bryostatin, retinoic acid, n-methylformamide, or interferons to induce differentiation of C M L cells more effectively in vivo. We are presently beginning our investigation of these possibilities in vivo as well as continuing the mechanistic studies in vitro.

Acknowledgements. This work was supported by grants from Rhode Island Foundation (to S.R.L. and C.E.) and National Institutes of Health (P30-CA13943 to the Cancer Research Center at Roger Williams General Hospital, RO1-CA50558 to S.R.L. and J.W.C. and RO1-CA50054 to M.R.P.). References

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