Identification of methotrexate as a heterochromatin-promoting drug

Heterochromatin is a tightly packed form of DNA involved in gene silencing, chromosome segregation, and protection of genome stability. Heterochromatin is becoming more recognized in tumor suppression and may thus serve as a potential target for cancer therapy. However, to date there are no drugs that are well established to specifically promote heterochromatin formation. Here, we describe a screening method using Drosophila to identify small molecule compounds that promote heterochromatin formation, with the purpose of developing epigenetic cancer therapeutics. We took advantage of a Drosophila strain with a variegated eye color phenotype that is sensitive to heterochromatin levels, and screened a library of 97 FDA approved oncology drugs. This screen identified methotrexate as the most potent small molecule drug, among the 97 oncology drugs screened, in promoting heterochromatin formation. Interestingly, methotrexate has been identified as a JAK/STAT inhibitor in a functional screen, causing reduced phosphorylation of STAT proteins. These findings are in line with our previous observation that unphosphorylated STAT (uSTAT) promotes heterochromatin formation in both Drosophila and human cells and suppresses tumor growth in mouse xenografts. Thus, Drosophila with variegated eye color phenotypes could be an effective tool for screening heterochromatin-promoting compounds that could be candidates as cancer therapeutics.

addition, it has been shown that BRCA1, a tumor suppressor gene well established to be associated with breast and ovarian cancer, prevents tumor formation through heterochromatin mediated silencing 7 . Retinoblastoma (Rb) tumor surpressor, a protein that suppresses transcription of E2F genes upon DNA damage in order to prevent the division of damaged cells, also mediates its effects through stabilizing heterochromatin [8][9][10][11][12] .
We have previously identified a non-canonical JAK-STAT pathway in which un-phosphorylated STAT promotes and stabilizes heterochromatin by binding to the HP1 chromodomain protein [13][14][15] . We have further shown that increasing global heterochromatin levels through expressing HP1a or unphosphorylatable STAT5A can suppress colon cancer growth using a mouse xenograph model. Interestingly, many genes down-regulated by STAT5A and HP1a in common have previously been shown to be overexpressed in or be drivers of human cancers, although genes involved in proliferation and cell death are not significantly affected 16 . These results support the idea that heterochromatin may offer a unique target for human cancers and could offer a treatment that is associated with less side effects compared with the cytotoxic oncology drugs.
For the purpose of identifying compounds that promote heterochromatin formation and for further research as candidates for cancer therapy, we designed and performed a simple screen for small-molecule compounds that can promote heterochromatin formation in Drosophila, which is an inexpensive in vivo system for drug discovery. These compounds will serve as lead molecules for developing epigenetic cancer drugs in the future.

Results
Screening of Oncology Set III using DX1 flies for heterochromatin-promoting drugs. To identify heterochromatin-promoting drugs, we sought to develop a screening method using Drosophila, an inexpensive in vivo system (Fig. 1). To this end, we obtained from the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) a small-molecule drug library, Oncology Set III, consisting of 97 FDA approved oncology drugs (Table S1). We screened this library using the DX1 strain of Drosophila melanogaster. DX1 flies contain seven tandem copies of a P[mini-white + ] reporter transgene inserted into a euchromatic region of the 2 nd chromosome, which induce heterochromatin formation at the insertion locus due to the repetitive nature of the transgene copies, resulting in variegated expression of the P[mini-white + ] reporter transgenes in white −/− (w −/− ) genetic background 17,18 . The white + gene encodes the transporter that carries precursors of the red and brown pigments into developing eyes during pupation, resulting in red colored eyes when adult flies emerge; flies lacking the white + gene have white eyes 19 . Thus the amount of red pigmentation in DX1 flies is proportional to the level of the white + gene products from the P[mini-white + ] transgene. Due to the metastable nature of heterochromatin induced at the P[mini-white + ] tandem repeats, transcription of P[mini-white + ] and thus the degree of variegation in the eye color of DX1 flies is sensitive to heterochromatin levels. Increasing heterochromatin levels leads to more variegation, less mini-white + expression and overall red eye pigmentation; decreasing heterochromatin levels has the opposite effects, resulting in increased red eye pigmentation 14,15,17,18 . Thus, the levels of red pigmentation in DX1 flies can serve as a convenient readout of heterochromatin levels in vivo.
We screened the Oncology Set III library based on the assumption that compounds that promote heterochromatin should cause more variegation in the eye color in DX1 flies and thus reduced red eye pigmentation, as has been previously reported 14,15,17,18 . For screening, we crossed DX1/CyO male flies to w 1118 , a commonly Identification of methotrexate as a heterochromatin-promoting drug. We scored each F1 w 1118 /Y; DX1/+ adult male fly on a scale from 1 to 5, 1 being most variegated and 5 being least variegated (Fig. 1). We then averaged the scores of all the F1 w 1118 /Y; DX1/+ males in each vial. Three trials were done per compound and were represented in a bar graph ( Fig. 2A). We identified hit drugs as compounds that received a score that was two standard deviations below the mean of the control, which were exposed to solvent only (33% DMSO in H 2 O). The control flies received a mean score of 3.82 with a standard deviation of 0.67. Therefore a drug that receives a score different from the control score by more than two standard deviations would be considered a hit. The drug that caused the most variegation in this set was methotrexate (4-aminopteroylglutamic acid), with a code name E7 in Plate 1 of the drug library, which received a score of 2.43, less than 2.48 or two standard deviations from the control mean of 3.82 (Fig. 2). Methotrexate was the only drug that received a score two standard deviations below that of the control among all the 97 Oncology Set III drugs screened. We thus pursued methotrexate further as a heterochromatin-promoting drug.

Methotrexate promotes heterochromatin formation and reduces overproliferation caused by JAK overactivation.
To confirm that methotrexate indeed promotes heterochromatin formation, we examined the levels of heterochromatin mark, histone H3 trimethylated at lysine 9 (H3K9me3), after treating larvae with methotrexate. Using immunostaining with anti-H3K9me3 antibodies, we found that H3K9me3 levels were higher in 3 rd instar larval salivary gland cells of methotrexate-treated larvae when compared with control larvae treated with solvent only (Figs 3A,B and S1A). Drosophila larval salivary glands contain large cells with prominent heterochromatin foci, which allow easy visualization and heterochromatin levels and morphology, as we have previously reported 14,15 . Heterochromatin levels can also be revealed by HP1 immunostaining, as we have previously shown 14 . Indeed, larval salivary gland nuclei show significantly larger HP1-positive heterochromatin area after methotrexate treatment than control (Figs 3C,D and S1B). The effects of methotrexate treatment on H3K9me3 levels are similar to those resulting from genetically altering JAK/STAT signaling, as we have previously shown 14,15 . Lastly, using Western blotting, we confirmed that methotrexate treatment causes an increase in levels of H3K9me3 in treated larvae (Fig. 3G). Thus, we are able to confirm at cellular and protein level, using immunostaining, that methotrexate promotes heterochromatin formation.
Methotrexate is one of the first chemotherapy agents used for cancer treatment, and it is also used as an immunosuppresant to treat rheumatoid arthritis 20,21 . Methotrexate is believed to be an antimetabolite that acts as a competitive inhibitor of dihydrofolate reductase (DHFR) 22 . Interestingly, in a screen for small molecules that  Table S1). (D) Representative pictures of the eyes of E7 (methotrexate) and control DX1 flies. modulate JAK/STAT signaling using cells, methotrexate was identified as a JAK/STAT pathway inhibitor, which reduces both STAT reporter gene expression in Drosophila cells and STAT5 phosphorylation in human cells 23 .
To test the effects of methotrexate on JAK/STAT signaling in flies, we used transgenic flies that overexpress the JAK/STAT ligand Unpaired (Upd) in the developing eye (GMR > Upd), resulting in larger eyes when adult flies emerge (Fig. 3E) 14,24 . We found that, treating the larvae with methotrexate at the same concentration that causes increased heterochromatin, reduced the eye size of GMR > Upd flies (Fig. 3F). This result is consistent with the idea that methotrexate reduces JAK/STAT signaling. However, we cannot rule out the possibility that the observed reduction in GMR > Upd fly eyes was due to decreased cell proliferation caused by inhibition of DHFR by methotrexate, as previously known 22 . Nonetheless, taken together with our previous finding that uSTAT promotes heterochromatin formation 13,15 , it is possible that methotrexate reduces pSTAT levels and thus increases relative uSTAT levels, resulting in higher levels of heterochromatin.

Discussion
Heterochromatin formation represents a potential epigenetic tumor suppression mechanism 2,5,7,16 . Compounds that promote heterochromatin formation are therefore desirable as candidate cancer therapeutic agents. In order to identify new heterochromatin-promoting small molecule drugs, we performed a simple screen using Drosophila, taking advantage of the DX1 strain that exhibits a heterochromatin-dependent eye color variegation phenotype (Fig. 1). Upon screening a small library of 97 drugs, we identified a small molecule drug, methotrexate, as causing the highest increase in variegation, which is significantly different from control (Fig. 2).
Interestingly, as a first-generation cancer chemotherapy drug and known to be a dihydrofolate reductase (DHFR) inhibitor, methotrexate has more recently been identified as a specific JAK/STAT pathway inhibitor, resulting in a reduced level of pSTAT without changing total STAT levels, therefore causing a relative increase in uSTAT levels 23 . We have previously shown that reducing pSTAT and increasing uSTAT levels promote heterochromatin formation as a non-canonical function of STAT 13,15,16 . Thus, the identification of methotrexate as a JAK/STAT inhibitor 23 and as a heterochromatin-promoting drug (this study) is line with our research demonstrating that uSTAT promotes heterochromatin formation 15,16 .
Our screening method is relatively straightforward and easy to carry out. However, there is a caveat when calculating the drug concentration used in the screening. We added drug solutions at 10 µM to the top of the food media, which is in semisolid state. The effective drug concentration is expected to remain in a steep gradient, with the top portion close to 10 µM and limited penetration to the bottom of the food. On the other hand, larvae mostly stay on and consume the top portion of the food. Thomas et al. have shown that methotrexate inhibits STAT phosphorylation in cultured cells at concentrations ranging from 0.01 to 50 µM with minimal toxicity 23 . Thus, we assume that our drugging regimen for methotrexate effectively inhibited STAT phosphorylation without strong toxic effects. Indeed, at 10 µM concentration, we did not observe much lethality in larvae treated with any of the 97 drugs we used in screening. However, when added at 100 µM concentration, methotrexate did cause lethality to the larvae. Thus, 10 µM is a tolerable concentration for screening these small-molecule drugs.
We will expand the screening effort for heterochromatin-promoting substances to a larger pool of small molecule compounds using the Drosophila variegated eye phenotype. Upon discovery of compounds that are suspected to promote heterochromatin formation, we will identify the mechanisms by which these drugs accomplish this in future research. We will also investigate the ability and the underlying mechanisms by which these compounds to suppress cancer.

Methods
Small-molecule drug library. Compound Libraries were obtained through the National Cancer Institute (NCI) Developmental Therapeutics Program. We screened Oncology Set III, a set of 97 oncology drugs that have been previously approved by the US Food and Drug Administration (FDA). The compounds were delivered in 96-well polypropylene (PP) microtiter plates with 60 compounds per plate. The plates were stored dry at −20 °C and contained 0.20 µMol of compound + 1 µL of glycerol. 19 µL of DMSO was added to each well to obtain 20 µl of 10 mM stock solutions.
Drosophila screening method. DX1 flies were kindly provided by James Birchler (University of Missouri) 17,25 . Three w 1118 /Y; DX1/CyO male flies and three virgin w 1118 female flies were crossed in a 25 × 95 mm food vial with 9 ml of standard food (Bloomington recipe). Flies were allowed to lay eggs for two days at room temperature. Parent flies were then removed from the vial and 60 µl of a 10 µM drug solution was pipetted onto food on day two, and another 60 µl of the same drug solution was added again on day four after crosses were set up (Fig. 1A). All drug compounds were prepared by diluting the original stock (10 mM in DMSO) to a 10 µM final concentration by dissolving in 33% DMSO in water. Since the fly food is in solid state, the drug solution is expected to remain in a deep gradient, with the top portion close to 10 µM and limited penetration to the bottom of the food. For control treatment, only 33% DMSO in water was used.
Scoring was carried out 2 days after the F1 flies emerge, all w 1118 /Y; DX1/+ heterozygous males, identified by lacking the CyO dominant curly wing phenotype, were scored for eye color on a scale of 1 to 5 (see Fig. 1B). (The choice of 1 to 5 scale was based on Sun Tzu's writing in the Art of War, "The colors do not exceed five, but the changes of the five colors can never be completely seen. ") Color scale is determined by the following rules.
1. Mostly white with light spots of red/orange scattered in <5% total surface area. 2. Patches of red spots in >5% but <25% of the surface area. 3. Red spots in >25% to <50% of surface area. 4. Red spots range from >50% to <75% of eye surface. 5. Red spots occupy >75% of the eye surface.
Triplicates of each cross were performed. Eye color for all F1 males of the w 1118 /Y; DX1/+ genotype were assigned from 1 to 5 as above. Color index was calculated as the average of all the males scored. Representative pictures were taken using a digital camera through microscope lens.
Eye pigmentation measurement. To measure red eye pigment levels, the heads of 40 male flies (2 days old, raised at 25 °C) of appropriate genotypes were severed and homogenized in methanol (1 ml, acidified with 0.1% HCl). Eye pigmentation was represented as the absorbance of the supernatant at 480 nm.
Salivary gland immunostaining and western blotting. The salivary gland dissection and whole-mount immunostaining procedure was adopted from published protocols 26 and as previously described 14 .
Briefly, salivary glands were dissected from wandering 3 rd instar larvae by grasping and pulling the mouth hook