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. 2021 Apr 23;11(1):8844.
doi: 10.1038/s41598-021-87734-6.

Transition state analogue of MTAP extends lifespan of APCMin/+ mice

Ross S Firestone #  1   2 , Mu Feng #  1 , Indranil Basu  3 , Karina Peregrina  4 , Leonard H Augenlicht  5 , Vern L Schramm  6
Affiliations

Transition state analogue of MTAP extends lifespan of APCMin/+ mice

Ross S Firestone et al. Sci Rep. .

Abstract

A mouse model of human Familial Adenomatous Polyposis responds favorably to pharmacological inhibition of 5'-methylthioadenosine phosphorylase (MTAP). Methylthio-DADMe-Immucillin-A (MTDIA) is an orally available, transition state analogue inhibitor of MTAP. 5'-Methylthioadenosine (MTA), the substrate for MTAP, is formed in polyamine synthesis and is recycled by MTAP to S-adenosyl-L-methionine (SAM) via salvage pathways. MTDIA treatment causes accumulation of MTA, which inhibits growth of human head and neck (FaDu) and lung (H359, A549) cancers in immunocompromised mouse models. We investigated the efficacy of oral MTDIA as an anti-cancer therapeutic for intestinal adenomas in immunocompetent APCMin/+ mice, a murine model of human Familial Adenomatous Polyposis. Tumors in APCMin/+ mice were decreased in size by MTDIA treatment, resulting in markedly improved anemia and doubling of mouse lifespan. Metabolomic analysis of treated mice showed no changes in polyamine, methionine, SAM or ATP levels when compared with control mice but indicated an increase in MTA, the MTAP substrate. Generation of an MTDIA-resistant cell line in culture showed a four-fold amplification of the methionine adenosyl transferase (MAT2A) locus and expression of this enzyme. MAT2A is downstream of MTAP action and catalyzes synthesis of the SAM necessary for methylation reactions. Immunohistochemical analysis of treated mouse intestinal tissue demonstrated a decrease in symmetric dimethylarginine, a PRMT5-catalyzed modification. The anti-cancer effects of MTDIA indicate that increased cellular MTA inhibits PRMT5-mediated methylations resulting in attenuated tumor growth. Oral dosing of MTDIA as monotherapy has potential for delaying the onset and progression of colorectal cancers in Familial Adenomatous Polyposis (FAP) as well as residual duodenal tumors in FAP patients following colectomy. MTDIA causes a physiologic inactivation of MTAP and may also have efficacy in combination with inhibitors of MAT2A or PRMT5, known synthetic-lethal interactions in MTAP-/- cancer cell lines.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The role of 5′-methylthioadenosine phosphorylase (MTAP) in the context of methionine metabolism and polyamine synthesis. MTAP catalyzes the phosphorolysis of 5′-methylthioadenosine (MTA) to form adenine and 5-methylthio-α-d-ribose 1-phosphate (MTR-1-P), which can be recycled to form ATP and methionine followed by conversion to S-adenosyl-l-methionine (SAM) by the MAT2A-catalyzed reaction. The S-methyl of SAM (red) is conserved in the MTA salvage reaction enabled by MTAP action. MTAP inhibitors interfere with MTA metabolism leading to elevated MTA concentration in cells and growth disruption.
Figure 2
Figure 2
(A) Kaplan–Meier analysis of APCMin/+ mouse survival (Study A). The majority of APCMin/+ untreated mice (N = 11) die before 170 days of life, consistent with prior reports. Oral MTDIA dosing at 10 mg/kg/day (N = 10) showed no significant effect on mouse survival. Oral MTDIA dosing at 20 mg/kg/day (N = 10) showed an approximate doubling in lifespan for APCMin/+ mice. (B,C) Histolopathologic assessment of APCMin/+ mouse intestinal tissue. The mean number of tumors (B) and average size (C) of intestinal adenomas was compared in control (N = 6) and 20 mg/kg/day treated (N = 6) mice. The change in the number of tumors per mouse following treatment was not statistically significant. The average tumor size for treatment mice was decreased by 43% in mice treated with 20 mg/kg/day MTDIA (p = 0.0086). Tumor size was measured in “AU” or arbitrary units.
Figure 3
Figure 3
Selected liver metabolites from APCMin/+ mice. Untreated (N = 6) and treated mice (N = 6) receiving 20 mg/kg/day MTDIA following weaning were euthanized at day 150 and livers were extracted to analyze metabolites. No significant changes were noted in polyamine levels (A), SAM or SAH levels (B), or ATP and methionine levels (C). A 4.2-fold increase in MTA was observed in mice receiving 20 mg/kg/day oral MTDIA (p = 0.001) (D). The anti-cancer effect of MTAP inhibition is coincident with MTA accumulation. Expanded metabolomics data is shown in the SI, Table S12.
Figure 4
Figure 4
(A) Microscopy images (100 x) with IHC staining for symmetric dimethylarginine (SDMA) in intestinal tissue from untreated APCMin/+ mice (left) and those treated with 20 mg/kg/day MTDIA (right) euthanized at 150 days. SDMA signal (brown) was reduced in MTDIA-treated mice. (B) Quantitative data from four control and seven MTDIA-treated mice from IHC staining experiments. The left panel shows quantitative signal intensity from all ROIs among all control and treated mice with an observed statistically significant decrease in SDMA signal among MTDIA treated mice (p < 0.0001). The right panel shows mean ROI signal intensity for each individual mouse, with mean signal intensity three-fold less in MTDIA treated mice compared to controls (p < 0.001).
Figure 5
Figure 5
Western blot analysis comparing parental FaDu cells (WT) compared to MTDIA-resistant FaDu cells (R). MAT2A expression is increased in FaDu resistant cells while MAT2B levels are unchanged. MTDIA resistance is linked to MAT2A overexpression and increased capacity for SAM production. The doublet appearance of the MAT2A band is consistent with prior reports. Unedited data is shown in Figure S5.
Figure 6
Figure 6
Proposed mechanism of action of MTDIA. In the absence of MTDIA, MTAP metabolizes MTA produced from polyamine synthesis. With MTAP inhibited by MTDIA, MTA accumulates, resulting in competitive inhibition of PRMT5-mediated histone methylation and intron splicing, slowing cancer cell proliferation. In MTDIA resistant cell lines, gene amplification of MAT2A increases SAM production to relieve the inhibition caused by excess MTA.
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