IMPDH inhibitors for antitumor therapy in tuberous sclerosis complex

Abstract

Recent studies in distinct preclinical tumor models have established the nucleotide synthesis enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) as a viable target for antitumor therapy. IMPDH inhibitors have been used clinically for decades as safe and effective immunosuppressants. However, the potential to repurpose these pharmacological agents for antitumor therapy requires further investigation, including direct comparisons of available compounds. Therefore, we tested structurally distinct IMPDH inhibitors in multiple cell and mouse tumor models of the genetic tumor syndrome tuberous sclerosis complex (TSC). TSC-associated tumors are driven by uncontrolled activation of the growth-promoting protein kinase complex mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), which is also aberrantly activated in the majority of sporadic cancers. Despite eliciting similar immunosuppressive effects, the IMPDH inhibitor mizoribine, used clinically throughout Asia, demonstrated far superior antitumor activity compared with the FDA-approved IMPDH inhibitor mycophenolate mofetil (or CellCept, a prodrug of mycophenolic acid). When compared directly to the mTOR inhibitor rapamycin, mizoribine treatment provided a more durable antitumor response associated with tumor cell death. These results provide preclinical support for repurposing mizoribine, over other IMPDH inhibitors, as an alternative to mTOR inhibitors for the treatment of TSC-associated tumors and possibly other tumors featuring uncontrolled mTORC1 activity.

Keywords: Cancer; Genetic diseases; Metabolism; Therapeutics; Tumor suppressors.

Figure 1. Mizoribine is the most selective IMPDH inhibitor for reducing the viability of TSC2-deficient cells in culture.

(A) Littermate-derived Tsc2^+/+ and Tsc2^–/– mouse embryonic fibroblasts (MEFs) or (B) Tsc2^–/– 105K renal tumor–derived cells stably reconstituted with empty vector or wild-type TSC2 were treated with vehicle or given concentrations of the indicated IMPDH inhibitors for (A) 72 hours or (B) 48 hours. Viable cells were counted by trypan blue exclusion and graphed as percentage of vehicle-treated cells. n = 3 independent experiments. (C) Cells in A were treated for 24 hours with vehicle, mizoribine (Miz: 1, 2, or 3 μM), mycophenolic acid (MPA: 125, 250, or 500 nM), ribavirin (Rib: 10, 20, or 30 μM), or AVN-944 (AVN: 100 or 250 nM) followed by immunoblotting for indicated proteins. Results are representative of at least 2 independent experiments. S6K, S6 kinase. (D) Annexin V (Ann V)/propidium iodide (PI) staining on cells in A treated for 72 hours with vehicle, 3 μM mizoribine, or 250 nM MPA. n = 3 independent experiments. Graphical data are presented as mean of indicated replicates ± SEM. *P < 0.05, ^#P < 0.01 by 2-tailed Student’s t test.

Figure 2. Distinct IMPDH inhibitors have similar effects on purine metabolism in vitro.

(A and B) Guanylate and adenylate nucleotides in littermate-derived Tsc2^+/+ and Tsc2^–/– MEFs measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) following 12-hour treatment, where indicated, with vehicle, mizoribine (2 μM), MPA (250 nM), ribavirin (20 μM), or AVN-944 (250 nM) and graphed relative to vehicle. n = 3 biological replicates from a single experiment. (C) AICAR levels relative to vehicle-treated cells in samples from A and B. (D) Viable cell counts in cells treated for 72 hours with indicated concentrations of mizoribine or MPA with or without addition of exogenous guanosine (guan, 50 μM). n = 3 independent experiments. Graphical data are presented as mean of indicated replicates ± SEM. *P < 0.05, ^#P < 0.01 by 2-tailed Student’s t test.

Figure 3. Mizoribine is superior to mycophenolate in TSC2-deficient 105K xenograft tumors at therapeutically relevant concentrations.

(A) Experimental design used in B–F. NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice bearing 105K cell xenograft tumors were treated for 20 days with vehicle, mizoribine (75 mg/kg/d by i.p. injection), MMF (100 mg/kg/d by oral gavage), or rapamycin (1 mg/kg MWF by i.p. injection). Treatments were discontinued after 20 days, and tumors in the mizoribine and rapamycin groups were allowed to regrow. (B) Tumor volume measured every third day. n = 6 mice per group. (C) Mizoribine and MPA concentrations in blood plasma collected 2.5 hours after the final treatment, measured by LC-MS/MS. n = 6 mice per group. (D) Representative H&E and IHC staining on tumors from vehicle- or MMF-treated mice resected 3 hours after the final treatment. (E) Immunoblotting of tumor extracts from vehicle- or MMF-treated mice resected 3 hours after final treatment. (F) Representative H&E staining of tumors from mizoribine- or rapamycin-treated mice resected after regrowth. Graphical data are presented as mean of indicated replicates ± SEM. *P < 0.05, ^#P < 0.01 by 2-tailed Student’s t test. Scale bars: 0.5 mm.

Figure 4. Mizoribine is superior to mycophenolate in TSC2-deficient ELT3 xenograft tumors with or without prior treatment with rapamycin.

(A) Experimental design used in B–F. NSG mice bearing ELT3 cell xenograft tumors were treated for 26 days with vehicle, mizoribine (75 mg/kg/d by i.p. injection), or MMF (100 mg/kg/d by oral gavage) or for 23 days with rapamycin (1 mg/kg MWF by i.p. injection). On day 23, rapamycin treatment was discontinued, and those mice were switched to vehicle, mizoribine, or MMF treatment, as above, for 17 days. (B) Tumor volume measured every third day. n = 6 mice per group. (C) Images of representative tumors resected at the end of the indicated treatments. Scale bars: 1 cm. (D) Mizoribine and MPA concentrations in blood plasma collected on day 26, 2.5 hours after the final treatment and measured by LC-MS/MS. n = 5 mice per group. (E and F) Peak area values of mizoribine and MPA in tumor (E) and liver (F) metabolite extracts were normalized to those in plasma from the same mice. The mean tumor/plasma (E) or liver/plasma (F) ratio of MPA is shown relative to mizoribine. n = 4 mice per group. Graphical data are presented as mean of indicated replicates ± SEM. *P < 0.05, ^#P < 0.01 by 2-tailed Student’s t test.

Figure 5. Effects of mizoribine and MMF on tumor metabolites.

(A and B) Steady-state LC-MS/MS–based metabolite profiling of ELT3 xenograft tumors collected from the mice in Figure 4B on day 26, 3 hours after final treatment with vehicle (n = 5), mizoribine (n = 6), or MMF (n = 4). Row normalized heat maps are shown for all significantly changed metabolites (P < 0.05) in tumors from (A) mizoribine- or (B) MMF-treated mice relative to vehicle. Metabolites are grouped as increasing or decreasing with treatment and then listed from lowest to highest P value. *Metabolites changed with both mizoribine and MMF. (C and D) Peak area values relative to vehicle of (C) adenylate and guanylate nucleotides in tumors and (D) AICAR in tumors and plasma from the treated mice in A and B. Graphical data are presented as mean of indicated replicates ± SEM. *P < 0.05, ^#P < 0.005 by 2-tailed Student’s t test.

Figure 6. Mizoribine is superior to the maximum tolerated dose of MMF in an immunocompetent syngeneic xenograft model of TSC2-deficient tumor growth.

(A and B) C57BL/6J mice were treated for 10 days with mizoribine (50 mg/kg/d by oral gavage) MMF (100, 300, or 500 mg/kg/d by oral gavage), or rapamycin (1 mg/kg every 2 days [Q2D] by i.p. injection). Blood and plasma were collected 2.5 hours after the final treatment for measurement of (A) mizoribine and MPA concentrations in plasma by LC-MS/MS and (B) white blood cell counts. n = 3 mice/group. (C) Experimental design used in D and in Figure 7. C57BL/6 mice bearing syngeneic 105K cell xenograft tumors were treated as in A and B, except rapamycin was administered on MWF, for 24 days, at which point mice were sacrificed or, for the mizoribine and rapamycin groups, treatments were discontinued and tumors were allowed to regrow. n = 6 mice/group. (D) Tumor volume during the treatment phase measured every third day. Graphical data are presented as mean of indicated replicates ± SEM. *P < 0.05 by 2-tailed Student’s t test.

Figure 7. MMF fails to induce cell death and mizoribine treatment elicits a more durable response than rapamycin in a syngeneic TSC tumor model.

(A and B) Representative H&E and IHC staining of tumors from Figure 6D resected 3 hours after the final treatment. Scale bars: 0.5 mm (A), 0.25 mm (B). (C) Quantification of Ki-67 staining in B. Staining-positive cells per field of view were counted in 4 tumors per group in a blinded fashion, with 3 nonoverlapping fields counted per tumor. (D) Fold change in tumor volume during the regrowth phase in mice from Figure 6, C and D, relative to the last day of treatment. (E) H&E staining of tumors from the indicated treatment groups, resected after regrowth (scale bars: 2 mm). Graphical data are presented as mean of indicated replicates ± SEM. *P < 0.05, ^#P < 0.01 by 2-tailed Student’s t test.

Figure 8. ADK, required for mizoribine action, is expressed in human TSC-associated pulmonary LAM and renal angiomyolipoma.

(A) Mizoribine is phosphorylated by ADK to produce mizoribine monophosphate (Miz-MP), the product that inhibits IMPDH. (B–D) 105K cells stably reconstituted with empty vector or wild-type TSC2 were transfected with control (siCtl) or ADK-targeting siRNAs (siADK) and treated for 48 hours with indicated concentrations of (B) mizoribine or (C) MPA. Viable cells were quantified by CellTiter-Glo and graphed as percentage of vehicle-treated cells. n = 3 independent experiments. (D) Immunoblots showing ADK knockdown and TSC2 reconstitution efficiency. Results are from a single experiment done alongside a replicate in panels B and C. (E and F) H&E and IHC staining of (E) 3 human pulmonary LAM and (F) 3 renal angiomyolipoma specimens from LAM and TSC patients. Graphical data are presented as mean of indicated replicates ± SEM. Scale bars: 100 μm.