Supplemental Figure 4. Life Span. Life span of the MNX strains was measured in days and compared to the vendor information (Jackson Laboratories for C57Bl/6 and FVB/NJ mice and Envigo Laboratories for C3H/HeN mice). N= 15 for the females. N=5 for the males.

Supplemental Figure 1. Validation of Methyl-Seq and RNA-Seq. Several hypo-/hyper-methylated and down-/up-regulated genes were selected for validation using methylation specific primers (64) or quantitative PCR, respectively. Graphs depict fold-change between MNX strains and corresponding wild-type mice are shown to the left. All gene expression data were normalized to beta actin. Each bar combines pools A and B and the experiments were performed in triplicate. Error bars represent standard error. Representative gels showing unmethylated (U) and methylated (M) DNA are shown. The table summarizes specific genes tested and whether the RNA-or Methyl-Seq data were and were not validated.

Supplemental Figure 2. Deduplication and alignment statistics. a. Bismark v0.10.1 was used for alignment and deduplication. Duplicates were discarded using deduplicate_bismark perl script. With total number of alignments analyzed (red), total number of duplicated alignments removed (green) and total count of de-duplicated sequences (blue). b. Total sequence pairs analyzed (yellow), number of paired-end alignments with a unique hit (green), sequence pairs with no alignment (light blue), sequence pairs that did not map (blue), sequence pairs that were discarded due to no genomic sequence extracted (pink)

Supplemental Figure 6. Cell Cycle Analysis. Cell cycle analysis using propidium iodide (PI) staining was performed on mouse embryo fibroblasts (MEFs) harvested from the following strains: FF, FC, BB, FB, CC, CH, HH, HC (as described in Table 1). Cell cycle was performed by flow cytometry and analyzed using FACS Diva.

Supplemental Figure 3. Integrative analysis of methylation and gene expression. The methylation level for the five most differentially expressed genes (listed at the top of each page), for which also methylation data was available, is shown in Panels A-E for each gene. Chromosomal position of the corresponding gene is shown on the x-axis, and the methylation in percentage is shown on the y-axis. Each star represents one CpG. CpG''s for BB are shown in green, while CpG''s for FB are shown in orange. For both types a LOWESS (Locally Weighted Scatterplot Smoothing) smoother was used to create a "regression line". The boxplot in panel F for each gene presents the normalized gene expression for both samples (BB and FB) for the five previously picked genes. Again, green indicates a BB-sample and orange indicates a FB-sample. Each box visualizes two normalized gene expressions (two replicates).

Supplemental Figure 7. Apoptosis Analysis. Annexin V-FITC/propidium iodide (PI) staining assay were used to measure apoptosis in MEFs harvested from the following strains: FF, FC, BB, FB, CC, CH, HH, HC (as described in Table 1). Apoptotic cells were detected by flow cytometry and analyzed using FACS Diva.

S1: Neither mitochondrial load nor membrane potential differ between MNX and wild-type strains. Mouse embryonic fibroblasts were isolated from each MNX and wild-type strain and stained with MitoTracker Green FM to identify overall mitochondrial load and with MitoTracker Red CMXRos to identify membrane potential. Probe fluorescence in each cell was analyzed by flow cytometry. A: Relative fluorescence for each probe showing mitochondrial load and raw membrane potential. B: MitoTracker Red is normalized to MitoTracker Green to show membrane potential per mitochondrion. Error bars represent standard error of the mean. S2: OCR and ECAR profiles vary between C57BL/6J and C3H/HeN MNX and wild-type strains. Mouse embryonic fibroblast from CC, CH, HH, and HC mouse strains were passaged once then analyzed for ECAR (Extracellular acidification rate) under serum starved conditions with no glucose to determine non-glycolytic acidification, after addition of glucose (glycolysis), after addition of oligomycin to determine glycolytic capacity (flux), and after addition of 2-DG (glycolytic reserve). * denotes p<0.05 and error bars represent standard error of the mean. CH and HH strains differ significantly in non-glycolytic acidification while CC and HC differ significantly in glycolytic reserve. S3: Mitochondrial flux of MNX and wild-type mice is variable. MNX and wild-type mouse embryonic fibroblasts from each strain were plated and the Seahorse analyzer was used to measure oxygen consumption and extracellular acidification prior to compound addition (Basal respiration), after oligomycin addition (ATP-Linked respiration), and after addition of the uncoupler FCCP (Maximal respiration). * denotes p<0.05 and error bars represent standard error of the mean. A: Basal respiration differs significantly between * FC and BB, and ** FF and BB. Maximal respiration differs significantly between * FC and BB, ** FC and FB, as well as ***FF and BB. B: Basal, ATP-linked, and Maximal respiration does not differ significantly between CC, CH, HH, and HC strains S4: Mitochondrial DNA content does not differ between C57BL/6J and C3H/HeN MNX and wild-type strains. Lungs were isolated from four-week old CC (n=5), CH (n=7), HH (n=5), and HC (n=8) mice. Total DNA was isolated from each lung taking care to preserve mtDNA. qPCR utilizing Taqman primers for mitochondrial encoded mtCO2 and ND1 as well as nuclear encoded 18s was performed and fold change for mitochondrial genes was calculated relative to nuclear 18s. Error bars represent standard error of the mean. S5. Analysis of Metastatic size and area. Images (10x) from Figure 5B were analyzed using Photoshop and Image J for relative size and number of metastases. Analysis of formalin-fixed paraffin-embedded experimental lung metastases derived from K1735-M2 Melanoma cell intravenous injections. DMSO vehicle (-) or MitoTEMPO (+) was IP injected into 4-week-old wild-type or MNX mice 24 hours and again 1 hour prior to intravenous injection of cancer cells. Mice were euthanized two weeks post cell injection and lungs were harvested. Data as medians (n = 9-16; One-way ANOVA and multiple comparisons tests). S6: MtDNA affects expression of select nuclear genes. RNA was isolated from lungs of 4-week old CC, CH, HH, and HC mice. qPCR was performed with Taqman primers to determine expression of nuclear genes normalized to the nuclear 18s housekeeping gene. Error bars represent standard error of the mean. Blue bars represent gene expression levels of HH and CH mice relative to CC while green bars represent gene expression levels of CC and HC mice relative to HH mice. S7: Mitochondrial superoxide scavenging selectively alters nuclear gene expression. RNA was isolated from lungs of 4-week old CC, CH, HH and HC mice treated with DMSO vehicle (-) or MitoTEMPO (+) 24 hours and 1 hour prior to euthanasia. qPCR was performed with Taqman primers to determine gene expression of A: miR199a B: miR125b C: dnmt1 D: ago2 E: sod2 F: fam120a G: rab6b H: sgtb and I: scai. All expression was normalized to 18s. S8: Mitochondria stromal haplotype does not alter cancer cell extravasation: Male and female wild-type (HH) and MNX (HC) mice were injected into the lateral tail vein with CFSE labeled K1735-M2 melanoma cells. Lungs were harvested 24 hours post-injection. A. The right lung lobes were analyzed by flow cytometry for CFSE positive cancer cells. Seven hundred thousand events were collected using the Acurri C6 cytometer and dead cells were identified using propidium iodide and excluded from further analysis. B. The left lung lobe was fixed in 10% formalin. Sections (3 x 20 µm) approximately 100 µm apart were analyzed from each mouse for detection of CFSE+ cancer cells. The number obtained from all three sections were then averaged and graphed in comparison to the uninjected controls

Supplementary Table S1: Primer sequences for RFLP sites and Her2 transgene

Supplemental Figure 5. Litter sizes and gender distribution. Female to male ratios with the average litter size and total number of mice used for each strain. For the female to male ratio the average litter size was calculated from over 20 litters generated. The number of females and males from each litter were counted.

Ciclopirox (CPX) is an FDA-approved topical antifungal agent that has demonstrated preclinical anticancer activity in a number of solid and hematologic malignancies. Its clinical utility as an oral anticancer agent, however, is limited by poor oral bioavailability and gastrointestinal toxicity. Fosciclopirox, the phosphoryloxymethyl ester of CPX (Ciclopirox Prodrug, CPX-POM), selectively delivers the active metabolite, CPX, to the entire urinary tract following parenteral administration. We characterized the activity of CPX-POM and its major metabolites in in vitro and in vivo preclinical models of high-grade urothelial cancer. CPX inhibited cell proliferation, clonogenicity and spheroid formation, and increased cell cycle arrest at S and G0/G1 phases. Mechanistically, CPX suppressed activation of Notch signaling. Molecular modeling and cellular thermal shift assays demonstrated CPX binding to γ-secretase complex proteins Presenilin 1 and Nicastrin, which are essential for Notch activation. To establish in vivo preclinical proof of principle, we tested fosciclopirox in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) mouse bladder cancer model. Once-daily intraperitoneal administration of CPX-POM for four weeks at doses of 235 mg/kg and 470 mg/kg significantly decreased bladder weight, a surrogate for tumor volume, and resulted in a migration to lower stage tumors in CPX-POM treated animals. This was coupled with a reduction in the proliferation index. Additionally, there was a reduction in Presenilin 1 and Hes-1 expression in the bladder tissues of CPX-POM treated animals. Following the completion of the first-in-human Phase 1 trial (NCT03348514), the pharmacologic activity of fosciclopirox is currently being characterized in a Phase 1 expansion cohort study of muscle-invasive bladder cancer patients scheduled for cystectomy (NCT04608045) as well as a Phase 2 trial of newly diagnosed and recurrent urothelial cancer patients scheduled for transurethral resection of bladder tumors (NCT04525131).

Abstract Background Metastatic burden is the leading cause of cancer deaths; however, it remains unclear why some patients are more susceptible to metastatic disease. While the nuclear genome's role in tumor progression and metastasis is known, the role of mitochondrial DNA (mtDNA) polymorphisms (SNP) has only recently been explored. Using mitochondrial nuclear exchange (MNX) mice, we previously showed that mtDNA strongly influences mammary carcinoma progression and metastasis both intrinsically and via non-cell autonomous mechanisms. We hypothesized that mtDNA SNP alter immune cell development/trafficking which, in turn, could influence metastasis efficiency. Methods Peritoneal exudate, and splenocytes were collected from male and female wild-type C57BL/6J (CC) and C3H/HeN (HH), and MNX mice - C57BL/6-mtMNX(C3H/HeN) (CH) and C3H/HeN-mtMNX(C57BL/6J) (HC) mice [first letter=nuclear; second letter=mitochondrial]. Lung metastases were established from tail-vein injection of E0771 (CC/CH) or K1735-M2 (HH/HC) cell lines. Results Lung metastases derived from histocompatible (i.e. nDNA matched) tumor cell injection into wild-type or MNX mice increased in C3H/HeN mtDNA backgrounds (HH and CH). No significant differences were observed in seeding, suggesting that mtDNA mediated differences in metastatic microenvironments likely impact metastatic outgrowth. No significant changes in broad immune cell populations were observed in naïve animals, but selective changes in differentiation markers were observed. The most significant change was lower CD11c+ peritoneal macrophages in HH (3%) versus CC (16%) mice (p < 0.001). C57BL/6 mtDNA (HC) increased the percentage of macrophages (9%) compared to wild-type (HH) (p < 0.001). We next confirmed a role for mitochondrial derived ROS mediating immune microenvironmental regulation of metastasis, as mtDNA mediated metastatic differences are abrogated upon treatment with the anti-oxidant MitoTEMPO. Tumor infiltrating CD8+ lymphocytes (TIL) increased 1.5-fold in CH (1.7%) compared to wild-type CC (1.2%) (p = 0.07) while no differences were observed between HH and HC mice. Consistent with changes in metastasis in the HH background, MitoTempo treatment reduced CD4+ TIL >1.5-fold (1.5%), compared to the vehicle control (2.8%) (p = 0.029). Discussion Our data support the hypothesis that mitochondrial SNP modulate immune cell development and/or trafficking, providing a plausible explanation for how metastatic potentials of syngeneic tumor cells injected into MNX mice are altered. That is, mtDNA contributions to immune function affect metastasis and may provide insight as to why some immune therapies succeed/fail. Support: DOD BCRP BC171381 and Kansas INBRE P20 GM103418 (TCB); Susan G. Komen for the Cure SAC110037; National Foundation for Cancer Research and NIH CA168524 (DRW) Citation Format: Thomas C. Beadnell, Amanda E. Brinker, Cori Fain, Carolyn J. Vivian, Danny R. Welch. Mitochondrial genetics appear to alter immune cell development/trafficking [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2662.

Abstract Mitochondria contribute to tumor growth through multiple metabolic pathways, regulation of extracellular pH, calcium signaling, and apoptosis. Using the Mitochondrial Nuclear Exchange (MNX) mouse models, which pair nuclear genomes with different mitochondrial genomes, we previously showed that mitochondrial SNPs regulate mammary carcinoma tumorigenicity and metastatic potential in genetic crosses. Here, we tested the hypothesis that polymorphisms in stroma significantly affect tumorigenicity and experimental lung metastasis. Using syngeneic cancer cells (EO771 mammary carcinoma and B16-F10 melanoma cells) injected into wild-type and MNX mice (i.e., same nuclear DNA but different mitochondrial DNA), we showed mt-SNP–dependent increases (C3H/HeN) or decreases (C57BL/6J) in experimental metastasis. Superoxide scavenging reduced experimental metastasis. In addition, expression of lung nuclear-encoded genes changed specifically with mt-SNP. Thus, mitochondrial–nuclear cross-talk alters nuclear-encoded signaling pathways that mediate metastasis via both intrinsic and extrinsic mechanisms. Significance: Stromal mitochondrial polymorphisms affect metastatic colonization through reactive oxygen species and mitochondrial–nuclear cross-talk.

Abstract Ciclopirox (CPX) is a FDA-approved topical antifungal agent that has demonstrated preclinical anticancer activity in solid and hematologic malignancies. It's clinical utility as an anticancer agent, however, is limited by poor oral bioavailability, gastrointestinal toxicity, and poor water solubility. Fosciclopirox, the phosphoryloxymethyl ester of CPX (Ciclopirox Prodrug, CPX-POM), is rapidly and completely metabolized to CPX, the active metabolite, which subsequently undergoes renal elimination resulting in urine concentrations of CPX that exceed in vitro IC50's several-fold. We characterized the activity of CPX-POM and its major metabolites in vitro utilizing authenticated human T24, HT-1376, and UM-UC-3 high-grade urothelial cancer cell lines. CPX inhibited cell proliferation, clonogenicity, and spheroid formation, and increased cell cycle arrest at S and G0/G1 phases. Mechanistically, CPX suppressed activation of Notch signaling, which was partially rescued by ectopic expression of the intracellular domain of Notch1. Molecular modeling and cellular thermal shift assays demonstrated CPX binding to γ-secretase complex proteins Presenilin1 and Nicastrin, which are essential for Notch activation. Interrogation of The Cancer Genome Atlas (TCGA) database demonstrated that both proteins were upregulated in bladder tumor tissue, and that higher levels of Presenilin1 and Nicastrin were significantly associated with lower overall survival in muscle invasive bladder cancer (MIBC) patients. To establish in vivo preclinical proof of principle, we tested fosciclopirox in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) mouse bladder cancer model in two separate studies. Intraperitoneal (IP) administration of CPX-POM once daily for four weeks at doses ranging from 25 to 200 mg/kg significantly decreased bladder weight and resulted in a migration to lower stage tumors in CPX-POM treated animals compared to untreated animals. This was coupled with a reduction in proliferation index, as well as reductions in Presenilin1 and Hey1 expression in bladder tumor tissues in CPX-POM treated animals. A similar anti-tumor response was observed following once daily versus three times weekly IP CPX-POM in this chemical carcinogen mouse model of bladder cancer. The safety, dose tolerance, pharmacokinetics and pharmacodynamics of intravenous (IV) CPX-POM were characterized in a US multi-center, First-in-Human, Phase 1, open-label, dose escalation study (NCT03348514). Eight cohorts of 19 patients received IV CPX-POM doses ranging from 30 to 1200 mg/m2 for as many as six 21-day treatment cycles. Adequate systemic and urinary tract CPX exposures were achieved at the maximum tolerated dose of 900 mg/m2 with evidence of Notch inhibition. An expansion cohort study in 12 cisplatin-ineligible MIBC patients receiving two treatment cycles of CPX-POM prior to radical cystectomy (RC) is underway. Evidence of pharmacologic activity is being characterized in bladder tumor tissues obtained at RC. Citation Format: Scott James Weir, Prasad Dandawate, Prabhu Ramamoorthy, Parthasarathy Ranjarajan, Robyn Wood, Amanda Brinker, Benjamin Woolbright, Mehmet Tanol, Tammy Ham, William McCulloch, Michael Dalton, Michael J. Baltezor, Roy A. Jensen, John A. Taylor, Shrikant Anant. Fosciclopirox suppresses growth of high-grade urothelial cancer by targeting Notch signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6405.

e14705 Background: Ciclopirox Prodrug (CPX-POM) is a novel anticancer agent currently being evaluated in patients with advanced solid tumors participating in a First-in-Human, Phase 1 safety, dose tolerance, pharmacokinetics (PK) and pharmacodynamics trial at four US sites. In vitro and in vivo preclinical proof of principle was established in high grade human urothelial cancer cell lines as well as a mouse model of bladder cancer.Methods: A series of in vivo PK studies were conducted in mice, rats and dogs to characterize the absolute bioavailability of CPX following intravenous (IV), subcutaneous (SC) and oral administration of CPX-POM. The single dose and steady-state plasma and urine pharmacokinetics of CPX-POM are also currently being characterized in patients participating in the ongoing Phase 1 trial. Plasma and urine concentrations of the prodrug and metabolites were determined by LC-MS/MS validated in each specie and matrix. Non-parametric pharmacokinetic parameters were generated from resultant plasma and urine drug and metabolite concentration-time data. Results: CPX-POM is rapidly and completely metabolized to CPX in blood via circulating phosphatases in animals and humans. CPX is completely bioavailable following IV CPX-POM administration in mice, rats and dogs. CPX and its major inactive glucuronide metabolite (CPX-G) are extensively eliminated in urine in all animal species. SC administration of CPX-POM demonstrated excellent bioavailability in rats and dogs. Following IV administration of 30-900 mg/m2CPX-POM to patients, the apparent elimination half-life of CPX ranged from 2 to 8 hours, CPX systemic exposure was dose-proportional and time-independent in cancer patients, and a major portion of the dose was eliminated as CPX-G. Conclusions: IV CPX-POM achieves plasma and urine CPX exposures that exceed in vitro IC50 values several-fold at well tolerated doses in animals and humans. CPX pharmacokinetics observed in animals were predictive of human systemic clearance based on allometric scaling. Clinical trial information: NCT03348514.

Pharmacokinetic studies in rats and dogs were performed to characterize the in vivo performance of a novel prodrug, fosciclopirox. Ciclopirox olamine (CPX-O) is a marketed topical antifungal agent with demonstrated in vitro and in vivo preclinical anticancer activity in several solid tumor and hematologic malignancies. The oral route of administration for CPX-O is not feasible due to low bioavailability and dose-limiting gastrointestinal toxicities. To enable parenteral administration, the phosphoryl-oxymethyl ester of ciclopirox (CPX), fosciclopirox (CPX-POM), was synthesized and formulated as an injectable drug product. In rats and dogs, intravenous CPX-POM is rapidly and completely metabolized to its active metabolite, CPX. The bioavailability of the active metabolite is complete following CPX-POM administration. CPX and its inactive metabolite, ciclopirox glucuronide (CPX-G), are excreted in urine, resulting in delivery of drug to the entire urinary tract. The absolute bioavailability of CPX following subcutaneous administration of CPX-POM is excellent in rats and dogs, demonstrating the feasibility of this route of administration. These studies confirmed the oral bioavailability of CPX-O is quite low in rats and dogs compared with intravenous CPX-POM. Given its broad-spectrum anticancer activity in several solid tumor and hematologic cancers and renal elimination, CPX-POM is being developed for the treatment of urothelial cancer. The safety, dose tolerance, pharmacokinetics, and pharmacodynamics of intravenous CPX-POM are currently being characterized in a United States multicenter first-in-human Phase 1 clinical trial in patients with advanced solid tumors (NCT03348514).

Abstract Introduction: Previous studies demonstrated that mitochondrial inheritance may contribute to aggressiveness of metastatic disease. Accumulating evidence suggests the mitochondrial genetic background may influence how certain cancers behave. We utilized Mitochondrial-Nuclear eXchange (MNX) female mice crossed with transgenic mice over-expressing the Her-2 gene and showed mitochondrial DNA (mtDNA)-dependent differences in tumor latency, lung metastasis number and lung metastasis size. We hypothesized that mtDNA mutations accumulate as mammary tumors progress and that the evolution is associated with mtDNA-nuclear DNA cross-talk. To test this hypothesis, we conducted next generation sequencing analyses to examine the spectra of mutations in the mitochondrial genome. Methods: Normal mammary gland, primary tumor and lung metastases [n=5 each] were obtained from FVB/NJ mice with FVB/NJ (designated FF), C57BL/6J (designated FC) or BALB/cJ mtDNA (designated FB). Epithelial cells from mammary gland or tumor cells were carefully isolated by laser capture microdissection in order to minimize contamination from surrounding stromal cell mtDNA. mtDNA was deep sequenced using three pools totaling 182 overlapping primers spanning the whole mitochondrial genome using the Ion TorrentTM PGM System. Sequences were compared to an FVB mtDNA reference sequence to detect variants. Results: Significant differences in the total number of mtDNA mutations were observed between the wild-type and MNX mice. Both FB and FC MNX cohorts exhibited increased mtDNA mutations compared to the wild-type (FF). As tumor progressed, the numbers of and distribution of mutations across the mitochondrial genome increased. ‘Hotspots' were observed in FB mice (S12 rRNA, COX I, ND4, CYTB) that were distinct from common mutations in the FC mice (16S rRNA, COX I). Discussion: As predicted, tumor cells accumulated more mutations in mtDNA as neoplastic cells from the primary tumor progressed to metastasis. Surprisingly, wild-type (FF) mice, even though more clinically aggressive (i.e., more metastases), accumulated fewer mtDNA mutations than tumors arising in the MNX mice. The mutations appear to occur in different sites, depending upon the nuclear-mitochondrial combination. Whether the mtDNA mutations function as contributors to metastatic efficiency has not yet been determined. Nonetheless, the data imply that nuclear-mitochondrial cross-talk influences mtDNA mutational spectra and metastasis and that defining the critical mtDNA genes most commonly involved may eventually be used to predict patient prognosis. Citation Format: Takae M. Brewer, Amanda E. Brinker, Sharon Manley, Carolyn J. Vivian, Danny R. Welch. Mitochondrial-nuclear crosstalk influences accumulation of mitochondrial DNA mutations in mammary tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 431.

Many inbred strains of mice develop spontaneous tumors as they age. Recent awareness of the impacts of mitochondrial DNA (mtDNA) on cancer and aging has inspired developing a mitochondrial-nuclear exchange (MNX) mouse model in which nuclear DNA is paired with mitochondrial genomes from other strains of mouse. MNX mice exhibit mtDNA influences on tumorigenicity and metastasis upon mating with transgenic mice. However, we also wanted to investigate spontaneous tumor phenotypes as MNX mice age. Utilizing FVB/NJ, C57BL/6J, C3H/HeN, and BALB/cJ wild-type inbred strains, previously documented phenotypes were observed as expected in MNX mice with the same nuclear background. However, aging nuclear matched MNX mice exhibited decreased occurrence of mammary tumors in C3H/HeN mice containing C57BL/6J mitochondria compared to wild-type C3H/HeN mice. Although aging tumor phenotypes appear to be driven by nuclear genes, evidence suggesting that some differences are modified by the mitochondrial genome is presented.

Ciclopirox (CPX) is contained in a number of FDA-approved topical antifungal drug products as the free acid and olamine salt. CPX possesses anticancer activity in a number of in vitro and in vivo preclinical models. Its clinical utility is limited as an oral anticancer agent, however. The oral bioavailability of CPX is quite low due to extensive first pass effect. The poor water solubility of CPX and its olamine salt prevent formulation as an injectable drug product. Thirdly, dose-limiting gastrointestinal toxicities were observed following four times daily oral dosing of CPX in patients with advanced hematologic malignancies. Ciclopirox Prodrug (CPX-POM), in contrast, has demonstrated excellent bioavailability via injectable routes of administration. Here we describe the preclinical characterization of CPX-POM, a novel anticancer agent being developed for the treatment of non-muscle invasive (NMIBC) and muscle invasive (MIBC) bladder cancer. Following IV, SQ and IP administration to mice, CPX-POM is rapidly and completely metabolized to CPX in blood via circulating phosphatases. CPX and its major, inactive glucuronide metabolite are extensively eliminated in urine. At well-tolerated doses, steady-state urine concentrations of CPX exceed in vitro IC50 values in mice by 15-30 fold. CPX inhibited cell proliferation, colony formation, and bladdosphere formation in vitro in T24 (NMIBC) and 253JBV (MIBC) human cell lines in both concentration- and time-dependent manners with IC50 values of 2-4 µM. CPX exposure increased the percentage of NMIBC and MIBC cells arrested at the S and G0/G1 phases, and induced cell death. CPX exposure significantly reduced expression of genes at the mRNA level involved in cancer stem cell signaling pathways including Notch, Wnt, and Hedgehog. CPX was shown to inhibit bladder cancer cell growth in vitro by inhibiting the Notch 1 signaling pathway. The validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) chemical carcinogen mouse model of bladder cancer was employed to establish in vivo preclinical proof of principle for CPX-POM. Over the once-daily IP dose range of 25-200 mg/kg, CPX-POM treatment resulted in significant decreases in bladder weight, a clear migration to lower stage tumors, dose-dependent reduction in Ki67 and PCNA staining, as well as a reduction in PCNA-expressing cells. All CPX-POM doses were well tolerated with no evidence of toxicity to the urinary tract based on blinded pathologic evaluation. There were also dose-dependent decreases in Notch 1, Presenilin 1, and Hey 1 in bladder cancer tissues obtained from CPX-POM treated animals. Tumor response was similar, in vivo, following once-daily and three-times weekly CPX-POM administration. CPX-POM has received FDA clearance to proceed to Phase I, and is currently being evaluated in a first-in-human trial in patients with advanced solid tumors. Citation Format: Scott J. Weir, Partha Ranjarajan, Robyn Wood, Karl Schorno, Prabhu Ramamoorthy, Lian Rajweski, Kathy Heppert, Michael J. McKenna, William McCulloch, Greg A. Reed, Amanda Brinker, Michael J. Baltezor, Roy A. Jensen, John A. Taylor, Shrikant Anant. Bench-to-bedside translation of ciclopirox prodrug for the treatment of non-muscle invasive and muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5882.

e14576 Background: Ciclopirox (CPX) is an antifungal agent contained in a number of FDA-approved topical drug products. CPX possesses anticancer activity in a number of in vitro and in vivo preclinical models, however, its clinical utility is limited due to poor oral bioavailability, gastrointestinal toxicity, and poor water solubility. Ciclopirox Prodrug (CPX-POM) is a novel anticancer agent being developed for the treatment of non-muscle invasive (NMIBC) and muscle invasive (MIBC) bladder cancer. Methods: In vitro were conducted in two high grade human bladder cancer (T24, 253JBV) cell lines to characterize the mechanisms of action for CPX. The pharmacokinetics of CPX-POM, CPX and its major, inactive glucuronide metabolite (CPX-G) were characterized in mouse, rat and dog studies. The effect of IP CPX-POM on progression from carcinoma in situ to muscle invasive bladder cancer was characterized in the mouse BBN model of bladder cancer. Preclinical safety of IV CPX-POM was characterized in 28-day GLP toxicology studies in rats and dogs. Results: CPX-POM is completely metabolized in vivo to its active metabolite, CPX. CPX and its major inactive glucuronide metabolite (CPX-G) are extensively eliminated in urine. CPX achieves steady-state urine concentrations exceeding in vitro IC50 values by 15 to 30-fold at well tolerated doses. CPX inhibited cell proliferation, colony formation, and bladdosphere formation in vitro with IC50 values of 2-4 µM. CPX arrested cells at the S and G0/G1 phases, and induced cell death. CPX significantly reduced expression of genes at the mRNA level involved in cancer stem cell signaling pathways including Notch, Wnt, and Hedgehog. In the BBN model, CPX-POM resulted in significant decreases in bladder weight, migration to lower stage tumors, dose-dependent reduction in Ki67 and PCNA staining, across a dose range of 25-200 mg/kg. Dose-dependent decreases in Notch 1, Presenilin 1, and Hey 1 were observed in bladder cancer tissues obtained from CPX-POM treated animals. Conclusions: The safety, dose tolerance, pharmacokinetics and pharmacodynamics of CPX-POM are currently being characterized in a first-in-human trial enrolling patients with advanced solid tumors.

Abstract Mitochondrial DNA (mtDNA) mutations and polymorphisms contribute to many complex diseases, including cancer. Using a unique mouse model that contains nDNA from one mouse strain and homoplasmic mitochondrial haplotypes from different mouse strain(s)—designated Mitochondrial Nuclear Exchange (MNX)—we showed that mtDNA could alter mammary tumor metastasis. Because retrograde and anterograde communication exists between the nuclear and mitochondrial genomes, we hypothesized that there are differential mtDNA-driven changes in nuclear (n)DNA expression and DNA methylation. Genome-wide nDNA methylation and gene expression were measured in harvested brain tissue from paired wild-type and MNX mice. Selective differential DNA methylation and gene expression were observed between strains having identical nDNA, but different mtDNA. These observations provide insights into how mtDNA could be altering epigenetic regulation and thereby contribute to the pathogenesis of metastasis. Cancer Res; 77(22); 6202–14. ©2017 AACR.