Development And Mechanistic Studies Of Novel Therapeutics Towards Pancreatic Ductal Adenocarcinoma By Combining PI3K,mTOR And ERK Inhibitors,or Targeting One-carbon Metabolism

Pancreatic cancer(Pa C)represents the 4th leading cause of cancer-related deaths in the US with a mortality rate of 99%.The 5-year overall survival rate for Pa C is currently 8%.Pancreatic ductal adenocarcinoma(PDAC)is a major subtype of Pa C which is in urgent need of newer therapeutic modalities.We explored two strategies to overcome this issue.The first one was to target KRAS downstream effectors PI3 K and m TOR.The majority of patients with PDAC have mutations in KRAS,which unfortunately remains an ineffectual target.Activating KRAS mutations occur in over 90% of PDAC cases and drive PDAC cell proliferation and survival.Inhibition of mutant KRAS has proven to be a difficult task,thus alternative therapies targeting downstream KRAS effectors have been exploited.The PI3K/m TOR pathway,which is downstream of KRAS,is commonly deregulated in many cancer types.The PI3K/m TOR pathway plays a key role in proliferation,migration,survival,and growth.The first inhibitors of the PI3K/m TOR signaling pathways were rapamycin and rapalogs,which targeted m TOR.Unfortunately,rapalogs have demonstrated limited clinical benefits due to drug-induced feedback loops,which cause hyperactivation of PI3K/AKT and enhance the proliferation rate of tumors.These findings have led to the development of dual PI3 K and m TOR inhibitors which can overcome feedback activation of PI3 K resulting from m TOR inhibition.However,efficacy can be hindered by activation of other pathways.The MEK/ERK pathway is another downstream pathway affected by KRAS.Cross-talk between the PI3K/m TOR and the MEK/ERK signaling pathways has been reported.MEK inhibition has been shown to cause PI3 K activation and PI3 K inhibition has been demonstrated to cause ERK activation.Thus,targeting both pathways simultaneously may be a promising approach to treat PDAC.In this study,we investigated the antitumor efficacy of the novel PI3 K and m TOR dual inhibitor VS-5584 in PDAC.Our data show that PI3K/m TOR dual inhibition causes ERK activation in all tested PDAC cell lines,including KRAS mutant cell lines(HPAC)and the KRAS wild-type cell line(Bx PC-3).Although the MEK inhibitor GSK1120212 could abrogate VS-5584-induced ERK activation,it did not substantially enhance cell death in all the PDAC cell lines tested.However,combination with the ERK inhibitor SCH772984 not only mitigated VS-5584-induced ERK activation but also enhanced VS-5584-induced cell death.In a xenograft model of HPAC,we observed 28% and 44% tumor inhibition for individual treatment with VS-5584 and SCH772984,respectively,while the combined treatment showed superior tumor inhibition(80%)compared to vehicle control treatment.Our findings support the clinical development of VS-5584 and ERK inhibitor combination for PDAC treatment.The second strategy we explored was to develop targeted therapeutics towards Pa C by targeting one-carbon(C1)metabolism which is frequently altered in cancer.For PDAC,TCGA data sets show that elevated expression of key enzymes involved in cytosolic [5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase(AICARFTase)and serine hydroxymethyltransferase(SHMT)1] and mitochondrial [SHMT2 and methylene tetrahydrofolate dehydrogenase 2(MTHFD2)] C1 metabolism is associated with poor survival.Antifolate therapeutics disrupt cytosolic C1 pathways required for syntheses of thymidylate,purines,and certain amino acids,and are a mainstay for therapy of several cancers.Antifolate uptake into tumors and tissues involves the reduced folate carrier(RFC),the major tissue folate transporter,and the proton-coupled folate transporter(PCFT),which shows a more limited tissue distribution but is widely expressed in human solid tumors and is active only at acidic p Hs characterizing the tumor microenvironment.We measured transcript levels for the PCFT and RFC by real-time PCR in 4 normal pancreas and 19 Pa C patient specimens(5 PDAC,9 neuroendocrine,4 islet cell,1 acinar cell).PCFT transcripts were highly expressed in Pa Cs over a 15-fold range with a median level approximating that in KB human tumor cells;there was no difference in PCFT levels between tumor and normal specimens.RFC was expressed(~1000-fold range)in Pa C specimens and was low and below the level in KB cells.PCFT proteins were measured by IHC on tissue microarrays with PCFT antibody,from a separate cohort of 91 primary PDACs and 4 normal pancreas specimens and were expressed over a 140-fold range.We identified a series of 6-substituted pyrrolo[2,3-d]pyrimidine 1st generation analogs with in vitro anti-tumor efficacies toward PDAC cell lines expressing PCFT transcripts and proteins at levels similar to those in primary patient specimens.PCFT proteins on Westerns and transport paralleled PCFT transcript levels.The highest PCFT levels were in Bx PC-3 and PANC-1 cells,with lower levels in ASPC-1 and MIA Pa Ca-2 cells.AGF94 and AGF272 inhibited proliferation of the PDAC sublines.AGF94 and AGF272 were cytotoxic,reflected in colony-forming assays,and inhibited de novo purine biosynthesis at glycinamide ribonucleotide(GAR)formyl-transferase(GARFTase;1st folate-dependent step),as analyzed by protection experiments with addition of adenosine(60 ?M)or 5-aminoimidazole-4-carboxamide(320 ?M),in situ assays of GARFTase with 14C-glycine labeling and targeted metabolomics.We discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine 2nd generation analogs(AGF347,AGF359)with substantial PCFT transport and that potently inhibited proliferation of PDAC cell lines(above),of which HPAC(KRAS mutant)and Bx PC-3(KRAS wild-type)cells were most sensitive.The PDAC cell lines were active for PCFT transport with 3H-AGF347 at acid p H.When HPAC cells were incubated with 3H-AGF347 over 48 h,drug accumulated in both cytosol and mitochondria.3H-AGF347 was extensively metabolized to polyglutamates in both compartments.Treatment of PDAC cells with AGF347 and AGF359 inhibited proliferation by inducing glycine and adenosine auxotrophy that was rescued by excess glycine and adenosine.This implied that both mitochondria and cytosolic C1 metabolism were inhibited.Tumor cell killing was confirmed(with HPAC and Bx PC-3)by colony-forming assays with AGF347 and AGF359 and drug-induced apoptosis with AGF347 and AGF359 was demonstrated(with HPAC)by annexin V-PI staining and flow cytometry.AGF347 and AGF359 depleted purine nucleotides and inhibited m TOR signaling via S6K1 at least in part(for Bx PC-3)via activation of AMPK,likely due to elevated ZMP accompanying suppression of AICARFTase.Collectively,our studies identify first-in-class inhibitors of C1 metabolism and establish the considerable therapeutic potential of dual-targeting mitochondrial and cytosolic C1 metabolism in PDAC independent of KRAS mutation status and reflecting cellular uptake by PCFT.In summary,we have developed promising therapeutic strategies towards very challenging PDAC with KRAS mutation,by either combining inhibitors for KRAS downstream PI3 K and m TOR,as well as ERK signaling pathways,or by developing novel antifolates targeting both cytosolic and mitochondrial C1 metabolism.