The proton-coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum. By real-time reverse transcription-polymerase chain reaction, PCFT was expressed in the majority of 53 human tumor cell lines, with the highest levels in Caco-2 (colorectal adenocarcinoma), SKOV3 (ovarian), and HepG2 (hepatoma) cells. A novel 6-substituted pyrrolo [2,3-d]pyrimidine thienoyl antifolate (compound 1) was used to establish whether PCFT can deliver cytotoxic drug under pH conditions that mimic the tumor microenvironment. Both 1 and pemetrexed (Pmx) inhibited proliferation of R1-11-PCFT4 HeLa cells engineered to express PCFT without the reduced folate carrier (RFC) and of HepG2 cells expressing both PCFT and RFC. Unlike Pmx, 1 did not inhibit proliferation of R1-11-RFC6 HeLa cells, which express RFC without PCFT. Treatment of R1-11-PCFT4 cells at pH 6.8 with 1 or Pmx inhibited colony formation with dose and time dependence. Transport of [ 3 H]compound 1 into R1-11-PCFT4 and HepG2 cells was optimal at pH 5.5 but appreciable at pH 6.8. At pH 6.8, [3 H]compound 1 was metabolized to 3 H-labeled polyglutamates. Glycinamide ribonucleotide formyltransferase (GARFTase) in R1-11-PCFT4 cells was inhibited by 1 at pH 6.8, as measured by an in situ GARFTase assay, and was accompanied by substantially reduced ATP levels. Compound 1 caused S-phase accumulation and a modest level of apoptosis. An in vivo efficacy trial with severe combined immunodeficient mice implanted with subcutaneous HepG2 tumors showed that compound 1 was active. Our findings suggest exciting new therapeutic possibilities to selectively deliver novel antifolate drugs via transport by PCFT over RFC by exploiting the acidic tumor microenvironment.

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We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine antifolates (compounds 5–10) with 1 to 6 bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analog and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Anti-proliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.

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Recent studies suggest that organophosphates and carbamates affect human fetal development, resulting in neurological and growth impairment. However, these studies are conflicting and the extent of adverse effects due to pesticide exposure warrants further investigation. In the present study, we examined the impact of the carbamate insecticide propoxur on zebrafish development. We found that propoxur exposure delays embryonic development, resulting in three distinct developmental stages: no delay, mild delay, or severe delay. Interestingly, the delayed embryos all physically recovered 5 days after exposure, but behavioral analysis revealed persistent cognitive deficits at later stages. Microarray analysis identified 59 genes significantly changed by propoxur treatment, and Ingenuity Pathway Analysis revealed that these genes are involved in cancer, organismal abnormalities, neurological disease, and hematological system development. We further examined hspb9 and hspb11 due to their potential roles in zebrafish development and found that propoxur increases expression of these small heat shock proteins in all of the exposed animals. However, we discovered that less significant increases were associated with the more severely delayed phenotype. This raises the possibility that a decreased ability to upregulate these small heat shock proteins in response to propoxur exposure may cause embryos to be more severely delayed.

Pediatric T-ALL typically has a poorer prognosis than B-precursor ALL, as T-ALL patients exhibit 5-event free survival rates approximating 70-75% compared to > 90% for B-precursor-ALL. Notch1 activating mutations occur in more than 50% of T-ALL cases. Notch1 mutations cluster within the heterodimerization (HD) and/or PEST domains and liberate activated intracellular Notch 1 (ICN1) by increasing susceptibility to HD domain cleavage by gamma-secretase (GS) and/or by increasing the half-life of ICN1, respectively. This results in increased expression of ICN1 target genes including hes1, deltex1, cmyc, igf1r, calcineurin, e2a, and il7r, which affect cell proliferation and survival at least in part by activating AKT. Notch1 increases Hes1 expression and transcriptional silencing of PTEN which antagonizes AKT activation. Mutational loss of PTEN, which occurs frequently in T-ALL, renders cells resistant to Notch1 inhibition with GS inhibitors (GSIs). This suggests that Notch1 favors AKT activation. However, Notch1 can exert inhibitory control over AKT signaling even in GSI-resistant, PTEN-null Jurkat cells, which have mutant activated Notch1. We found that both GSI treatment and Notch1 knockdown (N1KD) in Jurkat cells increased AKT activation loop (T308) phosphorylation and signaling, and protected cells from induction of apoptosis. This was not due to increased AKT phosphorylation by PI3K. Rather this was due to decreased dephosphorylation of AKT-T308 in the N1KD cells. PP2A is the major Ser/Thr phosphatase that dephosphorylates AKT at T308. Treatment of cells with the PP2A inhibitor okadaic acid increased AKT-T308 phosphorylation in non-targeted control (NTC) cells but had no overt effect in the N1KD cells, suggesting decreased PP2A activity. Notch1 primarily functions as a transcriptional regulator and could affect genes encoding the PP2A catalytic or regulatory subunits. However, neither the levels of the individual subunits nor PP2A catalytic activity were changed in N1KD cells. By immunoprecipitation, N1KD cells showed a decreased interaction between PP2A and AKT. This was accompanied by increased phosphorylation of AKT-T308 but also of other PP2A targets including AMPK and p70S6K. Increased phosphorylation of these targets also resulted from transient transfection with a dominant-negative MAML which interferes with ICN1 transcriptional effects. This was not mediated by cMyc, as established by cMyc transfections and treatment with a cMyc inhibitor. Conversely, transfection with Hes1 decreased phosphorylation of these PP2A targets in the N1KD cells. This suggests a causal role for Hes1, at least in part, in the Notch1 effects on PP2A and AKT-T308 phosphorylation. To our knowledge, these effects of Notch1 and Hes1 on PP2A and their impact on AKT and AMPK signaling have not been previously described. Citation Format: Eric Christopher Hales, Steven M. Orr, Amanda Larson Gedman, Jeffrey W. Taub, Larry H. Matherly. Notch1 regulates AKT-T308 dephosphorylation through modulation of the PP2A phosphatase in GSI-resistant T-cell acute lymphoblastic leukemia (T-ALL) cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4116. doi:10.1158/1538-7445.AM2013-4116

T-cell acute lymphoblastic leukemias (T-ALL) are frequently associated with mutations in Notch 1, many of which lead to increased susceptibilities to cleavage by gamma secretase and generation of ICN1, promoting enhanced Notch1 signaling. Increased Notch1 activity in T-ALL was reported to indirectly repress transcription of PTEN, the major tumor suppressor involved in antagonizing PIP3-dependent cell signaling and in maintaining tight control over the pro-survival AKT pathway. While use of gamma secretase inhibitors (GSIs) prevents Notch1-activating cleavage and restores PTEN protein levels, a link between GSI treatments and PTEN posttranslational inactivation was also implied (Haematologica 95: 674-678, 2010). Phosphorylation by casein kinase II (CK2) of the S380/T382/3 cluster in the carboxyl-terminal region of PTEN is an efficient means of regulating PTEN activity, and of maintaining tight control over cell viability. PTEN phosphorylation impacts its cytosolic versus membrane localization, and PTEN protein stability and levels. However, the relative impact of Notch1 and GSIs on PTEN levels resulting from effects on PTEN transcription versus potential PTEN posttranslational effects has not been established. To begin to address this important question, we used conditional ectopic PTEN expression under control of a tetracycline-inducible promoter in PTEN-null Jurkat cells (designated Jkt/tet-onPten) to assess whether Notch1 can regulate PTEN posttranslationally. Cells were treated with doxycycline to induce PTEN, then with compound E (CompE), a potent GSI. CompE treatment increased phosphorylation of PTEN and AKT (S473 and T308), leading to elevated phosphorylation of GSK3αβ and S6RP downstream, consistent with inactivation of PTEN. This was accompanied by a moderate increase in cell proliferation. Analogous results were seen in Jkt/tet-onPten cells in which Notch1 was knocked down with a shRNA lentiviral vector (Jkt/tet-onPtenKD cells). Knockdown of Notch1 was accompanied by increased cytosolic localization of PTEN, consistent with its phosphorylation and inactivation. Treatment of Jkt/tet-onPtenKD cells with a CK2 inhibitor (tetrabromobenzimidazole) reduced PTEN phosphorylation and restored AKT activation, establishing that the effects of Notch1 on PTEN phosphorylation were via CK2. Our results suggest a mechanism whereby reduced Notch1 signaling in T-ALL cells increases PTEN posttranslational inactivation and AKT signaling, through an effect on CK2-mediated phosphorylation of PTEN. Although GSI treatment should transcriptionally increase PTEN levels, our data suggest that GSI treatment could potentially result in a more aggressive T-ALL due to increased PTEN inactivation and increased AKT signaling. Thus, combined use of a GSI with a downstream inhibitor to CK2, AKT or mTOR could provide significant benefit in treating T-ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2881. doi:10.1158/1538-7445.AM2011-2881

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) has a variable prognosis, with many patients exhibiting high-risk features at diagnosis, including older age and high presenting white blood cell counts. Activating mutations involving the Notch1 heterodimerizeration and PEST domains occur in more than 50% of T-ALL cases. Increased Notch1 activity promotes survival and proliferation of leukemic cells, in part by transcriptionally repressing PTEN (via HES1). This provides compelling rationale for the use of gamma secretase inhibitors (GSIs) to block Notch1 activation and to restore PTEN levels, which antagonize AKT signaling. Further, PTEN mutations are implicated in GSI resistance. PTEN activity is also modulated by an assortment of posttranslational modifications and Notch1 may regulate PTEN at the posttranslational level. To investigate the impact of Notch1 on AKT signaling, independent of its transcriptional regulation of PTEN, we used PTEN-null Jurkat cells with constitutively active Notch1 and conditional ectopic PTEN expression under control of a tetracycline (tet)-inducible promoter. Induction of PTEN was accompanied by increased phosphorylation of AKTT308, increased insulin-like growth factor 1 receptor (IGF1R) protein and transcripts, and increased Notch1. Increased AKTT308 phosphorylation was blocked with an IGF1R inhibitor (GSK1904259A) and was abolished in Jurkat cells expressing a tet-inducible G129R PTEN phosphatase-inactive mutant. This suggests that PTEN can activate AKT signaling by modulating IGF1R levels. In Jurkat cells, inhibition of Notch1, either through GSI treatment or shRNA knockdown (KD), resulted in increased phosphorylation of AKTT308. Although this result could involve Notch1-dependent decreased cMyc, reflecting indirect effects of cMyc on mTOR1 and negative feedback on AKT, this was not impacted by treatment with a cMyc inhibitor (10058-F4) or by ectopic overexpression of cMyc, suggesting that other effectors and mechanisms are involved. Microarray analysis was performed on wild-type and KD Jurkat cells. Using a 1.5-fold cutoff, 1208 differentially regulated genes were identified. These include PH domain leucine-rich repeat protein phosphatase-1 (PHLPP1) and prokineticin-2 (PROK2), both of which could significantly impact AKT phosphorylation in response to Notch1. By real-time RT-PCR, PHLPP1 was significantly decreased (∼4-fold), and PROK2 was significantly increased (∼6-fold) in Notch1 KD cells compared to wild-type cells. Studies are underway to characterize the potential roles of PHLPP1 and PROK2 in mediating the inhibitory effects of Notch1 on AKT activation in T-ALL. Better understanding of the cross-talk between Notch1 and AKT in T-ALL may identify rational therapeutic strategies for combining GSIs with inhibitors of AKT, mTOR, or IGF1R, and/or for designing therapies based on capacities for constitutive Notch1 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2226. doi:1538-7445.AM2012-2226