Mark Stout

Synthesis, biological, and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase

2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1-3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with l-glutamate diethyl ester, followed by saponification, afforded 1-3. Compound 3 selectively inhibited the proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including KB and IGROV1 human tumor cells, much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1-, 2-, and 4-atom bridge lengths for the activity of this series.

MECHANISMS OF SYNERGISTIC ANTILEUKEMIC INTERACTIONS BETWEEN VALPROIC ACID AND CYTARABINE IN PEDIATRIC ACUTE MYELOID LEUKEMIA

Purpose: To determine the possibility of synergistic antileukemic activity and the underlying molecular mechanisms associated with cytarabine combined with valproic acid (VPA; a histone deacetylase inhibitor and a Food and Drug Administration–licensed drug for treating both children and adults with epilepsy) in pediatric acute myeloid leukemia (AML). Experimental Design: The type and extent of antileukemic interactions between cytarabine and VPA in clinically relevant pediatric AML cell lines and diagnostic blasts from children with AML were determined by MTT assays and standard isobologram analyses. The effects of cytarabine and VPA on apoptosis and cell cycle distributions were determined by flow cytometry analysis and caspase enzymatic assays. The effects of the two agents on DNA damage and Bcl-2 family proteins were determined by Western blotting. Results: We showed synergistic antileukemic activities between cytarabine and VPA in four pediatric AML cell lines and nine diagnostic AML blast samples. t(8;21) AML blasts were significantly more sensitive to VPA and showed far greater sensitivities to combined cytarabine and VPA than non-t(8;21) AML cases. Cytarabine and VPA cooperatively induced DNA double-strand breaks, reflected in induction of γH2AX and apoptosis, accompanied by activation of caspase-9 and caspase-3. Further, VPA induced Bim expression and short hairpin RNA knockdown of Bim resulted in significantly decreased apoptosis induced by cytarabine and by cytarabine plus VPA. Conclusions: Our results establish global synergistic antileukemic activity of combined VPA and cytarabine in pediatric AML and provide compelling evidence to support the use of VPA in the treatment of children with this deadly disease. Clin Cancer Res; 16(22); 5499–510. ©2010 AACR.

Targeting the proton-coupled folate transporter for selective delivery of 6-substituted pyrrolo[2,3-d]pyrimidine antifolate inhibitors of de novo purine biosynthesis in the chemotherapy of solid tumors.

The proton-coupled folate transporter (PCFT) is a folate-proton symporter with an acidic pH optimum, approximating the microenvironments of solid tumors. We tested 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with one to six carbons in the bridge region for inhibition of proliferation in isogenic Chinese hamster ovary (CHO) and HeLa cells expressing PCFT or reduced folate carrier (RFC). Only analogs with three and four bridge carbons (N-{4-[3-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)propyl]benzoyl}-l-glutamic acid (compound 2) and N-{4-[4-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)butyl]benzoyl}*-l-glutamic acid (compound 3), respectively) were inhibitory, with 2 ≫ 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [3H]methotrexate for PCFT transport in PCFT-expressing CHO (R2/hPCFT4) cells from pH 5.5 to 7.2; inhibition increased with decreasing pH. In Xenopus laevis oocytes microinjected with PCFT cRNA, uptake of 2, like that of Pmx, was electrogenic. Cytotoxicity of 2 toward R2/hPCFT4 cells was abolished in the presence of adenosine or 5-amino-4-imidazolecarboxamide, suggesting that glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis was the primary target. Compound 2 decreased GTP and ATP pools by ∼50 and 75%, respectively. By an in situ GARFTase assay, 2 was ∼20-fold more inhibitory toward intracellular GARFTase than toward cell growth or colony formation. Compound 2 irreversibly inhibited clonogenicity, although this required at least 4 h of exposure. Our results document the potent antiproliferative activity of compound 2, attributable to its efficient cellular uptake by PCFT, resulting in inhibition of GARFTase and de novo purine biosynthesis. Furthermore, they establish the feasibility of selective chemotherapy drug delivery via PCFT over RFC, a process that takes advantage of a unique biological feature of solid tumors.

Therapeutic targeting of a novel 6-substituted pyrrolo [2,3-d]pyrimidine thienoyl antifolate to human solid tumors based on selective uptake by the proton-coupled folate transporter

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 [3H]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, [3H]compound 1 was metabolized to 3H-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.

Myeloblasts from Down syndrome children with acute myeloid leukemia have increased in vitro sensitivity to cytosine arabinoside and daunorubicin.

Myeloblasts from Doen syndrome children with acute myeloid leukemia have increased in vitro sensitivity to cytosine arabinoside and daunorubicine

Functional loss of the reduced folate carrier enhances the antitumor activities of novel antifolates with selective uptake by the proton-coupled folate transporter.

Uptake of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with four or three bridge carbons [compound 1 (C1) and compound 2 (C2), respectively] into solid tumors by the proton-coupled folate transporter (PCFT) represents a novel therapeutic strategy that harnesses the acidic tumor microenvironment. Although these compounds are not substrates for the reduced folate carrier (RFC), the major facilitative folate transporter, RFC expression may alter drug efficacies by affecting cellular tetrahydrofolate (THF) cofactor pools that can compete for polyglutamylation and/or binding to intracellular enzyme targets. Human tumor cells including wild-type (WT) and R5 (RFC-null) HeLa cells express high levels of PCFT protein. C1 and C2 inhibited proliferation of R5 cells 3 to 4 times more potently than WT cells or R5 cells transfected with RFC. Transport of C1 and C2 was virtually identical between WT and R5 cells, establishing that differences in drug sensitivities between sublines were independent of PCFT transport. Steady-state intracellular [3H]THF cofactors derived from [3H]5-formyl-THF were depleted in R5 cells compared with those in WT cells, an effect exacerbated by C1 and C2. Whereas C1 and C2 polyglutamates accumulated to similar levels in WT and R5 cells, there were differences in polyglutamyl distributions in favor of the longest chain length forms. In severe combined immunodeficient mice, the antitumor efficacies of C1 and C2 were greater toward subcutaneous R5 tumors than toward WT tumors, confirming the collateral drug sensitivities observed in vitro. Thus, solid tumor-targeted antifolates with PCFT-selective cellular uptake should have enhanced activities toward tumors lacking RFC function, reflecting contraction of THF cofactor pools.

Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter

PurposeWe examined whether the novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate, compound 2, might be an effective treatment for malignant pleural mesothelioma (MPM), reflecting its selective membrane transport by the proton-coupled folate transport (PCFT) over the reduced folate carrier (RFC).MethodsHeLa sublines expressing exclusively PCFT (R1-11-PCFT4) or RFC (R1-11-RFC6) and H2452 MPM cells were assayed for transport with [3H]compound 2. [3H]Polyglutamate metabolites of compound 2 were measured in R1-11-PCFT4 and H2452 cells. In vitro cell proliferation assays and colony formation assays were performed. Inhibition of glycinamide ribonucleotide formyltransferase (GARFTase) was assayed by nucleoside protection assays and in situ GARFTase assays with [14C]glycine. In vivo efficacy was established with early- and advanced-stage H2452 xenografts in severe-combined immunodeficient (SCID) mice administered intravenous compound 2.Results[3H]Compound 2 was selectively transported by PCFT and was metabolized to polyglutamates. Compound 2 selectively inhibited proliferation of R1-11-PCFT4 cells over R1-11-RFC6 cells. H2452 human MPM cells were sensitive to the antiproliferative effects of compound 2. By colony-forming assays with H2452 cells, compound 2 was cytotoxic. Compound 2 inhibited GARFTase in de novo purine biosynthesis. In vivo efficacy was confirmed toward early- and advanced-stage H2452 xenografts in SCID mice administered compound 2.ConclusionsOur results demonstrate potent antitumor efficacy of compound 2 toward H2452 MPM cells in vitro and in vivo, reflecting its efficient membrane transport by PCFT, synthesis of polyglutamates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by tumor-targeted antifolates such as compound 2 presents an exciting new opportunity for treating solid tumors.

Role of USF1 in the differential expression of the human deoxycytidine kinase gene in acute myeloid leukemia

Role of USF1 in the differential expression of the human deoxycytidine kinase gene in acute myeloid leukemia

Abstract 3825: The impact of the reduced folate carrier on the collateral sensitivity of novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective uptake by the proton-coupled folate transporter

The 6-substituted pyrrolo[2,3-d]pyrimidine thienonyl antifolates with 3 (AG94) or 4 (AG71) carbon bridge lengths represent a new class of agents that inhibit α-glycinamide ribonucleotide (GAR) formyltransferase (GARFTase). AG71 and AG94 are selectively transported by the proton-coupled folate transporter (PCFT) over the reduced folate carrier (RFC), the major facilitative folate transporter in tissues and tumors. PCFT functions optimally at acidic pHs. Transport of AG71 and AG94 by PCFT and the acidic tumor microenvironment may further increase tumor selectivity. Although AG71 and AG94 are not RFC substrates, levels of RFC could nonetheless impact their anti-proliferative effects, via expansion or contraction of intracellular tetrahydrofolate (THF) pools. Studies were performed with wild-type (WT) and methotrexate resistant R5 HeLa cells in which RFC is deleted. In proliferation assays with AG71 and AG94, R5 cells were more sensitive than were WT cells (3.9- and 2.4-fold, respectively). RFC-transfected R5 cells had IC50s for growth inhibition similar to WT cells. To determine the mechanisms of the collateral sensitivities to AG71 and AG94, we measured steady-state intracellular [3H]THF accumulations at a physiologic concentration of [3H]5-formyl THF in WT and R5 cells. R5 cells had contracted intracellular [3H]THF pools compared to WT cells. The collateral antifolate sensitivities in R5 cells were independent of PCFT transport, since there were no differences in transport for AG71 and AG94 at any pH between the sublines. Intracellular THF cofactors may compete at the level of polyglutamylation by folylpolyglutamate synthetase and/or GARFTase binding. When incubated with radiolabeled AG71 and AG94, WT cells accumulated slightly lesser amounts of total drug forms (84 and 76% for AG71 and AG94, respectively) than did R5 cells, most of these as polyglutamates. This difference was greatest for the longest chain-length (Glu4-Glu6) polyglutamates (increased ∼2-fold for both AG71 and AG94). GARFTase activity was measured in WT and R5 cells by an in situ metabolic assay which measures incorporation of [14C]glycine into [14C]formyl GAR. R5 and WT cells were differentially sensitive to GARFTase inhibition by AG71 and AG94 and these differences were lost when RFC expression was restored to R5 cells. In an in vivo efficacy trial with subcutaneous WT and R5 cells in SCID mice, antitumor efficacies of both AG71 and AG94 were increased in R5 cells relative to WT cells and this effect was greater for AG71. These findings suggest that novel antifolates with selective membrane transport by PCFT over RFC would have enhanced activities toward tumors that have lost RFC function, and that levels of RFC may be an important biomarker of chemotherapy efficacy with this new class of solid tumor-selective drugs. 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 3825. doi:1538-7445.AM2012-3825

Abstract 4351: p53 is a critical determinant of the antileukemic interactions between histone deacetylase inhibitors and chemotherapy drugs in pediatric acute myeloid leukemia cells

Acute myeloid leukemia (AML) accounts for one-fourth of acute leukemias in children, but it is responsible for more than half of the leukemia deaths in this patient population. Resistance to ara-C/anthracycline-based chemotherapy is a major cause of treatment failure in this disease. Among the newer agents that have been investigated in high-risk AML, clofarabine and valproic acid (VPA) are particularly notable. Clofarabine is a new FDA-approved drug for treating pediatric relapsed acute lymphoblastic leukemia and is currently being tested in pediatric AML. VPA is a FDA-licensed drug for treating both children and adults with epilepsy that has been shown to act as a histone deacetylase inhibitor (HDACI). We hypothesized that VPA and clofarabine might exhibit synergistic antileukemic activity in pediatric AML. In this study, we demonstrated highly synergistic antileukemic activities of combined clofarabine and VPA in 9 diagnostic blast samples from children with de novo AML. In contrast, both additive-to-synergistic [in Kasumi-1 which harbors t(8;21) and MV4-11 which harbors t(4;11)] and antagonistic (in THP-1 and CMS) results were obtained in 4 pediatric AML cell lines. AML cells which harbored t(8;21) were substantially more sensitive to VPA and showed greater responses to the drug combination than non-t(8;21) AML cells. Essentially the same results were obtained when clofarabine was combined with SAHA (a FDA-licensed pan-HDACI) in the pediatric AML cell lines. Co-treatment with clofarabine and VPA resulted in synergistic induction of apoptosis in Kasumi-1 cells, accompanied by synergistic activation of caspase-3 and cleavage of PARP. Our mechanistic studies revealed cooperative induction of DNA double-strand breaks (as reflected by induction of gH2AX), phosphorylation and acetylation of p53, and activation of Bax by clofarabine and VPA. Automated DNA sequencing revealed a p53 deficiency in THP-1 and CMS cell lines which may account for the antagonistic cytotoxic interactions between clofarabine and VPA, whereas the rest of the cells harbored either wild-type or mutant p53. Interestingly, treatments of Kasumi-1 cells with ara-C and VPA also resulted in cooperative activation of p53 and Bax, which may explain the differential responses of pediatric AML cells to combined ara-C and VPA observed in our previous study (Xie C et al. Clin Cancer Res 2010). Collectively, our results provide compelling evidence that p53 plays a key role in the synergistic cytotoxic effects of HDACIs combined with chemotherapy drugs in pediatric AML cells. 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 4351. doi:10.1158/1538-7445.AM2011-4351