Steven Orr

Notch1 receptor regulates AKT protein activation loop (Thr308) dephosphorylation through modulation of the PP2A phosphatase in phosphatase and tensin homolog (PTEN)-null T-cell acute lymphoblastic leukemia cells.

Background: PTEN loss promotes resistance to γ-secretase inhibitors by increasing AKT signaling in T-cell acute lymphoblastic leukemia (T-ALL) with mutant activated Notch1. Results: Notch1 inhibition increases AKT phosphorylation and involves the PP2A phosphatase. Conclusion: Notch1 regulates PP2A dephosphorylation of AKT-Thr308 by impacting association of PP2A with AKT. Significance: Better understanding of regulation of AKT signaling by Notch1 may lead to new therapies for T-ALL. Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr308 phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr308 phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr308 phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr308 by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr172 and p70S6K-Thr389, both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K.

Discovery of 5-substituted pyrrolo[2,3- d ]pyrimidine antifolates as dual-acting inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis: Implications of inhibiting 5-aminoimidazole-4- carboxamide ribonucleotide formyltransferase to AMPK activation and antitumor activity

We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine antifolates (compounds 5-10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue 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. Antiproliferative 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.

6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors

2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or β (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and β over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.

Structure–Activity Profiles of Novel 6-Substituted Pyrrolo[2,3- d ]pyrimidine Thienoyl Antifolates with Modified Amino Acids for Cellular Uptake by Folate Receptors α and β and the Proton-Coupled Folate Transporter

Structure–activity relationships for cellular uptake and inhibition of cell proliferation were studied for 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates in which the terminal l-glutamate of the parent structure (7) was replaced by natural or unnatural amino acids. Compounds 7 and 10–13 were selectively inhibitory toward folate receptor (FR) α-expressing Chinese hamster ovary (CHO) cells. Antiproliferative effects of compounds 7 and 9–13 toward FRα- and FRβ-expressing CHO cells were only partly reflected in binding affinities to FRα and FRβ or in the docking scores with molecular models of FRα and FRβ. Compounds 7 and 11 were potent inhibitors of glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis in KB human tumor cells. These studies establish for the first time the importance of the α- and γ-carboxylic acid groups, the length of the amino acid, and the conformation of the side chain for transporter binding and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates.

Tumor-targeting with novel non-benzoyl 6-substituted straight chain pyrrolo[2,3-d ]pyrimidine antifolates via cellular uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis

A new series of 6-substituted straight side chain pyrrolo[2,3-d]pyrimidines 3a-d with varying chain lengths (n = 5-8) was designed and synthesized as part of our program to provide targeted antitumor agents with folate receptor (FR) cellular uptake specificity and glycinamide ribonucleotide formyltransferase (GARFTase) inhibition. Carboxylic acids 4a-d were converted to the acid chlorides and reacted with diazomethane, followed by 48% HBr to generate the α-bromomethylketones 5a-d. Condensation of 2,4-diamino-6-hydroxypyrimidine 6 with 5a-d afforded the 6-substituted pyrrolo[2,3-d]pyrimidines 7a-d. Hydrolysis and subsequent coupling with diethyl l-glutamate and saponification afforded target compounds 3a-d. Compounds 3b-d showed selective cellular uptake via FRα and -β, associated with high affinity binding and inhibition of de novo purine nucleotide biosynthesis via GARFTase, resulting in potent inhibition against FR-expressing Chinese hamster cells and human KB tumor cells in culture. Our studies establish, for the first time, that a side chain benzoyl group is not essential for tumor-selective drug uptake by FRα.

Novel 5-Substituted Pyrrolo[2,3- d ]pyrimidines as Dual Inhibitors of Glycinamide Ribonucleotide Formyltransferase and 5-Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase and as Potential Antitumor Agents

A new series of 5-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 6-11 with varying chain lengths (n = 1-6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds 6-11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8-10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure-activity relationship for transport and dual target inhibition.

Abstract 5493: Tumor-targeting with novel non-benzoyl 6-substituted pyrrolo[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis.

Interest has focused on folate receptor (FR) α for delivering cytotoxic folate-based therapeutics to solid tumors, as FRα is highly expressed in certain tumors, including ovarian and non-small cell lung cancers. While FRα is present in normal epithelial tissues, this is restricted to apical membranes and is inaccessible to the circulation. Selectivity of FR-targeted agents would be further enhanced if these were poor substrates for the ubiquitously expressed reduced folate carrier (RFC). Both 5- and 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl antifolates with 3 (AG19, AG126, respectively) and 4 (AG23, AG127, respectively) carbon bridge lengths potently inhibited proliferation of KB human tumor cells via uptake by FRα and inhibition of de novo purine nucleotide biosynthesis at β-glycinamide ribonucleotide formyltransferase (GARFTase) (all drugs) and 5-amino-4-imidazolecarboxamide ribonucleotide formyltransferase (AG126, AG127). Thienoyl-for-benzoyl B ring replacement in AG19 and AG23 increased inhibitory potencies, reflecting FRα uptake and increased GARFTase inhibition. To further examine the role in biological activities of the side chain benzoyl ring and terminal glutamate for this series, non-benzoyl 6-substituted pyrrolo[2,3-d]pyrimidine analogs were synthesized without a phenyl ring but with 5-8 carbon substituents with (AG143, AG145, AG147, AG149, respectively) and without a terminal glutamate. 5-Substituted analogs of AG145, AG147, and AG149 were also synthesized. Compounds were tested in proliferation assays with isogenic Chinese hamster ovary (CHO) sublines expressing RFC (PC43-10) or FRα (RT16). Additional testing was performed with KB human tumor cells. Analogs without a terminal glutamate and 5-substituted non-benzoyl pyrrolopyrimidine analogs were all inactive. AG143, AG145, AG147, and AG149 were also inactive with RFC- expressing CHO cells. For FRα-expressing RT16 and KB cells, growth inhibition was detectable in the order: AG147 ∼ AG149 > AG145 >> AG143. For AG147 and AG149, IC 50 s toward KB cells were ∼1 nM. Drug effects were abolished with excess folic acid, confirming FR uptake. Inhibition of cell proliferation was reflected in binding affinities to FRα, measured by competition with [ 3 H]folic acid. By protection experiments with excess thymidine or adenosine, de novo purine nucleotide biosynthesis was confirmed as the targeted pathway for the active analogs. 5-Amino-4-imidazolecarboxamide also reversed the growth inhibitory effects of this series, establishing GARFTase as the principal intracellular target. Collectively, our studies establish an important structure-activity relationship for FRα-targeted antifolates, namely that a side chain benzoyl group is not essential for tumor selective drug uptake by FRα and for inhibition of GARFTase. Citation Format: Christina Cherian, Yiqiang Wang, Shermaine Mitchell-Ryan, Steven Orr, Zhanjun Hou, Aleem Gangjee, Larry H. Matherly. Tumor-targeting with novel non-benzoyl 6-substituted pyrrolo[2,3- d ]pyrimidine antifolates via cellular uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis. [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 5493. doi:10.1158/1538-7445.AM2013-5493

Abstract 1620: Synthesis and preclinical evaluation of novel 6-substituted pyrrolo[2,3-d]pyrimidines as targeted antifolates

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Reduced folates are essential cofactors for biosynthesis of purines and pyrimidines. Since humans do not synthesize folate, it is necessary to obtain these cofactors from dietary sources. In mammals, three specialized systems exist that mediate membrane transport of folates and antifolates across biological membranes. These include the reduced folate carrier (RFC), the primary route for the uptake of folates and antifolates in mammalian cells, folate receptors (FRs) α and β, and the proton-coupled folate transporter (PCFT). Whereas RFC is ubiquitously expressed, FRs and PCFT show a more narrow pattern of tissue expression. Toxicity of clinically used antifolates occurs in part, to their lack of selectivity for tumor cells over normal cells due to RFC transport. Antifolates with tumor-specific FR and/or PCFT drug uptake would circumvent major toxicities of currently used antifolates. Our analog AG17F had shown selective FR and PCFT transport over RFC with excellent cell inhibitory activity in KB tumor cell line (IC50 = 1.7 nM). On the basis of AG17F, AG140F with sulfur in place of carbon at C10, AG182F with oxygen in place of carbon at C10 and AG183F with nitrogen in place of carbon at C10 were designed and synthesized. This isosteric replacement of carbon with heteroatoms in the bridge provides compounds with different chain lengths, angles, conformations and extra hydrogen bond donors and/or acceptors compared to the parent carbon chain analogues. AG17F had shown inhibitory activity toward the growth of a CHO cell line (hRFC) expressing human RFC (IC50 = 304 nM), the three heteroatom analogues AG140F, AG182F and AG183F were inactive in the CHO cell line expressing RFC transport at concentrations up to 1000 nM, but were highly active in inhibiting CHO cell lines (RT16) expressing human FRα (IC50s of 0.62, 1.10, and 2.3 nM respectively) and inhibiting PCFT-expressing CHO cells (R2/PCFT4) (IC50s of 267, 57.3, and 87.4 nM respectively). AG140F, AG182F, and AG183F were potently inhibitory toward KB tumor cells (IC50s of 2, 0.32 and 0.34 nM respectively) and IGROV1 tumor cells (IC50s of 0.51, 0.58 and 0.48 nM respectively). AG140F, AG182F, and AG183F showed binding affinities to FRα in RT16 cells exceeding that for AG17F, and comparable to folic acid. With R2/PCFT4 cells, all compounds showed high affinities with binding (reflected in Ki values) in the order of AG17F>AG182F∼AG183F>AG140F. In KB cells, adenosine and 5-aminoimidazole-4-carboxamide protected from growth inhibition suggesting de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase) and the likely targets. By in situ assays in KB cells, GARFTase inhibition was confirmed at concentrations similar to those which inhibit cell proliferation. AG183F was further evaluated for against early stage human IGROV tumor xenograft in female SCID mice models and was found to be remarkably efficacious (T/C = 12%) without any acute or long term toxicities. Citation Format: Lalit Kumar Golani, Aleem Gangjee, Christina Cherian, Steven Orr, Shermaine Mitchell-Ryan, Lisa Polin, Adrianne Wallace, Larry H. Matherly. Synthesis and preclinical evaluation of novel 6-substituted pyrrolo[2,3-d]pyrimidines as targeted antifolates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1620. doi:10.1158/1538-7445.AM2014-1620

Abstract 5492: Study the variation of the glutamate moiety of AG94, a targeted, potent GARFTase inhibitor, as antitumor agents with the 2-amino-6-substituted pyrrolo[2,3-d]pyrimidine scaffold.

We have been extensively involved in the elaboration of substituted pyrrolo[2,3-d]pyrimidines to obtain potential targeted agents without reduced folate carrier (RFC) activity. Lack of selectivity for RFC is a major cause of significant toxicity in the clinic associated with antifolate anticancer agents. These efforts have led to the discovery of several series of potent targeted agents, which showed specificity for folate receptors (FRs) and/or the proton coupled folate transporter (PCFT). These transport systems are two targets for selective cancer chemotherapy. One such targeted agent, AG94, was reported by us as the most efficacious targeted antifolate antitumor agent with PCFT and FR selectivity over RFC. AG94 is a GARFTase inhibitor and its targeted transport and inhibition of GARFTase was demonstrated to be responsible for its selective in vitro and in vivo antitumor activity. We were interested in carrying out a structure-activity relationship study (SAR) with AG94 as the lead compound. For this study we elected the glutamate moiety of AG94 for modification to determine the effect of this variation on transport as well as cell inhibition in culture. The glutamate moiety of AG94 was replaced with various natural and unnatural amino acids or completely deleted in an attempt to vary the chain length between the two carboxylic groups and retain one or none of the carboxylic moieties in the target compounds. The results showed that AG190 was relatively potent with an IC 50 of 9.0 nM in human tumor KB cells in culture. A complete set of cell proliferation assays in CHO sublines afforded a SAR study of the transporters with defined transport as well as for GARFTase inhibitory activity. With FRα expressing RT16 CHO cells, an order of decreasing sensitivities (AG94>AG189∼AG190>AG213>AG214>AG187) was obtained. For FRβ expressing D4 CHO cells, it showed that AG94>AG190>>AG189, whereas AG187, AG213 and AG214 are inactive. All target compounds are inactive in PCFT expressing R2/PCFT4 cells. In the binding assays towards PCFT and FRα with AG189 and AG190, we found that AG190 and AG189 still bound to PCFT although somewhat less avidly than that for AG94 towards PCFT, and AG189 along with AG190 exhibited very similar binding affinity towards FRα compared to AG94. In nucleoside protection assay, surprisingly neither adenosine nor AICA protect and suggested a change in intracellular target for AG189 and AG190 compared to AG94. Based on the profile of protection, we postulate that both AG189 and AG190 retain their targeted mechanism for transport but may not act as GARFTase inhibitors, a role previously established for AG94. Further study to elucitdate their exact role is currently underway. The ability to manipulate the intracellular target by variation in the glutamate moiety is novel and remarkable for this series. Citation Format: Aleem Gangjee, Sai Zhao, Lalit Kumar, Christina Cherian, Steven Orr, Jenny Huang, Zhanjun Hou, Larry H. Matherly. Study the variation of the glutamate moiety of AG94, a targeted, potent GARFTase inhibitor, as antitumor agents with the 2-amino-6-substituted pyrrolo[2,3- d ]pyrimidine scaffold. [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 5492. doi:10.1158/1538-7445.AM2013-5492

Abstract 4116: Notch1 regulates AKT-T308 dephosphorylation through modulation of the PP2A phosphatase in GSI-resistant T-cell acute lymphoblastic leukemia (T-ALL) cells.

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