Drug resistance is often a limiting factor in successful chemotherapy. Our laboratory has been interested in studying mechanisms of resistance to drugs that are targeted to the thymidylate biosynthesis pathway especially those that target thymidylate synthase (TS) and dihydrofolate reductase (DHFR). We have used leukemia as a model system to study resistance to methotrexate (MTX) and colorectal cancer as the model system to study 5-fluorouracil (5-FU) resistance. In leukemias, we and others have shown that transport, efflux, polyglutamylation and hydrolase activities are major determinants of MTX resistance. We have further reported that some leukemic cells have an increase in DHFR gene copy number possibly contributing to the resistant phenotype. Recently, we have begun to study in detail the molecular mechanisms that govern translational regulation of DHFR in response to MTX as an additional resistance mechanism. Studies thus far involving colorectal tumors obtained from patients have focused predominantly on the predictive value of levels of TS expression and p53 mutations in determining response to 5-FU. Although the predictive value of these two measures appears to be significant, given the variety of resistance to 5-FU observed in cell lines, it is not likely that these are the only measures predictive of response or responsible for acquired resistance to this drug. The enzyme uridine-cytidine monophosphate kinase (UMPK) is an essential and rate-limiting enzyme in 5-FU activation while dihydropyrimidine dehydrogenase (DPD) is a catabolic enzyme that inactivates 5-FU. Alterations in UMPK and DPD may therefore explain failure of 5-FU response in the absence of alterations in TS or p53. Transcription factors that regulate TS may also influence drug sensitivity. We have found that mRNA levels of the E2F family of transcription factors correlates with TS message levels and are higher in lung metastases than in liver metastases of colorectal cancers. Moreover, gene copy number of the E2F-1 gene appears to be increased in a significant number of samples obtained from metastases of colorectal cancer. We have also generated mutants of both DHFR and TS that confer resistance to MTX as well as 5-FU by random as well as site-directed mutagenesis. These mutants used alone or as fusion cDNAs of the mutants have proven to be useful in transplant studies where transfer of these mutant cDNAs to bone marrow cells have been shown to confer drug resistance to recipients. The fusion cDNAs of DHFR such as the DHFR-herpes simplex virus type 1 thymidine kinase (HSVTK) are also useful for regulation of gene expression in vivo using MTX as the small molecule regulator that can be monitored by positron emission tomography (PET) scanning or by optical imaging using a fusion construct such as DHFR-EGFP.

Thymidylate synthase plays an essential role in the synthesis of DNA. Recently, several new and specific thymidylate synthase inhibitors that occupy the folate binding site, including Tomudex®, BW1843U89, and Thymitaq, have demonstrated therapeutic activity in patients with advanced cancer. In order to find drugresistant forms of human thymidylate synthase for gene therapy applications, human sarcoma HT1080 cells were exposed to ethyl methanesulfonate and Thymitaq selection. Thymitaq-resistant clonal derived sublines were established, and analysis indicated that both gene amplification and point mutations contributed to drug resistance. Eight mutant cDNAs that were identified from Thymitaq-resistant sublines were generated by site-directed mutagenesis and transfected into thymidylate synthase-negative cells. Only K47E, D49G, or G52S mutants retain enzyme activity. Moreover, cytotoxicity studies demonstrated that D49G and G52S transfected cells, besides displaying resistance to Thymitaq with IC 50 values 40-and 12-fold greater than wild-type enzyme transfected cells, respectively, also lead to fluorodeoxyuridine resistance (26-and 97-fold in IC 50 values, respectively) but not to Tomudex or BW1843U89. Characterization of the purified altered enzymes obtained from expression in Escherichia coli is consistent with the cell growth inhibition results. We postulate that the D49G or G52S mutation leads to the structural perturbation of the highly conserved Arg 50 loop, decreasing the binding of thymidylate synthase to the inhibitors, Thymitaq and fluorodeoxyuridylate.Thymidylate synthase (TS, 1 EC 2.1.1.45) catalyzes the de novo biosynthesis of thymidylate, which is necessary for DNA synthesis and repair (1). The mechanism of TS activity involves the reductive methylation of the substrate, 2Ј-deoxyuridine 5Ј-monophosphate (dUMP) by transfer of a methylene group from the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 folate), to generate 2Ј-deoxythymidine 5Ј-monophosphate (dTMP) and 7,8-dihydrofolate. Human TS has been sequenced (2), purified (3, 4), and crystallized (5). As an attractive target for anti-cancer drug design, since the 1950s, many TS analogues of both the substrate, dUMP, and the cofactor, CH 2 H 4 folate, have been synthesized and tested as potential anti-cancer therapeutics. Until recently, 5-fluorouracil and fluorodeoxyuridine (FdUrd) were the sole TS-targeted drugs approved for clinical application. In vivo, 5-fluorouracil and FdUrd are metabolized to 5-fluoro-2-deoxyuridylate (FdUMP), a compound that subsequently occupies the pyrimidine binding site forming a ternary complex with TS and the folate cofactor, resulting in inhibition of enzyme function. The recent determination of the three-dimensional structure of human TS has allowed the design of highly specific inhibitors, leading to the emergence of novel folate analogues, such as Tomudex (ZD1694), BW1843U89, and Thymitaq (AG337) (Fig. 1) (6). These promising compounds have entered clinical trials in recent years (7).Previous studies h...

Gene transfer-based myeloprotection strategies against chemotherapy require the development of effective drug resistance genes or gene combinations. Our laboratory has previously generated drug-resistant mutants of dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S) for myeloprotection against methotrexate (MTX) and 5-fluorouracil (5-FU), respectively. For the purpose of conferring dual myeloprotection against both MTX and 5-FU, we have generated two retroviral constructs encoding both DHFR F/S and TS G52S as a fusion protein (DHFR F/S-TS G52S) or as individual proteins from a bicistronic gene. The DHFR F/S-TS G52S fusion protein is functional and exhibits kinetic properties similar to that of the individual mutant enzymes. NIH 3T3 cells and mouse bone marrow progenitors retrovirally transduced with the fusion DHFR F/S-TS G52S cDNA provided similar levels of resistance to MTX and 5-FU as cells expressing the individual mutant enzymes and higher levels of resistance to MTX than cells expressing DHFR F/S from the 3' end of a bicistronic gene. As MTX and 5-FU are used in combination therapy for diseases such as breast and colon cancer, this fusion gene may be useful in the clinic to reduce myelosuppressive toxicity associated with this drug combination.

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Background:The G52S mutation in the Arg 50 loop of thymidylate synthase leads to decreased binding of FdUMP. It has been suggested that the mutation affects the Arg 50 residue (within the Arg 50 loop) responsible for binding the phosphate of FdUMP. The binding of the methylguanidinium moiety as a model for Arg 50 to a methylphosphate entity as a model for FdUMP was investigated with theoretical calculations, as well as the structure of the Arg 50 -Thr 51 -Gly 52 tripeptide in comparison with the Arg 50 -Thr 51 -Ser 52 tripeptide. Methods: Gaussian-98 and PC Spartan programs were used to perform Hartree-Fock and Post-Hartree-Fock

CCAAT/Enhancer Binding Protein β (C/EBPβ) is a basic leucine zipper (bZIP) transcription factor that causes aberrant gene expression in many cancers. Upregulated or overactivated C/EBPβ drives oncogenesis by promoting tumor survival and proliferation and is a critical regulator of the immunosuppressive tumor microenvironment (TME). Specifically, C/EBPβ regulates macrophage differentiation, activating a transcriptional program driving macrophage polarization toward immunosuppressive M2-type myeloid-derived suppressor cells (MDSCs). Consistently, activation of C/EBPβ correlates with poor prognosis in several types of human cancer. Thus, targeting C/EBPβ to reprogram tumor-associated macrophages (TAMs) from the M2 toward the immune-promoting M1 phenotype represents an attractive strategy to enhance antitumor immunity. ST101 is a novel peptide antagonist of C/EBPβ dimerization that inhibits C/EBPβ-dependent gene expression. Here we evaluated the impact of ST101 on macrophage differentiation, cytotoxic T-cell activation, and in vivo anti-tumor activity. Primary human macrophages cultured from Peripheral Blood Mononuclear Cells (hPBMCs) were activated toward the M1 or M2 phenotype by LPS and IFNγ or IL-4, respectively. ST101 exposure dose-dependently suppressed expression of M2 markers (CD163, CD206) while inducing M1 markers (CD80, CD86) by flow cytometry and quantitative PCR, resulting in a 40-fold increase in the M1/M2 ratio without substantial impact on cell viability. Next, in co-cultures of T cells with M2 macrophage, ST101 exposure resulted in a 4-fold increase in T-cell activation compared to control M2/T cell co-cultures, as measured by intracellular IFN-γ staining. Importantly, ST101 did not suppress proliferation or activity of T cells cultured alone. Finally, in an orthotopic TNBC model in vivo, ST101 in combination with anti-PD-1 treatment enhanced anti-tumor activity compared to either single agent alone. The observed increase in tumor growth inhibition was accompanied by a reduced TAM fraction and increased intratumoral and peripheral M1/M2 ratios. ST101 is being evaluated in a Phase 1-2 clinical study in patients with advanced unresectable and metastatic solid tumors (NCT04478279). Initial gene expression analysis performed on 8 paired patient biopsies (prior to ST101 exposure and within 24 hrs of ST101 administration during cycle 2 of therapy) collected during dose escalation (4 mg/kg ST101 or greater) indicates a significant decrease in expression of multiple factors involved in M2 polarization, including CD209, SIGLEC5 and IL-24, and T cell suppression, including FoxP3 and inhibitory KIR proteins. The result is a decrease in intratumoral regulatory T cell (Treg) vs. TIL ratio, indicating a shift towards a more immunoactive TME. Overall, these results support a novel, macrophage-driven mechanism of action for ST101 as anticancer agent and suggest the exploration of ST101 in immune-oncology therapeutic strategies. Citation Format: Claudio Scuoppo, Rick Ramirez, Siok Leong, Lila Ghamsari, Gina Capiaux, Rob Michel, Steve Kaesshaefer, Gene Merutka, Barry Kappel, Abi Vainstein-Haras, Alice Bexon, Jim Rotolo. ST101, a peptide antagonist of novel I/O target CEBPβ,reprograms MDSCs and promotes an immunoactive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB236.

BACKGROUND C/EBPβ is a transcription factor that is active during embryofetal development but held in an inactive state in most mature cells (Zahnow 2009). C/EBPβ is upregulated or overactivated in multiple cancers, where it inversely correlates with disease prognosis and survival due to activation of a gene signature that promotes tumor cell proliferation and survival. ST101 is a cell-penetrating peptide antagonist of C/EBPβ. ST101 exposure leads to selective tumor cell death in multiple human cancer cell lines, including GBM, without impacting normal cell viability. In vivo, ST101 displays rapid uptake into multiple organs, the ability to cross the blood-brain barrier, and a long plasma half-life due to its resistance to degradations. It has potent anti-tumor activity in multiple GBM models, as a single agent or in combination, which supported moving into clinical development. TRIAL DESIGN This phase 1-2 study is enrolling patients ≥ 18 years of age with advanced, unresectable metastatic solid tumors refractory to or intolerant of other therapeutic options. We began recruitment in August 2020. The primary objective of phase 1 is to evaluate safety and tolerability of ST101. Secondary objectives include the recommendation of a dose and regimen of ST101 for further evaluation, pharmacokinetics, several pharmacodynamic measures, and preliminary efficacy. Patients receive intravenous ST101 once weekly in a standard 3 + 3 design. Enrollment is ongoing, and by 21 May 2021, 15 patients have been recruited in four dose-escalation cohorts up to 4 mg/kg; a 5th cohort (6 mg/kg) is ongoing. The recommended phase 2 dose will be used in a 15-30 patient GBM expansion cohort, with a Simon 2-stage design, which requires one response or two patients with PFS6 in the first cohort to continue the study. Up to 120 patients are planned in a total of four expansion cohorts, which should be enrolling by Q3 2021.

Introduction: The oncogenic transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is normally active in embryofetal development, but inactive and suppressed in mature cells. Upregulation or activation of C/EBPβ in cancer promotes tumor survival and proliferation while inhibiting its differentiation. ST101 is a peptide antagonist of C/EBPβ, with anti-tumor activity in glioblastoma (GBM), breast cancer (BC), prostate cancer (PC), melanoma, and other models. ST101 penetrates the blood-brain barrier, as demonstrated in a mouse model and biodistribution studies. We have also demonstrated the tumor-specificity of ST101 in numerous in vitro cell lines. Methods: We conducted the phase 1 portion of a phase 1-2 study in patients with refractory solid tumors. The primary objective was to evaluate safety and tolerability of ST101. Secondary and exploratory objectives included PK, preliminary efficacy, and PD from serial biopsies. The study used a 3+3 design, dosing ST101 IV at 0.5, 1, 2, 4, 6, and 8 (now modified to 9) mg/kg weekly (QW). Phase 2 will include four cohorts of patients with specific cancers: HR+ breast cancer, cutaneous melanoma, GBM, and castrate-resistant PC at the recommended phase 2 dose (RP2D). Results: As of July 2021, 18 pts were enrolled, and the last cohort (9 mg/kg) is underway. Patients received a median of 6 weeks’ treatment (range 2 – 45). There were no dose-limiting toxicities, dose modifications, or SAEs related to ST101. The only adverse events (AEs) of note were G1-2 histaminergic infusion-related reactions (IRRs), largely pruritis and urticaria, managed with antihistamines, montelukast, and interruption/slowing of infusion. IRRs affected 100% pts on the 1st dose of ST101 at 4mg/kg. Montelukast was added to the antihistamine premedication regimen in the 6 mg/kg cohort, which attenuated IRRs. 66% of patients in the 6 mg/kg experienced an IRR on the first dose. The intensity and frequency of IRRs decreased with repeat dosing across all cohorts. No other AEs were consistently reported. PK was dose-proportionate, with continued exposure. There was no evidence of accumulation, and no anti-drug antibodies were detected. Tumor immunohistochemistry showed dose-proportionate staining for ST101 and a trend of decreased Ki67 staining (proliferation marker) after ST101 exposure. One confirmed partial response in a patient with metastatic cutaneous melanoma refractory to standard therapy is still on study, and 3 pts with varied histologies have had stable disease lasting 18-45 weeks (1 ongoing). Conclusions: ST101 demonstrated safety at all doses explored and evidence of efficacy across dose levels, particularly higher doses. PK and PD support a dose relationship for efficacy, and we will select a QW RP2D for the phase 2 expansion cohorts by September 2021. Citation Format: Nehal J. Lakhani, Hendrik-Tobias Arkenau, Stefan N. Symeonides, Jeffry Evans, Meredith A. McKean, Elisa Fontana, Manojkumar Bupath, Alistair McLaren, Sreenivasa Chandana, Tze-en Ding, Emerson A. Lim, Jim Rotolo, Gina Capiaux, Rob Michel, Stephen Kaesshaefer, Alice S. Bexon, Gerald S. Falchook. ST101, a peptide targeting oncogenic transcription factor C/EBPβ: initial safety, efficacy, pharmacokinetic (PK) and pharmacodynamic (PD) data from an ongoing phase 1 dose escalation study in patients with advanced, metastatic solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P06-03.