Nucleoside Analog Gemcitabine Research Articles

Abstract 6565: Improving survival of atypical teratoid/rhabdoid tumor orthotopic xenografts through the combination of PI3K inhibitor paxalisib and nucleoside analog gemcitabine

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are an aggressive infantile brain tumor with a dismal four-year event-free survival of 37%. We have previously identified high activation of mTORC1 and mTORC2 within AT/RT and shown that the highly brain penetrant PI3K inhibitor paxalisib is capable of simultaneously inhibiting mTORC1/2 by acting upstream of both complexes. In two orthotopic xenograft models, paxalisib slowed tumor growth and significantly extended survival (CHLA-06: 40 to 54 days, p=0.001; BT-12: 21 to 35 days, p=0.02). RNAseq analysis following mTOR inhibition in AT/RT cell lines showed an upregulation of genes involved in the activation of the integrated stress response (ISR) (ATF4, CHOP, PPP1R15A t-test p<0.05). Analysis of primary pediatric brain tumors through the Children’s Brain Tumor Network showed increased levels of ATF4 mRNA and eIF2α protein and mRNA in AT/RT compared to other indolent pediatric brain tumors. Increased expression of ISR components suggests this pathway may be a dependency in AT/RT. Activation of this pathway is known to be cytoprotective, however persistent over-activation drives cell death through induction of pro-apoptotic factors. Gemcitabine is a pyrimidine analog functioning to inhibit DNA synthesis and induce DNA damage; notably, recent literature has correlated DNA damage to activation of the ISR with downstream phosphorylation of eIF2α. We hypothesize that due to high baseline activation of ISR for AT/RT, paxalisib combined with gemcitabine will result in an over-activation of the ISR leading to AT/RT cell death. Western blot analysis of combination therapy compared to DMSO control showed induction of phospho-eIF2α, ATF4, and CHOP, denoting an activation of the ISR. Combination therapy in AT/RT cells induces cell death (Western blot analysis cPARP, MUSE Annexin V assay, SynergyFinder BLISS score [viability] CHLA-06: 21.50, CHLA-266: 22.60) and synergizes to decrease cell growth (SynergyFinder BLISS score [inhibition] CHLA-06: 16.80, BT-12: 14.30, CHLA-266: 13.90, BT-37: 11.40, CHLA-05: 10.40). Paxalisib/gemcitabine combination therapy in murine orthotopic xenograft models of AT/RT has slowed tumor growth (bioluminescent imaging) and led to significant extension of survival. Mice bearing BT-37 orthotopic xenografts and treated with vehicle control had a median survival of 56 days, while all of the paxalisib/gemcitabine treated mice were alive at 100 days of treatment (p<0.0001). In CHLA-06 orthotopic tumors combination therapy extended median survival from 22 days for control to 82.5 days (p<0.0001). Based on these findings, the Pacific Pediatric Neuro-Oncology Consortium (PNOC) is planning to include paxalisib/gemcitabine combination therapy in the next international clinical trial for patients with relapsed/recurrent AT/RT. Citation Format: Tyler Findlay, Kristen Malebranche, Anupa Geethadevi, Charles Eberhart, Jeffrey Rubens, Eric Raabe. Improving survival of atypical teratoid/rhabdoid tumor orthotopic xenografts through the combination of PI3K inhibitor paxalisib and nucleoside analog gemcitabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6565.

Abstract 3368: Combination of a chk1/2 dual inhibitor (prexasertib) and gemcitabine reveals a novel intrinsic synergy mechanism in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) presents a challenging clinical scenario due to its recurrence propensity despite existing treatments. Altered intrinsic signaling pathways significantly contribute to therapeutic resistance, demanding exploration of novel strategies to induce tumor cell demise. Dysregulated cell cycle and DNA damage repair constitute pivotal events driving HNSCC progression, offering potential therapeutic targets.CHK1 and CHK2, pivotal regulators of the DNA replication checkpoint, are promising targets in HNSCC. The convergence of DNA-damaging agents with DNA checkpoint inhibitors represents a burgeoning paradigm in cancer management. Our study delves into elucidating the intrinsic signaling mechanisms triggered by the synergistic action of the CHK1/2 dual inhibitor (Prexasertib) and the nucleoside analog (Gemcitabine) in HNSCC.Our study unveiled a robust synergistic effect between Prexasertib and Gemcitabine, inducing substantial cell death in multiple HNSCC cell lines and mouse model. Remarkably, the combination rapidly induced DNA damage and reduced tumor growth in vivo. To unravel the underlying intrinsic signaling mechanism provoked by the Prexasertib/Gemcitabine combination, we conducted comprehensive molecular analyses.Proteomic profiling uncovered distinct alterations in key signaling pathways upon combination treatment. We identified unique overexpression of an embryonic stem cell-specific transcription factor pivotal in genomic stability regulation, solely induced by single-agent Prexasertib. Specifically, the combination therapy showcased unique modulation of critical genes involved in DNA repair, replication, and cell cycle regulation. These alterations included overexpression or suppression of key genes associated with DNA stability and cell proliferation pathways, accompanied by evidence of CHK1/2 inhibition-mediated DNA damage response accompanied by cleavage of CHK-2 and caspase-3 proteins, validated through Western blots.Additionally, functional assays including the colony formation assays demonstrated markedly reduced survival rates with the combination treatment compared to single-agent therapy. Cell cycle analysis revealed a replication catastrophe and increased pH2AX across multiple HNSCC cell lines following combination treatment.Our findings highlight the significance of CHK1/2 inhibition coupled with nucleoside analog-induced DNA damage in unveiling novel intrinsic signaling mechanisms in HNSCC. These insights offer a promising avenue for targeted therapeutic strategies to enhance treatment efficacy and outcomes in HNSCC.This work has been supported by the MGC, FC, P&MC, and BBSR at the H. Lee Moffitt Cancer Center & Research Institute, a comprehensive cancer center designated by the National Cancer Institute (P30-CA076292). Citation Format: Vinayak C. Palve, Hannah L. Walker-Mimms, Ritu Chaudhary, Victoria Izumi, Neelkamal Chaudhary, Bin Fang, John Koomen, Jose Alejandro Guevara, Paulo C. Rodriguez, Christine H. Chung, Derek R. Duckett. Combination of a chk1/2 dual inhibitor (prexasertib) and gemcitabine reveals a novel intrinsic synergy mechanism in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3368.

Addressing the Clinical Importance of Equilibrative Nucleoside Transporters in Drug Discovery and Development.

The US Food and Drug Administration (FDA), European Medicines Agency (EMA), and Pharmaceuticals and Medical Devices Agency (PMDA) guidances on small-molecule drug-drug interactions (DDIs), with input from the International Transporter Consortium (ITC), recommend the evaluation of nine drug transporters. Although other clinically relevant drug uptake and efflux transporters have been discussed in ITC white papers, they have been excluded from further recommendation by the ITC and are not included in current regulatory guidances. These include the ubiquitously expressed equilibrative nucleoside transporters (ENT) 1 and ENT2, which have been recognized by the ITC for their potential role in clinically relevant nucleoside analog drug interactions for patients with cancer. Although there is comparatively limited clinical evidence supporting their role in DDI risk or other adverse drug reactions (ADRs) compared with the nine highlighted transporters, several in vitro and in vivo studies have identified ENT interactions with non-nucleoside/non-nucleotide drugs, in addition to nucleoside/nucleotide analogs. Some noteworthy examples of compounds that interact with ENTs include cannabidiol and selected protein kinase inhibitors, as well as the nucleoside analogs remdesivir, EIDD-1931, gemcitabine, and fialuridine. Consequently, DDIs involving the ENTs may be responsible for therapeutic inefficacy or off-target toxicity. Evidence suggests that ENT1 and ENT2 should be considered as transporters potentially involved in clinically relevant DDIs and ADRs, thereby warranting further investigation and regulatory consideration.

Development and validation of simultaneous quantification method for gemcitabine and betulinic acid: augmenting industrial application.

Combinatorial treatment utilizing a nucleoside analogue gemcitabine (GEM), with a characteristic pentacyclic triterpenoid betulinic acid (BET), has exhibited empowering adequacy in the therapy of cancer. It lessens the advancement of collagen and upgrades the saturation of tumour medicines. With the advancement in nanotechnology, the co-loaded formulation urges for a validated method of estimation. The purposed work entails a robust, simple, and economical analytical method for the simultaneous estimation of GEM and BET through RP-HPLC. Orthophosphoric acid (0.1%)-acetonitrile was considered as the mobile phase for the detection of GEM and BET at 248nm and 210nm with retention times of 5min and 13min, respectively. The method was further validated as per the regulatory guidelines with all the parameters found within the limit. The developed method with adequate resolution and quantification was found to be linear, accurate, precise, robust, and stable with an intra- and inter-day variability of less than 2%. The method was found specific for GEM and BET with no matrix interference of drug-spiked FBS samples. To demonstrate the applicability of the developed method, a nano-formulation containing GEM and BET was prepared and assessed for various parameters including encapsulation efficiency, loading efficiency, drug release, and drug stability. The method developed can be a possible tool for the simultaneous quantification of GEM-BET in analytical and biological samples.

CDK4/6 inhibition confers protection of normal gut epithelia against gemcitabine and the active metabolite of irinotecan

ABSTRACT Cancer chemotherapy relies on a high ratio of toxicity toward cancer cells vs. nonmalignant cells, making it desirable to protect normal cells. Among the nonmalignant cells, epithelia of the gut belong to the most vulnerable ones toward chemotherapeutics. Here, we use a murine intestinal organoid model to assess a strategy for protecting such epithelia against chemotherapy. Cell cycle progression was first stalled by palbociclib, a clinically established cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. Washout of the drug allowed subsequent outgrowth of gut organoids. This transient cell cycle arrest conferred near-complete protection of the cells toward the nucleoside analogue gemcitabine. Moreover, pre-treatment with palbociclib protected the organoids against SN-38, the topoisomerase I-inhibiting metabolite of irinotecan, which is otherwise known for its severe gastrointestinal toxicities. In contrast, RB1-mutated cancer cells were not protected against gemcitabine or SN-38 when pre-treated with palbociclib. Taken together, these results outline a strategy for protecting nonmalignant cells against the toxicities of chemotherapeutics commonly used to treat advanced colorectal and pancreatic cancer. We propose that this strategy is particularly promising to protect the gut when treating RB1-deficient tumors that fail to arrest the cell cycle in response to CDK4/6 inhibitors.

Increased renal elimination of endogenous and synthetic pyrimidine nucleosides in concentrative nucleoside transporter 1 deficient mice

Concentrative nucleoside transporters (CNTs) are active nucleoside influx systems, but their in vivo roles are poorly defined. By generating CNT1 knockout (KO) mice, here we identify a role of CNT1 in the renal reabsorption of nucleosides. Deletion of CNT1 in mice increases the urinary excretion of endogenous pyrimidine nucleosides with compensatory alterations in purine nucleoside metabolism. In addition, CNT1 KO mice exhibits high urinary excretion of the nucleoside analog gemcitabine (dFdC), which results in poor tumor growth control in CNT1 KO mice harboring syngeneic pancreatic tumors. Interestingly, increasing the dFdC dose to attain an area under the concentration-time curve level equivalent to that achieved by wild-type (WT) mice rescues antitumor efficacy. The findings provide new insights into how CNT1 regulates reabsorption of endogenous and synthetic nucleosides in murine kidneys and suggest that the functional status of CNTs may account for the optimal action of pyrimidine nucleoside analog therapeutics in humans.

Abstract 5490: Combination of the PI3k inhibitor Paxalisib with the nucleoside analog Gemcitabine over-activates the integrated stress response and induces atypical teratoid/rhabdoid tumor cell death

Abstract We have previously identified high activation of both mTORC1 and mTORC2 in the aggressive infantile brain tumor, atypical teratoid/rhabdoid tumor (AT/RT). Paxalisib is a highly brain penetrant PI3k inhibitor which acts upstream of mTOR to simultaneously inhibit mTORC1/2. Paxalisib is well-tolerated in ongoing pediatric clinical trials. In murine orthotopic xenograft models of AT/RT, Paxalisib significantly extended survival (CHLA-06: 40 to 54 days, p=0.001; BT12: 21 to 35 days, p=0.02). However, to improve the durability of this single agent therapy, we evaluated the efficacy of rational therapeutic partners. Analysis of RNAseq following mTOR inhibition (mTORi) of 4 cell lines representative of AT/RT showed an upregulation of genes associated with the integrated stress response (ISR), (ATF4: CHLA-06, t-test p<0.05; CHOP: CHLA-02, CHLA-06 t-test p<0.001). The activation of the ISR is further suggested through induction of PPP1R15A, a gene whose activation is closely associated with stress events and leads to downstream cell growth arrest and apoptosis (BT37 t-test p<0.001, CHLA-02, CHLA-06, CHLA-266, t-test p<0.01). Gemcitabine is a pyrimidine nucleoside prodrug, which inhibits DNA synthesis and induces DNA damage. As a result, Gemcitabine also induces phosphorylation of eIF2α and the activation of the ISR. While the ISR can be protective, intense or prolonged activation leads to cell death. We therefore hypothesized that Paxalisib would combine with Gemcitabine to hyper-activate the ISR and drive AT/RT cell death. Through Western blot analysis, we demonstrate that combination therapy increases phospho-eIF2α, ATF4, and CHOP compared to DMSO control. Furthermore, we demonstrate that combination therapy increases apoptosis (Western blot cPARP, MUSE Annexin V Assay) and synergizes to decrease AT/RT cell growth (SynergyFinder BLISS score CHLA-06: 16.8, BT12: 14.3, CHLA-266: 13.9, BT37: 11.4, CHLA-05: 10.4). Ongoing treatment of mice bearing CHLA06 orthotopic tumors demonstrate that combination therapy further extends median survival. Our studies suggest that pharmacologically activating the ISR may be an effective strategy to target AT/RT and combination of Paxalisib and Gemcitabine may be an effective treatment to help extend survival in this deadly disease. Citation Format: Tyler Findlay, Kristen Malebranche, Anupa Geethadevi, Eric Raabe, Charles Eberhart, Jeffrey Rubens. Combination of the PI3k inhibitor Paxalisib with the nucleoside analog Gemcitabine over-activates the integrated stress response and induces atypical teratoid/rhabdoid tumor cell death. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5490.

A ferrocene-containing nucleoside analogue targets DNA replication in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is a disease that remains refractory to existing treatments including the nucleoside analogue gemcitabine. In the current study we demonstrate that an organometallic nucleoside analogue, the ferronucleoside 1-(S,Rp), is cytotoxic in a panel of PDAC cell lines including gemcitabine-resistant MIAPaCa2, with IC50 values comparable to cisplatin. Biochemical studies show that the mechanism of action is inhibition of DNA replication, S-phase cell cycle arrest and stalling of DNA-replication forks, which were directly observed at single molecule resolution by DNA-fibre fluorography. In agreement with this, transcriptional changes following treatment with 1-(S,Rp) include activation of three of the four genes (HUS1, RAD1, RAD17) of the 9-1-1 check point complex clamp and two of the three genes (MRE11, NBN) that form the MRN complex as well as activation of multiple downstream targets. Furthermore, there was evidence of phosphorylation of checkpoint kinases 1 and 2 as well as RPA1 and gamma H2AX, all of which are considered biochemical markers of replication stress. Studies in p53-deficient cell lines showed activation of CDKN1A (p21) and GADD45A by 1-(S,Rp) was at least partially independent of p53. In conclusion, because of its potency and activity in gemcitabine-resistant cells, 1-(S,Rp) is a promising candidate molecule for development of new treatments for PDAC.

MiR-198 sensitizes pancreatic cancer to gemcitabine treatment through downregulation of VCP-mediated autophagy maturation.

e16290 Background: The mechanism of human pancreatic ductal adenocarcinoma (PDAC) resistance to nucleoside analog and first-line chemotherapy drug gemcitabine is not clearly understood, but some studies have associated it with increased autophagy. In cancer biology, autophagy plays dual roles as it can promote tumor suppression during early stages, but in established tumors, it plays a crucial role in tumor growth by enhancing survival under metabolic and therapeutic stress. We have previously found that miR-198 acts as a tumor suppressor in PDAC through the targeting of a network of tumorigenic factors, including the Valosin-containing protein (VCP), which has been reported to play an important role in autophagy. In this study, we investigate whether the repression of VCP through miR-198 replacement disrupts the autophagy process and sensitizes PDAC cells to gemcitabine treatment. Methods: MIAPaCa2 cells with forced overexpression of mesothelin (MSLN) and AsPC-1 cell lines and, CDX and PDX mouse models were used for gemcitabine sensitization studies. For miR-198 replacement, a miR-198 expression vector-loaded lactic co-glycolic-acid-modified polyethylenimine polyplex (LPNP-p198) was used. Autophagy disruption experiments were run over miR-198 and/or VCP overexpressing cell lines. Nude mice were used for subcutaneous and orthotopic CDX models and SCID/Beige were used for the subcutaneous PDX model. Results: Cell lines were treated with LPNP-p198 in combination with gemcitabine and cell growth was significantly inhibited when compared to gemcitabine alone. Additionally, it was determined that miR-198 disrupts the autophagy maturation process and, that it is then restored by overexpression of VCP. LPNP-p198 can effectively enter tumor cells and induce tumor sensitization in vivo, resulting in an 80-90% reduction of tumor burden and metastatic spread in the LPNP-p198 plus gemcitabine group when compared to controls, with the concomitant downregulation of VCP expression in the tumor tissue. In addition, this group had the fewest mice with jaundice, ascites, and metastases to the abdominal cavity, spleen, liver, and kidney. Finally, to address the potential of the observed effect in the context of the heterogenic nature of PDAC, we confirmed our findings in a PDX mouse model, where a marked reduction in tumor burden was observed in the LPNP-p198 plus gemcitabine group compared to controls. Conclusions: Our findings indicate that miR-198 replacement disrupts the autophagy maturation process and sensitizes PDAC cells to gemcitabine through VCP repression, indicating a potential therapeutic strategy for targeting gemcitabine-resistant PDAC and, establishing the use of LPNPs as a prototype for effective nucleic acid delivery in vitro and in vivo, with potential to be used from bench to clinic.

Abstract P063: Novel prodrugs coupled with albumin nanoparticle encapsulation improves antitumor effects of the nucleoside analog gemcitabine

Abstract Nucleoside analogs such as gemcitabine have been widely used to treat many solid tumors. Despite their near universal use and well understood mechanisms of action, this class of chemotherapeutic agents has notable shortcomings including rapid metabolism, limited systemic exposure, acquired resistance, and dose-limiting toxicities. Albumin-based nanoparticles offer several advantages for drug delivery including direct tumor targeting, low risk of immunogenicity, and ease of synthesis and manufacturing. We have modified numerous nucleoside drugs of clinical significance to form new prodrugs and use nanoparticle encapsulation in an attempt to improve the therapeutic index of the parent drugs. Different prodrugs of gemcitabine were designed to evade drug resistance mechanisms, and methods were developed to form stable lyophilized nanoparticle formulations. Nanoparticles were characterized by particle size (~40-65 nm) and polydispersity, and dose-dependent effects on the proliferation of human tumor cell lines were compared to the response to the parent drug gemcitabine. To evaluate effects in vivo, nanoparticle formulations were dosed intravenously (IV) to immunocompromised mice bearing patient-derived xenografts (PDx). The prodrug JTX-836 (IC50=0.46 nM) was ~10x more potent against BxPC-3 pancreatic adenocarcinoma cells than the parent gemcitabine (IC50 = 5.2 nM) in vitro. IV dosing of JTX-836 albumin nanoparticle formulation improved tumor growth inhibition in PDx models of both pancreatic ductal adenocarcinoma (CTG-0282 TumorGraft, Champions Oncology) and ovarian cancer (CTG-1180, Champions Oncology) when compared to IV gemcitabine dosing. These results demonstrate the potential for nanoparticle encapsulation of prodrugs to improve the antitumor activity of existing nucleoside analog therapeutics and support continued development of albumin-based delivery of gemcitabine for the treatment of human cancer. Citation Format: Neil Raheja, Jason Kahana, Robin Jackman, Curtis Monnig. Novel prodrugs coupled with albumin nanoparticle encapsulation improves antitumor effects of the nucleoside analog gemcitabine [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 P063.

Efficacy and safety of gemcitabine plus doxorubicin in patients with renal medullary carcinoma.

Renal medullary carcinoma (RMC) is a rare and lethal renal cell carcinoma characterized by the loss of tumor suppressor SMARCB1. Molecular profiling studies have suggested that RMC cells may be vulnerable to therapies that generate DNA damage, such as the combination of the nucleoside analog gemcitabine, and topoisomerase inhibitor doxorubicin. We retrospectively analyzed the records of patients with RMC treated with gemcitabine plus doxorubicin at our institution between January 2005 and September 2020. Best radiographic response and disease progression (RECIST v1.1) were assessed by a blinded radiologist. Sixteen patients were included in the study. All but 1 patient (93.8%) received prior platinum-based chemotherapy. Gemcitabine was given intravenously at 900-1200 mg/m2 and doxorubicin at 40-50 mg/m2 intravenously every 2 weeks. Three patients (18.8%) achieved partial response and 7 (43.8%) patients achieved stable disease. The median progression-free survival was 2.8 months (95% CI, 0-6.0). Median overall survival (OS) from gemcitabine plus doxorubicin initiation was 8.1 months (95% CI, 4.6-11.7) and OS from diagnosis was 15.5 months (95% CI, 4.2-26.8 months). There were no grade ≥ 4 AEs; grade 3 AEs were cytopenias (18.8%), nausea (12.5%), fatigue (12.5%), and cardiotoxicity (6.2%). No somatic alterations were detected in the 9 patients tested by targeted next generation sequencing assays. Gemcitabine plus doxorubicin was well tolerated and demonstrated clinical activity in patients with platinum-refractory RMC, with a subset of patients experiencing durable responses lasting longer than 6 months. Further investigation is warranted to determine biomarkers of sensitivity and target mechanisms of resistance.

Abstract 1003: NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells

Abstract Background: NUC-1031 is a ProTide transformation of the nucleoside analog gemcitabine, designed to overcome key cancer resistance mechanisms and generate significantly higher levels of the active metabolite, dFdCTP. Its incorporation into DNA interrupts cell replication and induces DNA damage, leading to cell death. NUC-1031 has shown broad clinical activity across a range of solid tumors as both a single agent and in combination with platinum agents. Increasing evidence in the clinical setting in both ovarian and biliary tract cancer populations suggests potential synergy between NUC-1031 and platinum agents, which may lead to increased anti-tumor activity. The aim of this study was to investigate the dose and time relationships between the incorporation of dFdCTP into DNA, the effect on the cell cycle and DNA damage response in biliary tract cancer cells treated with NUC-1031. Methods: Human intrahepatic cholangiocarcinoma HuCCT1 cells were treated with 500 nM (half-IC50 dose) or 1 µM (IC50 dose) NUC-1031 for 24h and samples were collected every 24h up to 96h. The intracellular conversion of NUC-1031 to its active metabolite dFdCTP was assessed by liquid chromatography mass spectrometry (LC-Q-TOF). This technique was also used to determine the incorporation of dFdCTP into DNA, using the dFdC signature as a surrogate and deoxyguanosine (2dG) for normalization. Flow cytometry was used to measure cell cycle populations and to analyse histone variant H2AX phosphorylation (γH2AX) as a marker for response to DNA damage, where increased signal is related to increase in response. Results: NUC-1031 generated dFdCTP in HuCCT1 cells. The incorporation of this fluorinated deoxycytidine into DNA was dose-dependent over time. At 48h post-treatment, the ratio of dFdC:2dG was 0.70 at half-IC50 dose, with comparable levels at IC50 dose. After 48h, the ratio continued to rise for the 1 µM dose but decreased with 500 nM. The incorporation of the active metabolite was accompanied by an increase in cells in S phase, up to 46% for 500 nM and 64% for 1 µM. This dose-dependent increase coincides with H2AX phosphorylation over time up to 48h. Active metabolite incorporation, S phase population and yH2AX signals reduced towards the 96h time point. Conclusion: NUC-1031 is a potent cytotoxic agent that causes DNA damage through the incorporation of its active metabolite into DNA, in a cell cycle dependant manner. This incorporation induces S phase arrest and evokes the DNA damage response via generation of double-strand breaks. The cytotoxic effect of NUC-1031 is prolonged post-treatment. Ongoing studies are investigating NUC-1031 in combination with cisplatin, where relative levels of dFdCTP incorporated in tumor DNA may act as a pharmacodynamic biomarker to determine synergy in patients who receive the combination treatment. Citation Format: Dillum Patel, Alison L. Dickson, Oliver J. Read, Clarissa M. Czekster, Essam A. Ghazaly, David J. Harrison, Jennifer Bré. NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1003.

Preclinical efficacy of prexasertib in acute lymphoblastic leukemia

Preclinical efficacy of prexasertib in acute lymphoblastic leukemia

Single Diastereomers of the Clinical Anticancer ProTide Agents NUC-1031 and NUC-3373 Preferentially Target Cancer Stem Cells In Vitro.

A 3'-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. The in vitro cytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.

Phase II study of eribulin in combination with gemcitabine for the treatment of patients with locally advanced or metastatic triple negative breast cancer (ERIGE trial). Clinical and pharmacogenetic results on behalf of the Gruppo Oncologico Italiano di Ricerca Clinica (GOIRC)

BackgroundThe combination of a microtubule inhibitor (eribulin) with a nucleoside analog (gemcitabine) may synergistically induce tumor cell death, particularly in triple negative breast cancer (TNBC) characterized by high cell proliferation, aggressive behavior, and chemo-resistance.Patients and methodsThis is an open-label, multicenter phase II study evaluating the combination of eribulin (0.88 mg/m2) plus gemcitabine (1000 mg/m2) on days 1 and 8 of a 21-day cycle as either first- or second-line treatment of locally advanced or metastatic TNBC. The primary endpoint was the objective response for evaluable patients. A prospective, molecular correlative study was carried out to assess the role of germinal BRCA pathogenic variants and single nucleotide polymorphisms (SNPs) in predicting efficacy and toxicity of the combination regimen.ResultsFrom July 2013 to September 2016, 83 evaluable patients were enrolled. They received a median number of six cycles of treatment. An overall response rate (ORR) of 37.3% (31 patients) was observed, with a complete response rate of 2.4% and a partial response rate of 34.9%; the clinical benefit rate was 48.8%. With a median follow-up of 28.8 months, the median response duration was 6.6 months, the median progression-free survival (PFS) was 5.1 months, and the median overall survival (OS) was 14.5 months. The most common grade 3-4 adverse events were aminotransferase elevation (in 25% of the patients) and neutropenia (in 23.8%). Women with BRCA1/2 pathogenic variants were associated with worse ORR, PFS, and OS than BRCA1/2 wild-type carriers. CYP3A4 and FGD4 SNPs were associated with increased risk of liver toxicity. Three different SNPs in CDA∗2, RRM1, and CYP2C8 genes were significantly associated with poorer OS.ConclusionsThe combination of eribulin and gemcitabine showed promising activity and a moderate toxicity profile in metastatic TNBC. BRCA status and pharmacogenetics tests may help identify patients with high probability of response with negligible toxicity.EudraCT number2012-003505-10.

ATRT-18. SHH-SUBTYPE ATYPICAL TERATOID/RHABDOID TUMORS ARE SELECTIVELY SENSITIVE TO GEMCITABINE TREATMENT

Atypical Teratoid Rhabdoid Tumors (ATRT) are highly malignant embryonal tumors of the central nervous system with a dismal prognosis. ATRT can be divided into three molecular subgroups of which the Sonic Hedgehog (SHH) subgroup is most prevalent. In this study, we developed and validated a novel patient-derived ATRT model, which we used along a panel of other primary ATRT models for large scale drug discovery assays. We found that ATRTs are selectively sensitive to the nucleoside analogue gemcitabine, with SHH-subtype ATRTs being the most sensitive subgroup. Gene expression profiles and protein analysis indicated that gemcitabine treatment causes degradation of Sirtuin 1 (SIRT1), which causes ATRT specific cell-death through NF-kB and p53 activation. Furthermore, we found that this gemcitabine induced loss of SIRT1 results in a nucleus-to-cytoplasm shift of the SHH signaling activator Gli, explaining the additional gemcitabine sensitivity in SHH-subtype ATRT. Treatment of SHH-subgroup ATRT xenograft-bearing mice resulted in a >40% increase in median survival (p<0.01, log-rank test) and long-term survivors in two independent models. To prepare translation of our findings to the clinic, we investigated potential gemcitabine induced resistance mechanisms by conducting kinome-wide CRISPR/Cas9 knockout screens in primary ATRT cells. Through these experiments we found that low-dose gemcitabine treatment combined with inhibition of protein kinase C zeta (PKCζ) prevents regrowth of resistant ATRT subclones. Together, these findings show that ATRT are highly sensitive to gemcitabine treatment; and as such we suggest that gemcitabine may be rapidly incorporated into future treatment regimens for SHH-ATRT.

Targeting interleukin-17 receptor B enhances gemcitabine sensitivity through downregulation of mucins in pancreatic cancer

Pancreatic cancer is the fourth leading cause of death worldwide due to its poorest prognoses with a 7% 5-year survival rate. Eighty percent of pancreatic cancer patients relapse after chemotherapy and develop early metastasis and drug resistance. Resistance to nucleoside analog gemcitabine frequently used in first-line therapy is an urgent issue in pancreatic cancer treatment. Expression of mucin (MUC) glycoproteins has been shown to enhance chemoresistance via increased cell stemness. Here we show interlukine-17 receptor B (IL-17RB) expression is positively correlated with MUC1 and MUC4 expression in pancreatic cancer cells and tumor tissue. Moreover, IL-17RB transcriptionally up-regulates expression of MUC1 and MUC4 to enhance cancer stem-like properties and resistance to gemcitabine. These results suggest IL-17RB can be a potential target for pancreatic cancer therapy. Indeed, treatment with IL-17RB-neutralizing antibody has a synergistic effect in combination with gemcitabine for killing pancreatic cancer cells. Altogether, these findings provide feasible applications for IL-17RB-targeting therapy in pancreatic cancer treatment.

Abstract 1840: NUC-1031 causes release of DAMPs and upregulates PD-L1 expression in lung cancer cells

Abstract Background: The nucleoside analog gemcitabine is used for the treatment of patients with lung cancer. Its cytotoxic effect is largely attributed to generation of the active metabolite dFdCTP, which causes DNA damage. NUC-1031, a ProTide transformation of gemcitabine, is specifically designed to overcome the key cancer resistance mechanisms associated with poor survival following gemcitabine treatment. NUC-1031 has shown high response rates in clinical studies, and efficacy in patients who were refractory to or had relapsed on gemcitabine. Certain cytotoxic agents are known to promote the release of damage-associated molecular patterns (DAMPs) from cancer cells, including cell surface exposure of calreticulin (CRT) and re-localization of nuclear high mobility group box protein 1 (HMGB1). DAMPs enhance cancer cell recognition by the immune system, promoting immunogenic cell death (ICD). Conversely, studies have shown that cancer cells can upregulate programmed death-ligand 1 (PD-L1) as an adaptive, protective response to cell stress resulting from drug exposure, potentially leading to evasion from immune cell-mediated cytotoxicity. This study investigates the effect of NUC-1031 on the release of DAMPs and modulation of PD-L1 in lung cancer cells. Methods: Non-small cell lung cancer (NSCLC) cell lines were treated with NUC-1031 for 24h and cytotoxicity was assessed at 96h post-treatment to determine half-maximal inhibitory concentrations (IC50). Cells were treated with increasing concentrations of NUC-1031 and cell surface expression of CRT and PD-L1 were analyzed by flow cytometry and complemented with RT-qPCR. IFN-γ was used as a positive control for PD-L1 induction. Localization of HMGB1 was assessed by immunofluorescence microscopy. Results: NUC-1031 induced the release of HMGB1 from the nucleus and increased cell surface expression of CRT in a dose-dependent manner. We confirmed that IFN-γ strongly and uniformly induced cell surface PD-L1 (270% increase). By comparison, NUC-1031-induced cell surface expression of PD-L1 was modest (67% increase). This effect was dose-dependent, with no further increase in PD-L1 expression above 1 μM. Conclusions: NUC-1031 is a potent cytotoxic agent that directly causes DNA damage through generation of dFdCTP. In addition, these data demonstrate the NUC-1031 promotes adaptive changes that alter the recognition of cancer cells by the immune system. The release of DAMPs by NUC-1031 and increase in PD-L1 is predicted to engender an immune response, which might be further potentiated by immune checkpoint inhibition. Citation Format: Jennifer Bré, Oliver J. Read, David J. Harrison. NUC-1031 causes release of DAMPs and upregulates PD-L1 expression in lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1840.

Mre11 exonuclease activity removes the chain-terminating nucleoside analog gemcitabine from the nascent strand during DNA replication.

The Mre11 nuclease is involved in early responses to DNA damage, often mediated by its role in DNA end processing. MRE11 mutations and aberrant expression are associated with carcinogenesis and cancer treatment outcomes. While, in recent years, progress has been made in understanding the role of Mre11 nuclease activities in DNA double-strand break repair, their role during replication has remained elusive. The nucleoside analog gemcitabine, widely used in cancer therapy, acts as a replication chain terminator; for a cell to survive treatment, gemcitabine needs to be removed from replicating DNA. Activities responsible for this removal have, so far, not been identified. We show that Mre11 3' to 5' exonuclease activity removes gemcitabine from nascent DNA during replication. This contributes to replication progression and gemcitabine resistance. We thus uncovered a replication-supporting role for Mre11 exonuclease activity, which is distinct from its previously reported detrimental role in uncontrolled resection in recombination-deficient cells.

Mdm4 supports DNA replication in a p53-independent fashion.

The Mdm4 (alias MdmX) oncoprotein, like its paralogue and interaction partner Mdm2, antagonizes the tumor suppressor p53. p53-independent roles of the Mdm proteins are emerging, and we have reported the ability of Mdm2 to modify chromatin and to support DNA replication by suppressing the formation of R-loops (DNA/RNA-hybrids). We show here that the depletion of Mdm4 in p53-deficient cells compromises DNA replication fork progression as well. Among various deletion mutants, only full-length Mdm4 was able to support DNA replication fork progression. Co-depletion of Mdm4 and Mdm2 further impaired DNA replication, and the overexpression of each partially compensated for the other's loss. Despite impairing replication, Mdm4 depletion only marginally hindered cell proliferation, likely due to compensation through increased firing of replication origins. However, depleting Mdm4 sensitized p53-/- cells to the nucleoside analog gemcitabine, raising the future perspective of using Mdm4 inhibitors as chemosensitizers. Mechanistically, Mdm4 interacts with members of the Polycomb Repressor Complexes and supports the ubiquitination of H2A, thereby preventing the accumulation of DNA/RNA-hybrids. Thus, in analogy to previously reported activities of Mdm2, Mdm4 enables unperturbed DNA replication through the avoidance of R-loops.