The elevated level of replication stress is an intrinsic characteristic of cancer cells. Targeting the mechanisms that maintain genome stability to further increase replication stress and thus induce severe genome instability has become a promising approach for cancer treatment. Here, we identify histone deacetylase 8 (HDAC8) as a drug target whose inactivation synergized with the inhibition of checkpoint kinases to elicit substantial replication stress and compromise genome integrity selectively in cancer cells. We showed that simultaneous inhibition of HDAC8 and checkpoint kinases led to extensive replication fork collapse, irreversible cell-cycle arrest, and synergistic vulnerability in various cancer cells. The efficacy of the combination treatment was further validated in patient tumor-derived organoid (PDO) and xenograft mouse (PDX) models, providing important insights into patient-specific drug responses. Our data revealed that HDAC8 activity was essential for reducing the acetylation level of structural maintenance of chromosomes protein 3 (SMC3) ahead of replication forks and preventing R loop formation. HDAC8 inactivation resulted in slowed fork progression and checkpoint kinase activation. Our findings indicate that HDAC8 guards the integrity of the replicating genome, and the cancer-specific synthetic lethality between HDAC8 and checkpoint kinases provides a promising replication stress-targeting strategy for treating a broad range of cancers.

BACKGROUND:Premature aging has been identified as a global risk factor for cancer. Causes of premature aging are multifactorial, including inflammation, infection, chronic stress, and lifestyle factors. METHOD:We evaluated whether premature aging in people living with HIV (PLWH) was associated with antiretroviral therapy (ART) or the diagnosis of cancer. We used well-established DNA methylation patterns to assess premature aging, using Horvath et al., in individuals with HIV located in Cleveland, Ohio and compared these to standardized datasets of US historical blood samples. Some of the PLWH developed cancer over time. RESULTS:We found that DNA methylation analysis identified accelerated aging in PLWH whereas ART therapy mitigated the advancement of DNA methylation age. A variety of cancers were observed in this population, but a cancer diagnosis was not significantly associated with more advanced DNA methylation age. CONCLUSION:We find that the age acceleration detected in PLWH is mitigated by ART therapy and is not further accelerated by a diagnosis of cancer.

Supplementary Figure Legends

Figure S2 shows UDG depletion by two other different shRNAs selectively sensitizes p53 KO cells to 5-FdU.

Figure S1 shows the effect of irradiation on p21 induction in various cancer cells with different p53 status.

PDF file, 1661K, Supplemental Figure S1- Chemical Structures. Supplemental Figure S2-Oxygen Consumption in MCF-7 Cells treated with or without betalapchone and dicoumarol. Supplemental Figure S2 legend. Supplemental Figure S3-Survival assay for normal mammary epithelial cells treated with betalapachone with or without dicoumarol. Supplemental Figure S3 legend. Supplemental Figure S4-Survival assay for MDA-MB231 cells treated with or without quinones and ROS scavengers. Supplemental Figure S4 legend. Supplemental Figure S5-Westernblot of MCF-7 cells treated with betalapachone with or without catalase. Supplemental Figure S5 legend. Supplemental Figure S6-Proteolysis of PARP in MCF-7 cells treated with betalapachone or staurosporine. Supplemental Figure S6 legend. Supplemental Figure S7- NQO1 mediated futile cycle of betalapachone. Supplemental Figure S7 legend. Supplemental Figure S8- Western blot and analysis of NQO1 to Catalase ratios in Breast Cancer Cell lines. Supplemental Table 1- NQO1 enzymatic activity in breast cancer cell lines. Supplemental Table 1 legend.

Abstract Purpose: TRC102, a small-molecule base-excision repair inhibitor, potentiates the cytotoxicity of pemetrexed and reverses resistance by binding to chemotherapy-induced abasic sites in DNA. We conducted a phase I clinical trial combining pemetrexed and TRC102 with cisplatin–radiation in stage III nonsquamous non–small cell lung cancer (NS-NSCLC). Patients and Methods: Fifteen patients were enrolled from 2015 to 2019. The primary objective was to determine the dose-limiting toxicity and maximum tolerated dose of TRC102 in combination with pemetrexed, cisplatin, and radiotherapy. Secondary objectives were to assess toxicity, tumor response, and progression-free survival at 6 months. Based on our preclinical experiments, pemetrexed–TRC102 was given on day 1, and cisplatin/radiotherapy was initiated on day 3. This schedule was duplicated in the second cycle. After completion, two additional cycles of pemetrexed–cisplatin were given. Toxicities were assessed using NCI CTACAE versions 4/5. Results: The median age was 69 years (45–79) with the median follow-up of 25.7 months (range, 7.9–47.4). No dose-limiting toxicities and no grade 5 toxicity were seen. Hematologic and gastrointestinal toxicities were the most common side effects. No clinical radiation pneumonitis was seen. Of 15 evaluable patients, three had complete response (20%), and 12 had partial response (80%). The 6-month progression-free survival was 80%, and the 2-year overall survival was 83%. Conclusions: Pemetrexed–TRC102 combined with cisplatin/radiotherapy in NS-NSCLC is safe and well tolerated. The recommended phase II dose is 200 mg TRC102 along with cisplatin–pemetrexed. No additional safety signal was seen beyond the expected CRT risks. A phase II trial, integrating post-CRT immunotherapy with this aggressive DNA-damaging regimen, is warranted.

Human uracil DNA-glycosylase (UDG) is the prototypic and first identified DNA glycosylase with a vital role in removing deaminated cytosine and incorporated uracil and 5-fluorouracil (5-FU) from DNA. UDG depletion sensitizes cells to high APOBEC3B deaminase and to pemetrexed (PEM) and floxuridine (5-FdU), which are toxic to tumor cells through incorporation of uracil and 5-FU into DNA. To identify small-molecule UDG inhibitors for pre-clinical evaluation, we optimized biochemical screening of a selected diversity collection of >3,000 small-molecules. We found aurintricarboxylic acid (ATA) as an inhibitor of purified UDG at an initial calculated IC50 < 100 nM. Subsequent enzymatic assays confirmed effective ATA inhibition but with an IC50 of 700 nM and showed direct binding to the human UDG with a KD of <700 nM. ATA displays preferential, dose-dependent binding to purified human UDG compared to human 8-oxoguanine DNA glycosylase. ATA did not bind uracil-containing DNA at these concentrations. Yet, combined crystal structure and in silico docking results unveil ATA interactions with the DNA binding channel and uracil-binding pocket in an open, destabilized UDG conformation. Biologically relevant ATA inhibition of UDG was measured in cell lysates from human DLD1 colon cancer cells and in MCF-7 breast cancer cells using a host cell reactivation assay. Collective findings provide proof-of-principle for development of an ATA-based chemotype and "door stopper" strategy targeting inhibitor binding to a destabilized, open pre-catalytic glycosylase conformation that prevents active site closing for functional DNA binding and nucleotide flipping needed to excise altered bases in DNA.

Despite advances in melanoma treatment, more than 70% of patients with distant metastasis die within 5 years. Proactive treatment of early melanoma to prevent metastasis could save lives and reduce overall healthcare costs. Currently, there are no treatments specifically designed to prevent early melanoma from progressing to metastasis. We used the Connectivity Map to conduct an in silico drug screen and identified 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) as a drug class that might prevent melanoma metastasis. To confirm the in vitro effect of statins, RNA sequencing was completed on A375 cells after treatment with fluvastatin to describe changes in the melanoma transcriptome. Statins induced differential expression in genes associated with metastasis and are used in commercially available prognostic tests for melanoma metastasis. Finally, we completed a chart review of 475 patients with melanoma. Patients taking statins were less likely to have metastasis at the time of melanoma diagnosis in both univariate and multivariate analyses (24.7% taking statins vs. 37.6% not taking statins, absolute risk reduction = 12.9%, P = 0.038). These findings suggest that statins might be useful as a treatment to prevent melanoma metastasis. Prospective trials are required to verify our findings and to determine the mechanism of metastasis prevention.

Abstract The key epigenetic regulator DNA methyltransferase 1 (DNMT1) is a scientifically validated target in p53-null chemorefractory cancers like pancreatic ductal adenocarcinoma (PDAC) since DNMT1-depletion effects cancer cell cycle exits by p53-independent epithelialization. DNMT1 can be depleted by the pyrimidine nucleoside analog pro-drugs decitabine (Dec) or 5-azacytidine (5Aza). However, PDAC clinical trials with Dec/5Aza disappointed. In pre-clinical and clinical analyses, we found resistance was caused by configurations of pyrimidine metabolism in PDAC cells that forestall Dec or 5Aza processing into DNMT1-depleting nucleotide: high expression of cytidine deaminase (CDA) that rapidly catabolizes Dec/5Aza; and suppression of deoxycytidine kinase (DCK) and uridine kinase 2 (UCK2) that rate limit Dec/5Aza pro-drug processing respectively. Accordingly, combination of Dec with a CDA clinical inhibitor, tetrahydrouridine (THU), enabled DNMT1-depletion and PDAC cytoreduction in vitro and in Dec/gemcitabine-refractory PDAC pre-clinical in vivo models. We then conducted a pilot clinical trial in 13 patients with chemorefractory PDAC given oral THU ~10 mg/kg/day combined with decitabine ~0.2 mg/kg/day, for 5 consecutive days, then twice weekly. This Phase 2 was based on several PK/PD studies in human subjects showing potent non-cytotoxic DNMT1-targeting in myeloid cells. Yet again, there were no meaningful clinical responses in the patients. A reason for this was a surprising lack of neutropenia, the most sensitive indicator of systemic DNMT1-targeting. Upon measuring plasma CDA enzyme activity, we found a >10-fold increase in patients with metastatic vs resectable PDAC. Thus, CDA activity is increased not only locally but also systemically in metastatic PDAC, suggesting a need for higher THU doses. We have also observed DCK downregulation, necessary for Dec/gemcitabine uptake and processing, as a cause of PDAC resistance to Dec/gemcitabine. To counter this mechanism, we discovered that 5Aza upregulates DCK as an adaptive response to 5Aza-mediated decrease in dCTP, while Dec upregulates UCK2 (that mediates 5Aza uptake) as an adaptive response to Dec mediated reductions in dTTP. Thus, we alternated Dec with 5Aza in an in vivo model of gemcitabine-resistant PDAC, to exploit their mutual cross-priming, together with THU to inhibit CDA: median vehicle control tumor measurements 972 mm3(range 726-1267.5); median THU-Dec/THU-5Aza 16 mm3 (range 0-87.5); P<0.00001). A non-cytotoxic, epithelial-differentiation based mechanism was confirmed by significant increases in pancreatic epithelial markers while apoptosis markers were unchanged. In sum, metabolism-based resistance to Dec/5Aza can be countered by clinically relevant modifications to treatment, such as alternating doses of THU/Dec and THU/5Aza, for non-cytotoxic p53-independent therapy, a modality distinct from chemoradiation. Citation Format: Rita Tohme, Francis Enane, Caroline Schuerger, Xiaorong Gu, Melissa Fishel, John Pink, Daniel Lindner, Davendra Sohal, Yogen Saunthararajah. Advancing non-cytotoxic DNMT1-targeting to treat chemorefractory pancreatic cancer [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 1088.

9027 Background: About 35% of all NSCLC presents with locally advanced disease and chemo-radiation results in 5-year OS of only ~31%. PEM-platinum combination is approved in stage IV NSCLC and has similar efficacy to platinum-etoposide in stage 3 NSCLC and a favorable toxicity profile (Proclaim trial). TRC102 is an oral small molecule inhibitor of BER. TRC102 potentiates the cytotoxicity of antimetabolites and alkylators and reverses chemotherapy resistance by rapidly and covalently binding to chemotherapy-induced abasic sites in DNA. TRC102 increased radio-sensitization by PEM of NSCLC cell lines and H1299 and A549 xenografts. Methods: Between 11/2015 and 5/2019, 15 patients were enrolled in a 3+ 3 design: 12 with stage III and 3 with oligometastatic stage IV NS-NSCLC. The primary objective was to determine dose-limiting toxicities (DLT’s) and recommended Phase 2 dose (RP2D) of TRC102 in combination with PEM, cisplatin and radiotherapy. Secondary objectives were to assess toxicity, tumor response and PFS at 6 months. Based on pre-clinical data, PEM-TRC102 was given on day 1, and cisplatin/radiotherapy was initiated on day 3. This schedule was duplicated on day 21 and day 23 of the second cycle. After completion of radiotherapy, two additional cycles of PEM-cisplatin were given. Toxicities were assessed by NCI CTACAE version 4 and 5. Results: Median patient age was 69 years (45-79) and median follow up was 16.6 months (3.1-38.6). There were no DLTs or grade 5 toxicity. Hematologic and GI toxicities were the most common adverse events (Table) and radiation pneumonitis was not seen. The RP2D of TRC102 was 200 mg when given with cisplatin/radiotherapy and PEM. Of 15 evaluable patients, 3 had CR (20%) and 12 had PR (80%). The 2-year PFS rate was 49%. Conclusions: PEM-TRC102 combined with cisplatin/radiotherapy in non-squamous NSCLC was safe and well tolerated, and did not cause safety signals beyond those expected from CRT. Preliminary response data and PFS in this cohort was encouraging. A phase 2 trial, integrating post-CRT immunotherapy with this aggressive DNA-damaging regimen is warranted. Clinical trial information: NCT02535325. [Table: see text]

DNA methyltransferase 1 (DNMT1) is scientifically validated as a molecular target to treat chemo-resistant pancreatic ductal adenocarcinoma (PDAC). Results of clinical studies of the pyrimidine nucleoside analog decitabine to target DNMT1 in PDAC have, however, disappointed. One reason is high expression in PDAC of the enzyme cytidine deaminase (CDA), which catabolizes decitabine within minutes. We therefore added tetrahydrouridine (THU) to inhibit CDA with decitabine. In this pilot clinical trial, patients with advanced chemorefractory PDAC ingested oral THU ~10 mg/kg/day combined with oral decitabine ~0.2 mg/kg/day, for 5 consecutive days, then 2X/week. We treated 13 patients with extensively metastatic chemo-resistant PDAC, including 8 patients (62%) with ascites: all had received ≥ 1 prior therapies including gemcitabine/nab-paclitaxel in 9 (69%) and FOLFIRINOX in 12 (92%). Median time on THU/decitabine treatment was 35 days (range 4-63). The most frequent treatment-attributable adverse event was anemia (n=5). No deaths were attributed to THU/decitabine. Five patients had clinical progressive disease (PD) prior to week 8. Eight patients had week 8 evaluation scans: 1 had stable disease and 7 PD. Median overall survival was 3.1 months. Decitabine systemic exposure is expected to decrease neutrophil counts; however, neutropenia was unexpectedly mild. To identify reasons for limited systemic decitabine effect, we measured plasma CDA enzyme activity in PDAC patients, and found a > 10-fold increase in those with metastatic vs resectable PDAC. We concluded that CDA activity is increased not just locally but also systemically in metastatic PDAC, suggesting a need for even higher CDA-inhibitor doses than used here.

Temozolomide (TMZ) generates DNA adducts that are repaired by direct DNA and base excision repair mechanisms. Methoxyamine (MX, TRC-102) potentiates TMZ activity by binding to apurinic and apyrimidinic (AP) sites after removal of N3-methyladenine and N7-methylguanine, inhibiting site recognition of AP endonuclease. We conducted a phase I trial to determine the maximum tolerated dose and dose-limiting toxicities (DLTs) of intravenous MX when given with oral TMZ. Patients with advanced solid tumors and progression on standard treatment were enrolled to a standard 3 + 3 dose escalation trial assessing escalating doses of TMZ and MX. Tumor response was assessed per RECIST and adverse events (AEs) by CTCAEv3. Pharmacokinetics (PK) of MX and COMET assays on peripheral blood mononuclear cells were performed. 38 patients were enrolled—median age 59.5 years (38–76), mean number of cycles 2.9 [1–13]. No DLTs were observed. Cycle 1 grade 3 AEs included fatigue, lymphopenia, anemia, INR, leukopenia, neutropenia, allergic reaction, constipation, psychosis and paranoia. Cycle 2–13 grade 4 AEs included thrombocytopenia and confusion. A partial response was seen in 1 patient with a pancreatic neuroendocrine tumor (PNET) and six additional patients, each with different tumor types, demonstrated prolonged stable disease. MX PK was linear with dose and was not affected by concomitant TMZ. TMZ 200 mg/m2 daily × 5 may be safely administered with MX 150 mg/m2 intravenously once on day 1 with minimal toxicity. Further studies assessing this drug combination in select tumor types where temozolomide has activity may be warranted.

Ribonucleotide reductase (RR) catalyses the rate-limiting step of dNTP synthesis, establishing it as an important cancer target. While RR is traditionally inhibited by nucleoside-based antimetabolites, we recently discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH) that binds reversibly to the catalytic site (C-site). Here we report the synthesis and in vitro evaluation of 13 distinct compounds (TP1-13) with improved binding to hRR over NSAH (TP8), with lower KD’s and more predicted residue interactions. Moreover, TP6 displayed the greatest growth inhibiting effect in the Panc1 pancreatic cancer cell line with an IC50 of 0.393 µM. This represents more than a 2-fold improvement over NSAH, making TP6 the most potent compound against pancreatic cancer emerging from the hydrazone inhibitors. NSAH was optimised by the addition of cyclic and polar groups replacing the naphthyl moiety, which occupies the phosphate-binding pocket in the C-site, establishing a new direction in inhibitor design.

One of the major health concerns on long‐duration space missions will be radiation exposure to the astronauts. Outside the earth's magnetosphere, astronauts will be exposed to galactic cosmic rays (GCR) and solar particle events that are principally composed of protons and He, Ca, O, Ne, Si, Ca, and Fe nuclei. Protons are by far the most common species, but the higher atomic number particles are thought to be more damaging to biological systems. Evaluation and amelioration of risks from GCR exposure will be important for deep space travel. The hematopoietic system is one of the most radiation‐sensitive organ systems, and is highly dependent on functional DNA repair pathways for survival. Recent results from our group have demonstrated an acquired deficiency in mismatch repair (MMR) in human hematopoietic stem cells (HSCs) with age due to functional loss of the MLH1 protein, suggesting an additional risk to astronauts who may have significant numbers of MMR deficient HSCs at the time of space travel. In the present study, we investigated the effects gamma radiation, proton radiation, and 56Fe radiation on HSC function in Mlh1+/+ and Mlh1‐/‐ marrow from mice in a variety of assays and have determined that while cosmic radiation is a major risk to the hematopoietic system, there is no dependence on MMR capacity. Stem Cells Translational Medicine 2018;7:513–520

Abstract Thymidylate synthase (TS) inhibitors including fluoropyrimidines [e.g., 5-Fluorouracil (5-FU) and 5-Fluorodeoxyuridine (5-FdU, floxuridine)] and antifolates (e.g., pemetrexed) are widely used against solid tumors. Previously, we reported that shRNA-mediated knockdown (KD) of uracil DNA glycosylase (UDG) sensitized cancer cells to 5-FdU. Because p53 has also been shown as a critical determinant of the sensitivity to TS inhibitors, we further interrogated 5-FdU cytotoxicity after UDG depletion with regard to p53 status. By analyzing a panel of human cancer cells with known p53 status, it was determined that p53-mutated or -deficient cells are highly resistant to 5-FdU. UDG depletion resensitizes 5-FdU in p53-mutant and -deficient cells, whereas p53 wild-type (WT) cells are not affected under similar conditions. Utilizing paired HCT116 p53 WT and p53 knockout (KO) cells, it was shown that loss of p53 improves cell survival after 5-FdU, and UDG depletion only significantly sensitizes p53 KO cells. This sensitization can also be recapitulated by UDG depletion in cells with p53 KD by shRNAs. In addition, sensitization is also observed with pemetrexed in p53 KO cells, but not with 5-FU, most likely due to RNA incorporation. Importantly, in p53 WT cells, the apoptosis pathway induced by 5-FdU is activated independent of UDG status. However, in p53 KO cells, apoptosis is compromised in UDG-expressing cells, but dramatically elevated in UDG-depleted cells. Collectively, these results provide evidence that loss of UDG catalyzes significant cell death signals only in cancer cells mutant or deficient in p53. Implications: This study reveals that UDG depletion restores sensitivity to TS inhibitors and has chemotherapeutic potential in the context of mutant or deficient p53. Mol Cancer Res; 16(2); 212–21. ©2017 AACR.

Abstract Our research aims to develop therapeutically active inhibitors of the human uracil-DNA glycosylase (UNG). UNG, a highly conserved enzyme, plays a pivotal role in preventing abnormal bases, uracil and 5-fluorouracil, from incorporation into DNA as a result of treatment with chemotherapeutic drugs, floxuridine (5-FdU) and pemetrexed (PEM). Our previous publication reports that the depletion of UNG by shRNA causes uracil and 5-FU incorporation into DNA following 5-FdU or PEM exposure, and highly sensitizes certain human cancer cells to these chemotherapeutic drugs. These data suggest that UNG specific inhibitors could improve the anticancer effect of 5-FdU or PEM. However, a few known inhibitors have not been shown to function at therapeutic concentrations in human cells. Therefore, in order to identify a novel, small-molecule compound targeting the human UNG enzymatic activity, we have previously optimized a biochemically high-throughput screening (HTS) assay that measures the removal of uracil from a DNA hairpin based on the un-quenching of fluorescence. This assay has been successfully employed in a pharmacologically active compound collection screen (>3,000 small-molecules+ approved drugs). In 384-well plate runs using control wells in which enzyme is not added, the average assay provides signal to background ratios of 3.0 to 4.0. Typical Z factors range from 0.6-0.7, indicating a robust assay. We have identified five bioactive compounds with IC50 values < 1uM and have selected a nanoMolar inhibitor of the human UNG enzyme termed, UNGi-A. By using an in vitro DNA glycosylase activity assay, this compound displays a significant inhibitory activity towards the purified human UNG enzyme over time, in a dose-dependent manner. Further characterization shows a significant effect of this inhibitor on UNG activity in cellular extract that expresses endogenous UNG from DLD1 human colon cancer cell line. The result suggests that UNGi-A compound diminishes UNG activity in cell extracts. In addition, thermal shift assay (TSA) data suggest that UNGi-A binds directly to the human UNG enzyme. Together, this compound shows potential activity that blocks the human UNG activity and we are currently evaluating the effect of UNGi-A in combination with 5-FdU on UNG activity in human cancer cells. Citation Format: Mya T. Nguyen, Yan Yan, Yuriy Fedorvo, John Pink, Drew Adams, Stanton Gerson. Identification and characterization of a small-molecule inhibitor of the human uracil-DNA glycosylase for use in combination with DNA damage-based anticancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1663.

Ribonucleotide reductase (RR), an established cancer target, is usually inhibited by antimetabolites, which display multiple cross-reactive effects. Recently, we discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH or E-3a) of human RR (hRR) binding at the catalytic site (C-site) and inhibiting hRR reversibly. We herein report the synthesis and biochemical characterization of 25 distinct analogs. We designed each analog through docking to the C-site of hRR based on our 2.7 Å X-ray crystal structure (PDB ID: 5TUS). Broad tolerance to minor structural variations preserving inhibitory potency is observed. E-3f (82% yield) displayed an in vitro IC50 of 5.3 ± 1.8 μM against hRR, making it the most potent in this series. Kinetic assays reveal that E-3a, E-3c, E-3t, and E-3w bind and inhibit hRR through a reversible and competitive mode. Target selectivity toward the R1 subunit of hRR is established, providing a novel way of inhibition of this crucial enzyme.

Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.

Purpose: We determined the safety, pharmacokinetics, pharmacodynamics and recommended phase II dose of the base excision repair blocker methoxyamine combined with fludarabine. Materials and Methods: This was a phase I study with intravenous fludarabine (25 mg/m(2), days 1-5), and methoxyamine (15 mg/m(2)-120 mg/m(2), once). A maximum of six cycles were given. Adult patients with relapsed/refractory hematologic malignancies, excluding acute myeloid leukemia, were eligible. Results: Twenty patients were treated; diagnoses included CLL/SLL (n = 10), follicular lymphoma (n = 3), DLBCL (n = 3), mantle cell lymphoma (n = 1), anaplastic large cell lymphoma (n = 1) and plasma cell myeloma (n = 2). No DLTs were observed and dose escalation reached the maximum planned dose. Hematologic toxicity was frequent; most common grade 3-4 toxicities were lymphopenia (70%), neutropenia (60%), leukopenia (50%) and anemia (40%). Four patients achieved a partial remission and 8 achieved stable disease. The drug combination resulted in increased DNA damage measured with the Comet assay. Conclusions: Methoxyamine combined with fludarabine was safe and well tolerated. Hematologic toxicity was comparable to single agent fludarabine. Activity appears to correlate with increased levels of DNA damage. Further studies will examine use of this combination of as part conditioning regimens of stem cell transplant and use of methoxyamine as fludarabine dose-sparing agent.