Repair of DNA damage induced by the novel nucleoside analogue CNDAG through homologous recombination.

PURPOSE: We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-ß-D-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-ß-D-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-ß-D-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG. METHODS: Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines. RESULTS: The 6-NH2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH2, compared to the wild type. Furthermore, CNDAG 6-NH2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase. CONCLUSION: CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.

CNDAC-Induced DNA Double-Strand Breaks Cause Aberrant Mitosis Prior to Cell Death.

Incorporation of the clinically active deoxycytidine analogue 2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosyl-cytosine (CNDAC) into DNA generates single-strand breaks that are subsequently converted to double-strand breaks (DSB). Here, we investigated the cellular manifestations of these breaks that link these mechanisms to cell death, and we further tested the relevance of DNA repair pathways in protection of cells against CNDAC damage. The present investigations demonstrate that following exposure to CNDAC and a wash into drug-free medium, chromosomal aberrations, DNA strand breaks, and multinucleate cells arose. These portended loss of viability and were dependent upon exposure time, CNDAC concentration, and passage through mitosis. Following a pulse incubation with CNDAC, live cell imaging using GFP-tagged histone H2B as a marker demonstrated a normal rate of progression to mitosis, but a concentration-dependent delay in passage to a second mitosis. Progression through mitosis was also delayed and accompanied by formation of multinucleate cells. CNDAC-treated cells lacking XPF-ERCC1 nuclease function showed a 16-fold increase in chromosome aberrations. Chromosomal damage in Rad51D-mutant cells (homologous recombination repair deficient) were even more severely affected with extensive aberrations. Rodent or human Polq (POLQ) mutant cells, defective in Pol θ-mediated alternative end joining, did not show enhanced cellular sensitivity to CNDAC. These findings are consistent with formation of DSBs in the second S-phase following exposure, resulting in chromosome aberrations, aberrant mitoses, and subsequent apoptosis.

Targeting BRCA1/2 deficient ovarian cancer with CNDAC-based drug combinations.

PURPOSE: The mechanism of action of CNDAC (2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosyl-cytosine) is unique among deoxycytidine analogs because upon incorporation into DNA it causes a single strand break which is converted to a double strand break after DNA replication. This lesion requires homologous recombination (HR) for repair. CNDAC, as the parent nucleoside, DFP10917, and as an oral prodrug, sapacitabine, are undergoing clinical trials for hematological malignancies and solid tumors. The purpose of this study is to investigate the potential of CNDAC for the therapy of ovarian cancer (OC). METHODS: Drug sensitivity was evaluated using a clonogenic survival assay. Drug combination effects were quantified by median effect analysis. RESULTS: OC cells lacking function of the key HR genes, BRCA1 or BRCA2, were more sensitive to CNDAC than corresponding HR proficient cells. The sensitization was associated with greater levels of DNA damage in response to CNDAC at clinically achievable concentrations, manifested as chromosomal aberrations. Three classes of CNDAC-based drug combinations were investigated. First, the PARP1 inhibitors, rucaparib and talazoparib, were selectively synergistic with CNDAC in BRCA1/2 deficient OC cells (combination index < 1) at a relatively low concentration range. Second, cisplatin and oxaliplatin had additive combination effects with CNDAC (combination index ~ 1). Finally, paclitaxel and docetaxel achieved additive cell-killing effects with CNDAC at concentration ranges of the taxanes similar for both BRCA1/2 deficient and proficient OC cells. CONCLUSIONS: This study provides mechanistic rationales for combining CNDAC with PARP inhibitors, platinum compounds and taxanes in ovarian cancer lacking BRCA1/2 function.

Mechanism-Based Drug Combinations with the DNA Strand-Breaking Nucleoside Analog CNDAC.

CNDAC (2'-C-cyano-2'-deoxy-1-ß-d-arabino-pentofuranosyl-cytosine, DFP10917) and its orally bioavailable prodrug, sapacitabine, are undergoing clinical trials for hematologic malignancies and solid tumors. The unique action mechanism of inducing DNA strand breaks distinguishes CNDAC from other deoxycytidine analogs. To optimize the clinical potentials of CNDAC, we explored multiple strategies combining CNDAC with chemotherapeutic agents targeting distinct DNA damage repair pathways that are currently in clinical use. The ability of each agent to decrease proliferative potential, determined by clonogenic assays, was determined in paired cell lines proficient and deficient in certain DNA repair proteins. Subsequently, each agent was used in combination with CNDAC at fixed concentration ratios. The clonogenicity was quantitated by median effect analysis, and a combination index was calculated. The c-Abl kinase inhibitor imatinib had synergy with CNDAC in HCT116 cells, regardless of p53 status. Inhibitors of PARP1 that interfere with homologous recombination (HR) repair or base excision repair (BER) and agents such as temozolomide that cause DNA damage repaired by the BER pathway were also synergistic with CNDAC. The toxicity of the nitrogen mustards bendamustine and cytoxan, or of platinum compounds, which generate DNA adducts repaired by nucleotide excision repair and HR, was additive with CNDAC. An additive cell killing was also achieved by the combination of CNDAC with taxane mitotic inhibitors (paclitaxel and docetaxel). At concentrations that allow survival of the majority of wild-type cells, the synergistic or additive combination effects were selective in HR-deficient cells. This study provides mechanistic rationales for combining CNDAC with other active drugs. Mol Cancer Ther; 15(10); 2302-13. ©2016 AACR.

Phase I clinical, pharmacokinetic, and pharmacodynamic study of the Akt-inhibitor triciribine phosphate monohydrate in patients with advanced hematologic malignancies.

Akt, a serine/threonine protein kinase, is constitutively phosphorylated and hyperactivated in multiple cancers, including acute myeloid leukemia. High levels are linked to poor survival and inferior responses to chemotherapy, making Akt inhibition an attractive therapeutic target. In this phase I/II study of TCN-PM, a small-molecule Akt inhibitor, TCN-PM therapy was well tolerated in patients with advanced hematological malignancies, and reduced levels of phosphorylation of Akt and its substrate Bad were shown, consistent with inhibition of this survival pathway and induction of cell death. Further investigation of TCN-PM alone or in combination in patients with high Akt levels is warranted.

Sapacitabine, the prodrug of CNDAC, is a nucleoside analog with a unique action mechanism of inducing DNA strand breaks.

Sapacitabine is an orally bioavailable prodrug of the nucleoside analog 2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosylcytosine (CNDAC). Both the prodrug and active metabolite are in clinical trials for hematologic malignancies and/or solid tumors. CNDAC has a unique mechanism of action: after incorporation into DNA, it induces single-strand breaks (SSBs) that are converted into double-strand breaks (DSBs) when cells go through a second S phase. In our previous studies, we demonstrated that CNDAC-induced SSBs can be repaired by the transcription-coupled nucleotide excision repair pathway, whereas lethal DSBs are mainly repaired through homologous recombination. In the current work, we used clonogenic assays to compare the DNA damage repair mechanism of CNDAC with two other deoxycytidine analogs: cytarabine, which is used in hematologic malignacies, and gemcitabine, which shows activity in solid tumors. Deficiency in two Rad51 paralogs, Rad51D and XRCC3, greatly sensitized cells to CNDAC, but not to cytarabine or gemcitabine, indicating that homologous recombination is not a major mechanism for repairing damage caused by the latter two analogs. This study further suggests clinical activity and application of sapacitabine that is distinct from that of cytarabine or gemcitabine.

Multifaceted actions of 8-amino-adenosine kill BCR-ABL positive cells.

Survival of chronic myelogenous leukemia (CML) cells is dependent on BCR-ABL kinase, the activity of which is contingent on the level of BCR-ABL protein and the availability of adenosine triphosphate (ATP). We hypothesized that 8-amino-adenosine (8-amino-Ado)-mediated reduction in cellular ATP level and inhibition of mRNA synthesis leading to a decrease in protein level would result in a multifaceted targeting of BCR-ABL. Using K562 cells, we demonstrated that there was a dose- and time-dependent increase in 8-amino-ATP accompanied by a > 95% decline in the endogenous ATP pool. In parallel, 8-amino-Ado inhibited RNA synthesis and resulted in a depletion of BCR-ABL transcript. Consistent with this, BCR-ABL and ABL protein levels were also decreased. These effects were associated with the initiation of cell death as visualized by poly(ADP-ribose) polymerase (PARP) cleavage, decreased clonogenicity and greater than additive interaction with imatinib. In imatinib-sensitive and -resistant KBM5 cells, 8-amino-Ado treatment augmented the imatinib effect on growth inhibition.

Inhibition of ATP synthase by chlorinated adenosine analogue.

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; F(O) intermembrane base and F1 domain, containing alpha and beta subunits. Crystal structures of both alpha and beta subunits that bind to the substrate, ADP, are known in tight binding (alpha(dp)beta(dp)) and loose binding (alpha(tp)beta(tp)) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the alpha(tp)beta(tp) state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (alpha(dp)beta(dp)) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF(3)(-). Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase.

Clinical and pharmacokinetic study of clofarabine in chronic lymphocytic leukemia: strategy for treatment.

PURPOSE: Based on its mechanistic similarity to fludarabine and cladribine and the success of these analogues for treatment of chronic lymphocytic leukemia (CLL), we hypothesized that clofarabine would be effective for indolent leukemias. The present study was conducted to determine the efficacy and cellular pharmacology during clinical trials of single-agent clofarabine in CLL. EXPERIMENTAL DESIGN: Previously treated patients with relapsed/refractory CLL were eligible for this study. Clofarabine was infused over 1 hour daily for 5 days. Most patients received 3 or 4 mg/m2/d x 5 days, whereas the other two were treated with 15 mg/m2/d x 5 days. Clinical outcome and associated pharmacologic end points were assessed. RESULTS: Myelosuppression limited the maximum tolerated dose of clofarabine to 3 mg/m2/d on this schedule. Cellular pharmacokinetic studies showed a median clofarabine triphosphate concentration in CLL lymphocytes of 1.5 micromol/L (range, 0.2-2.3 micromol/L; n = 9). In the majority of cases, >50% of the analogue triphosphate was present 24 hours after infusion, indicating prolonged retention of the triphosphate in CLL cells. Although cytoreduction was observed, no patients achieved a response. In vitro clofarabine incubation of leukemic lymphocytes from 29 CLL patients showed that clofarabine monophosphate accumulated to a higher concentration compared with the triphosphate. Nonetheless, the triphosphate increased in a dose-dependent fashion and upon successive clofarabine infusions, suggesting benefit from greater doses given at less frequent intervals. CONCLUSION: Levels of clofarabine triphosphate at higher doses and prolonged maintenance of clofarabine triphosphate in leukemic lymphocytes provide a rationale to treat CLL in a weekly clofarabine schedule.

Bis(pivaloyloxymethyl) thymidine 5'-phosphate is a cell membrane-permeable precursor of thymidine 5'-phosphate in thymidine kinase deficient CCRF CEM cells.

Bis(pivaloyloxymethyl) thymidine 5-phosphate (POM(2)-dTMP) has been investigated as a membrane-permeable prodrugs of dTMP. The growth inhibitory activity of POM(2)-TMP has been compared with thymidine (TdR) in wild type CCRF CEM cells (CEM) and a strain that lacks TdR kinase (CEM tk-). After 72 h incubation at 37 degrees C, TdR showed significant antiproliferative activity (IC(50)=27 microM) against CEM cells but was weakly effective (IC(50)=730 microM) against the mutant cell line. By comparison, bis(pivaloyloxymethyl) thymidine 5'-monophosphate (POM(2)-dTMP) was equally inhibitory (IC(50)=5 microM) to both cell lines. The growth inhibitory effects were reversed by deoxycytidine. Cellular [methyl-(3)H]dTTP pools increased linearly over 2h during incubation of CEM or CEM tk- with 5 microM POM(2)-[methyl-(3)H]dTMP. The incorporation of [methyl-(3)H]TdR into HClO(4)-insoluble cell residue by CEM tk- was <0.1% that of CEM and did not increase over 1h. In contrast, CEM tk- incorporated radioactivity from POM(2)-dTMP into acid insoluble residue at a rate 59% that of CEM. These results demonstrate that POM(2)-dTMP can penetrate into cells and serve as a source of dTMP.

Biochemical modulation of cytarabine triphosphate by clofarabine.

PURPOSE: Clofarabine has proven to be effective in the treatment of adult and pediatric acute myelogenous leukemia (AML). To investigate if clofarabine could be used with success in biochemical modulation strategies, we investigated the biochemical modulation of cytarabine triphosphate (ara-CTP) by clofarabine in a myeloid leukemia cell line and the effect of this combination on cytotoxicity. EXPERIMENTAL DESIGN: K562 cells were incubated with clofarabine and ara-C either sequentially or simultaneously to evaluate the combination effect on their phosphorylated metabolites. Clonogenic assays were used to determine the cytotoxicity of each agent alone and in combination. Deoxynucleotide analysis was performed to assess the effect of clofarabine on dNTPs. RESULTS: Clofarabine added either simultaneously or in sequence increased ara-CTP accumulation. The maximal modulation of ara-CTP accumulation occurred with 1 microM clofarabine. This level was achieved at the maximum tolerated dose for adult and pediatric patients with AML. With 10 microM ara-C alone, 86 microM ara-CTP had accumulated after 3 h. The optimal sequence for the drug combination, i.e., clofarabine followed 4 h later by ara-C, resulted in 248 microM ara-CTP at 3 h. Clofarabine accumulated maximally in the monophosphate form. Preincubation with ara-C did not affect the triphosphate form, but it lowered clofarabine monophosphate. Clofarabine resulted in the intracellular decrease of dATP and dGTP levels. Clonogenic assays revealed that the combination of clofarabine and ara-C produced synergistic killing of myeloid leukemia cells. CONCLUSIONS: These findings demonstrate that combination of clofarabine followed by ara-C results in a biochemical modulation of ara-CTP and synergistic cell kill. These studies provide a compelling rationale for clinical trials using this combination regimen for adult and pediatric patients with AML.

In vitro assessment of nucleoside analogs in multiple myeloma.

PURPOSE: To identify nucleoside analogs that may be effective for multiple myeloma (MM), we tested fludarabine, clofarabine, arabinosylguanine, cytarabine, troxacitabine, and gemcitabine in MM cell lines. METHODS: We employed biologic and biochemical assays in MM cell lines to evaluate the clinical potential of these nucleoside analogs. RESULTS: Among these purine and pyrimidine nucleoside analogs, fludarabine, clofarabine and gemcitabine were the most potent. MM cell lines, resistant to commonly used chemotherapeutic agents for this disease, were more sensitive to gemcitabine with an IC50 in the nanomolar range. The greater cytotoxicity of gemcitabine in MM cells was consistent with greater accumulation of gemcitabine triphosphate, the major cytotoxic metabolite of this drug. MM.1S cells accumulated >100 microM gemcitabine triphosphate but accumulated <20 microM of the other analogs as the respective triphosphates. In addition incubation with gemcitabine resulted in inhibition of DNA synthesis. Incubation with 25, 50 or 100 nM gemcitabine resulted in a dose- and time-dependent increase in the cell population with a subG1 DNA content indicative of apoptosis. CONCLUSIONS: These results suggest that gemcitabine is a potent nucleoside analog in MM cell lines including cell types resistant to other chemotherapeutic agents. The greater activity of gemcitabine compared to other analogs seems to be due to favorable metabolism of this agent.