Enhancing Selectivity and Inhibitory Effects of Chemotherapy Drugs Against Myelogenous Leukemia Cells with Lippia alba Essential Oil Enriched in Citral.

Acute myelogenous leukemia (AML) is one of the most lethal cancers, lacking a definitive curative therapy due to essential constraints related to the toxicity and efficacy of conventional treatments. This study explores the co-adjuvant potential of Lippia alba essential oils (EO) for enhancing the effectiveness and selectivity of two chemotherapy agents (cytarabine and clofarabine) against AML cells. EO derived from L. alba citral chemotype were produced using optimized and standardized environmental and extraction protocols. Rational fractionation techniques were employed to yield bioactive terpene-enriched fractions, guided by relative chemical composition and cytotoxic analysis. Pharmacological interactions were established between these fractions and cytarabine and clofarabine. The study comprehensively evaluated the cytotoxic, genotoxic, oxidative stress, and cell death phenotypes induced by therapies across AML (DA-3ER/GM/EVI1+) cells. The fraction rich in citral (F2) exhibited synergistic pharmacological interactions with the studied chemotherapies, intensifying their selective cytotoxic, genotoxic, and pro-oxidant effects. This shift favored transitioning from necrosis to a programmed cell death phenotype (apoptotic). The F2-clofarabine combination demonstrated remarkable synergistic anti-leukemic performance while preserving cell integrity in healthy cells. The observed selective antiproliferative effects may be attributed to the potential dual prooxidant/antioxidant behavior of citral in L. alba EO.

In Vivo Cytotoxic, Genotoxic and Radiosensitizing Effects of Clofarabine.

BACKGROUND/AIM: ClFdA is a second-generation antineoplastic agent that has demonstrated significant anticancer activity, particularly against acute lymphoblastic leukemia and has been shown to have radiosensitizing activity. The aim of the study was to explore the genotoxic, cytotoxic and radiosensitizing effects of clofarabine (ClFdA) on bone marrow cells (BMCs), normoblasts and leukocytes of mice in vivo. MATERIALS AND METHODS: Cytotoxicity was determined by the reduction in reticulocytes (RET), and genotoxicity was determined by the induction of micronucleated reticulocytes (MN-RET) in the peripheral blood and by DNA break induction in leukocytes determined by single-cell gel electrophoresis (SCGE). The radiosensitizing capacity of ClFdA was determined in leukocytes and BMCs by SCGE. RESULTS: Two mechanisms of MN-RET induction were identified according to the antecedents, that could be due to inhibition of DNA synthesis and demethylation of G-C regions, and subsequent chromosome fragility. ClFdA cytotoxicity causes two contiguous peaks, an early peak that seems to inhibit MN-RET induction and a second peak that seems to be caused by ribonucleotide reductase (RR) and/or DNA synthesis inhibitions. ClFdA induced early DNA damage in noncycling leukocytes, and also radiosensitizes leukocytes immediately after treatment. ClFdA-ionizing radiation (IR) causes two time-dependent episodes of DNA damage, the latest after 80 min triggers a major breakage of DNA. In terms of the number of damaged cells, leukocytes and BMCs are similarly sensitive to ionizing radiation; BMCs are slightly more sensitive than leukocytes to ClFdA, but BMCs are doubly sensitive to combined treatment. CONCLUSION: ClFdA causes early DNA damage and radiosensitivity in non-proliferating leukocytes, which rules out the most favored hypotheses of the participation of RR and DNA polymerase inhibition.

The PPP2R1A cancer hotspot mutant p.R183W increases clofarabine resistance in uterine serous carcinoma cells by a gain-of-function mechanism.

PURPOSE: Uterine serous carcinoma (USC) is generally associated with poor prognosis due to a high recurrence rate and frequent treatment resistance; hence, there is a need for improved therapeutic strategies. Molecular analysis of USC identified several molecular markers, useful to improve current treatments or identify new druggable targets. PPP2R1A, encoding the Aα subunit of the tumor suppressive Ser/Thr phosphatase PP2A, is mutated in up to 40% of USCs. Here, we investigated the effect of the p.R183W PPP2R1A hotspot variant on treatment response to the nucleoside analogue clofarabine. METHODS AND RESULTS: USC cells stably expressing p.R183W Aα showed increased resistance to clofarabine treatment in vitro and, corroborated by decreased clofarabine-induced apoptosis, G1 phase arrest, DNA-damage (γH2AX) and activation of ATM and Chk1/2 kinases. Phenotypic rescue by pharmacologic PP2A inhibition or dicer-substrate siRNA (dsiRNA)-mediated B56δ subunit knockdown supported a gain-of-function mechanism of Aα p.R183W, promoting dephosphorylation and inactivation of deoxycytidine kinase (dCK), the cellular enzyme responsible for the conversion of clofarabine into its bioactive form. Therapeutic assessment of related nucleoside analogues (gemcitabine, cladribine) revealed similar effects, but in a cell line-dependent manner. Expression of two other PPP2R1A USC mutants (p.P179R or p.S256F) did not affect clofarabine response in our cell models, arguing for mutant-specific effects on treatment outcome as well. CONCLUSIONS: While our results call for PPP2R1A mutant and context-dependent effects upon clofarabine/nucleoside analogue monotherapy, combining clofarabine with a pharmacologic PP2A inhibitor proved synergistically in all tested conditions, highlighting a new generally applicable strategy to improve treatment outcome in USC.

A cocktail therapeutic strategy based on clofarabine-containing aptamer-PROTAC for enhanced cancer therapy.

By introducing a therapeutic nucleoside analogue tail to the parent Aptamer-PROTACs, a PROTAC-cocktail system (ApTCs-3X) was designed and evaluated. ApTCs-3X exhibited improved nuclease resistance and efficiently degraded target protein with subcellular localization preference. This cocktail therapy results in enhanced therapeutic outcomes, making it suitable for advancing PROTAC in combination therapy.

Clofarabine and total body irradiation (TBI) as conditioning regimen for allogeneic stem cell transplantation in high-risk acute leukemia patients: a two-center retrospective cohort study.

Clofarabine (Clo) is an immunosuppressive purine analog that may have better anti-leukemic activity than fludarabine (Flu). The addition of total body irradiation (TBI) to conditioning regimens has been widely investigated. However, the use of single agent Clo in combination with intermediate doses of TBI ranging from 4 to 8 Gy has not been studied yet. This study is a double center, observational, retrospective study of patients with high-risk hematological malignancies diagnosed from 2012 to 2021, treated at the American University of Beirut Medical Center in Beirut (AUBMC), Lebanon, and Saint-Antoine Hospital (SAH) in Paris, France. It aims to identify the outcome of patients with high-risk hematological malignancies who underwent allogeneic stem cell transplant (allo-SCT) and received Clo and TBI (4-8 Gy) before transplant. Data regarding patient baseline characteristics, disease-related factors, and transplant outcomes including graft-versus-host disease (GVHD), Non-relapse mortality (NRM), progression-free survival (PFS), and overall survival (OS), were collected. We identified 24 high-risk patients diagnosed with a hematological malignancy. The median age at transplant was 37 years (range 22-78). At the time of the transplant, only 15 patients (63%) were in complete remission (CR). All patients received Clo/TBI (4-8 Gy). After a median follow-up of 40 months, the cumulative incidences of grade II-III acute GVHD, grade IV acute GVHD, and chronic GVHD were 50%, 4%, and 8%, respectively. NRM at 100 days, and 1 year after transplant was 4% and 25%, respectively. 17% of the patients had a relapse or progression of the disease by the end of the study. The 2-year PFS and OS were 50% and 56%, respectively. The median PFS and OS were 66 and 68 months respectively. As a conclusion, Clo/TBI (4-8 Gy) as a conditioning regimen for allo-SCT in high-risk patients confers disease control with an acceptable toxicity profile.

Design, synthesis and biological evaluation of Nucleosidic CD99 inhibitors that selectively reduce Ewing sarcoma viability.

Ewing Sarcoma (ES) is a cancer of bone and soft tissues affecting mostly children and young adults. Aggressive progression and poor prognosis of this malignancy call for novel and targeted treatments. CD99 is a transmembrane protein that is abundantly expressed on ES cells and is a diagnostic marker for the disease. ES cells are selectively sensitive to CD99 inhibition compared to most normal cells and other tumors. Therefore, CD99 is a good molecular target for ES treatment. Clofarabine and cladribine are two FDA approved drugs that are administered for their inhibitory acts on DNA synthesis to treat relapsed or refractory acute lymphoblastic and myeloid leukemia. They have also been shown to directly bind to CD99 and inhibit ES growth through a distinct mechanism. In the current study, we designed, synthesized and tested new ES specific derivatives of both drugs that would continue to target CD99 but with expected reduction in cellular membrane permeability and rendered unsuitable for inhibiting DNA synthesis. By using commercially available clofarabine and cladribine purine nucleoside analogs, we modified the primary alcohol moiety at the deoxyribose C-5' terminal site to suppress phosphorylation and thus inhibition of subsequent DNA synthesis pathways. In addition, we incorporated a variety of polar groups in the ribose and purine rings to reduce membrane permeability and investigated the effects of configurational changes in the sugar moiety. Among 26 new derivatives, we identified two compounds, BK50164 and BK60106, that cause cell death specifically in ES primarily due to inhibition of CD99 but not via inhibition of DNA synthesis. These findings provide a road map for the future development selective CD99 inhibitors for targeted treatment of ES.

Aptamers Entirely Built from Therapeutic Nucleoside Analogues for Targeted Cancer Therapy.

Owing to the specific and high binding affinity of aptamers to their targets, aptamer-drug conjugates (ApDCs) have emerged as a promising drug delivery system for targeted cancer therapy. However, in a conventional ApDC, the aptamer segment usually just serves as a targeting moiety, and only a limited number of drug molecules are sequentially conjugated to the oligonucleotide, giving a relatively low drug loading capacity. To address this challenge, herein we employ four clinically approved nucleoside analogues, including clofarabine (Clo), ara-guanosine (AraG), gemcitabine (Ge), and floxuridine (FdU), to replace all natural nucleosides in aptamer sequences, generating a series of whole drug-constituted DNA-like oligomers that are termed drugtamers. Similar to their parent aptamers, the obtained drugtamers maintain the targeting capability and can specifically bind to the target receptors overexpressed on the cancer cell surface. With 100% drug loading ratio, active targeting capability, and enzyme-mediated release of active therapeutics, our drugtamers can strongly induce the apoptosis of cancer cells and inhibit the tumor progression, which enables a new potential for a better targeted cancer therapy.

Targeting Ribonucleotide Reductase Induces Synthetic Lethality in PP2A-Deficient Uterine Serous Carcinoma.

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30% to 40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in Aα results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of Aα mutant uterine serous tumors were sensitive to clofarabine, an RNR inhibitor (RNRi). Aα-mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild-type (WT) cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized WT USC cells to RNRi. Analysis of The Cancer Genome Atlas data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of patients with USC harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second- or later-line therapy revealed a trend for improved outcomes in patients with USC treated with RNRi gemcitabine compared with patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC. SIGNIFICANCE: A drug repurposing screen identifies synthetic lethal interactions in PP2A-deficient uterine serous carcinoma, providing potential therapeutic avenues for treating this deadly endometrial cancer.

Clofarabine induces ERK/MSK/CREB activation through inhibiting CD99 on Ewing sarcoma cells.

Clofarabine, an FDA approved purine analog, is used in the treatment of relapsed or refractory acute lymphoblastic leukemia. Clofarabine acts by inhibiting DNA synthesis. We demonstrated that clofarabine may have a novel function though inhibiting CD99, a transmembrane protein highly expressed on Ewing Sarcoma (ES) cells. CD99 is a validated target in ES whose inhibition may lead to a high therapeutic index for patients. Here we present additional data to support the hypothesis that clofarabine acts on CD99 and regulates key signaling pathways in ES. Cellular thermal shift assay indicated a direct interaction between clofarabine and CD99 in ES cell lysates. Clofarabine induced ES cell death does not require clofarabine's conversion to its active form by deoxycytidine kinase. A phosphokinase array screen with clofarabine and a CD99 blocking antibody identified alterations in signaling pathways. CD99 inhibition with clofarabine in ES cells caused rapid and sustained phosphorylation of ERK, MSK, and CREB. However, activation of this pathway did not correlate with clofarabine induced ES cell death. In summary, we demonstrated that clofarabine may activate ERK, MSK, and CREB phosphorylation through CD99 within minutes, however this paradoxical activation and subsequent ES cell death requires additional investigation.

Long-term results of low-intensity chemotherapy with clofarabine or cladribine combined with low-dose cytarabine alternating with decitabine in older patients with newly diagnosed acute myeloid leukemia.

The treatment of older patients with newly diagnosed acute myeloid leukemia (AML) using intensive chemotherapy is associated with treatment intolerance and poor survival. We evaluated two new lower-intensity regimens with clofarabine (n = 119) or cladribine (n = 129) combined with low-dose cytarabine (LDAC) alternating with decitabine. We reviewed response rates by subgroup and long term outcomes of 248 patients with newly diagnosed non core-binding-factor AML treated on two clinical trials investigating double nucleoside-analogue therapy (DNT) alternating with HMA from October, 2008 to April, 2018. Of 248 patients with a median age of 69 years (range, 49-85 years), 102 patients (41%) were ≥ 70 years, and 108 (44%) had adverse karyotype. Overall, 164 patients (66%) responded: 147 (59%) complete remission (CR) and 17 (7%) CR with incomplete count recovery (CRi). With a median follow up of 60 months, median relapse-free and overall survival (OS) were 10.8 and 12.5 months, respectively. The 2-year OS was 29%. Among patients with normal karyotype, the CR/CRi rate was 79% and the median OS 19.9 months. High response rates and OS were observed in patients with mutations in NPM1, FLT3, IDH2, and RUNX1. The 4- and 8-week mortality rates were 2% and 11%, respectively. The backbone of clofarabine or cladribine and LDAC alternating with decitabine was effective and safe for the treatment of older patients with newly diagnosed AML. Incorporating targeted therapies could extend the efficacy of this approach and provide more curative therapeutic options in this AML population.

Venetoclax and alvocidib are both cytotoxic to acute myeloid leukemia cells resistant to cytarabine and clofarabine.

BACKGROUND: Cytarabine (ara-C) is the major drug for the treatment of acute myeloid leukemia (AML), but cellular resistance to ara-C is a major obstacle to therapeutic success. The present study examined enhanced anti-apoptosis identified in 3 newly established nucleoside analogue-resistant leukemic cell line variants and approaches to overcoming this resistance. METHODS: HL-60 human AML cells were used to develop the ara-C- or clofarabine (CAFdA)-resistant variants. The Bcl-2 inhibitor venetoclax and the Mcl-1 inhibitor alvocidib were tested to determine whether they could reverse these cells' resistance. RESULTS: A 10-fold ara-C-resistant HL-60 variant, a 4-fold CAFdA-resistant HL-60 variant, and a 30-fold CAFdA-resistant HL-60 variant were newly established. The variants demonstrated reduced deoxycytidine kinase and deoxyguanosine kinase expression, but intact expression of surface transporters (hENT1, hENT2, hCNT3). The variants exhibited lower expression of intracellular nucleoside analogue triphosphates compared with non-variant HL-60 cells. The variants also overexpressed Bcl-2 and Mcl-1. Venetoclax as a single agent was not cytotoxic to the resistant variants. Nevertheless, venetoclax with nucleoside analogs demonstrated synergistic cytotoxicity against the variants. Alvocidib as a single agent was cytotoxic to the cells. However, alvocidib induced G1 arrest and suppressed the cytotoxicity of the co-administered nucleoside analogs. CONCLUSIONS: Three new nucleoside analogue-resistant HL-60 cell variants exhibited reduced production of intracellular analogue triphosphates and enhanced Bcl-2 and Mcl-1 expressions. Venetoclax combined with nucleoside analogs showed synergistic anti-leukemic effects and overcame the drug resistance.

Ultralong-Circulating and Self-Targeting "Watson-Crick A = T"-Inspired Supramolecular Nanotheranostics for NIR-II Imaging-Guided Photochemotherapy.

A carrier-free theranostic nanodrug directly coassembled using a NIR probe and a chemotherapeutic drug is a promising alternative for cancer theranostics. Nevertheless, this nanodrug still faces the limitations of short blood circulation and inefficient tumor accumulation/tumoral cellular uptake in vivo. Meanwhile, most exogenous targeting ligands and poly(ethylene glycol) have no therapeutic effect. Herein, we designed an ultralong-circulating and self-targeting nanodrug by an ordered supramolecular coassembly of indocyanine green (ICG), methotrexate (MTX, chemotherapeutic drug and cancer-cell-specific ligand), and clofarabine (CA). Notably, CA, as a surfactant-like chemotherapeutic drug, was introduced into the initial ICG-MTX coassembly by "Watson-Crick A = T-inspired" hydrogen-bond-driven sequential assembly with MTX. This carrier-free theranostic nanodrug with exceptionally high drug payload (100 wt %) not only showed superior serum stabilities but also displayed ultralong blood circulation (>7 days), enabling efficient accumulation at tumor sites. Moreover, our nanodrugs could be self-recognized by cancer cells and release the drugs on demand through lysosomal acidity and external laser stimulus. Under NIR-II imaging guidance, high-efficiency tumor ablation via synergistic photothermal-chemotherapy could be achieved in one treatment cycle while preventing the tumor recurrence. Our ultralong-circulating and self-recognizing carrier-free theranostic nanodrug based on the "drug-delivering-drug" strategy might have the potential for clinical theranostic application.

The effects of clofarabine in ALL inhibition through DNA methylation regulation.

Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF), a second-generation 2'-deoxyadenosine analog, possesses manifold anti-cancer activities. Our previous reports and some of others demonstrate the potential capacity of ClF to regulate the epigenetic machinery. The study presented here is the first to investigate the influence of ClF on modulators of the DNA methylation machinery, including DNMT1 and CDKN1A, in acute lymphoblastic leukemia (ALL) cells. ClF effects on promoter methylation and transcriptional activity of hypermethylated and silenced tumor suppressor genes (TSGs), including APC, CDKN2A, PTEN, and RARB, have been tested as well. Methylation level of the proximal promoter region of APC, CDKN2A, PTEN, and RARB, as well as expression of those TSGs, DNMT1 and CDKN1A, were estimated by using a methylation-sensitive restriction analysis and qPCR, respectively. The Nalm-6 cell line was used as an experimental in vitro model of ALL cells. We observed ClF-mediated inhibition of cellular viability and apoptosis induction of Nalm-6 cells with an increased percentage of cells positive for active Caspase-3. Interestingly, exposure of Nalm-6 cells to CIF at 20 nM concentration for three days has led to a significant DNMT1 downregulation, accompanied by robust CDKN1A upregulation. ClF caused hypomethylation of APC, CDKN2A, and PTEN, with a concomitant increase in their transcript levels. Taken together, our results demonstrate the ability of ClF to reactivate DNA methylation-silenced TSGs in ALL cells. This may implicate translational significance of our findings and support ClF application as a new epigenetic modulator in the anti-leukemia therapy.

Clofarabine Commandeers the RNR-α-ZRANB3 Nuclear Signaling Axis.

Ribonucleotide reductase (RNR) is an essential enzyme in DNA biogenesis and a target of several chemotherapeutics. Here, we investigate how anti-leukemic drugs (e.g., clofarabine [ClF]) that target one of the two subunits of RNR, RNR-α, affect non-canonical RNR-α functions. We discovered that these clinically approved RNR-inhibiting dATP-analogs inhibit growth by also targeting ZRANB3-a recently identified DNA synthesis promoter and nuclear-localized interactor of RNR-α. Remarkably, in early time points following drug treatment, ZRANB3 targeting accounted for most of the drug-induced DNA synthesis suppression and multiple cell types featuring ZRANB3 knockout/knockdown were resistant to these drugs. In addition, ZRANB3 plays a major role in regulating tumor invasion and H-rasG12V-promoted transformation in a manner dependent on the recently discovered interactome of RNR-α involving select cytosolic-/nuclear-localized protein players. The H-rasG12V-promoted transformation-which we show requires ZRANB3-supported DNA synthesis-was efficiently suppressed by ClF. Such overlooked mechanisms of action of approved drugs and a previously unappreciated example of non-oncogene addiction, which is suppressed by RNR-α, may advance cancer interventions.

Optimization of a clofarabine-based drug combination regimen for the preclinical evaluation of pediatric acute lymphoblastic leukemia.

BACKGROUND: The aim of this study was to improve the predictive power of patient-derived xenografts (PDXs, also known as mouse avatars) to more accurately reflect outcomes of clofarabine-based treatment in pediatric acute lymphoblastic leukemia (ALL) patients. PROCEDURE: Pharmacokinetic (PK) studies were conducted using clofarabine at 3.5 to 15 mg/kg in mice. PDXs were established from relapsed/refractory ALL patients who exhibited good or poor responses to clofarabine. PDX engraftment and response to clofarabine (either as a single agent or in combinations) were assessed based on stringent objective response measures modeled after the clinical setting. RESULTS: In naïve immune-deficient NSG mice, we determined that a clofarabine dose of 3.5 mg/kg resulted in systemic exposures equivalent to those achieved in pediatric ALL patients treated with clofarabine-based regimens. This dose was markedly lower than the doses of clofarabine used in previously reported preclinical studies (typically 30-60 mg/kg) and, when scheduled consistent with the clinical regimen (daily × 5), resulted in 34-fold lower clofarabine exposures. Using a well-tolerated clofarabine/etoposide/cyclophosphamide combination regimen, we then found that the responses of PDXs better reflected the clinical responses of the patients from whom the PDXs were derived. CONCLUSIONS: This study has identified an in vivo clofarabine treatment regimen that reflects the clinical responses of relapsed/refractory pediatric ALL patients. This regimen could be used prospectively to identify patients who might benefit from clofarabine-based treatment. Our findings are an important step toward individualizing prospective patient selection for the use of clofarabine in relapsed/refractory pediatric ALL patients and highlight the need for detailed PK evaluation in murine PDX models.

Clofarabine­phytochemical combination exposures in CML cells inhibit DNA methylation machinery, upregulate tumor suppressor genes and promote caspase­dependent apoptosis.

Clofarabine (2­chloro­2'­fluoro­2'­deoxyarabinosyladenine, CIF), a second­generation 2'­deoxyadenosine analog, possesses a variety of anti­cancer activities, including the capacity to modulate DNA methylation marks. Bioactive nutrients, including resveratrol (RSV) and all­trans retinoic acid (ATRA) have been indicated to regulate epigenetic machinery in malignant cells. The purpose of the current study was to evaluate whether the tested phytochemicals, RSV or ATRA, can improve the therapeutic epigenetic effects of CIF in chronic myeloid leukemia (CML) cells. The present study investigates, to the best of our knowledge, for the first time, the influence of CIF in combination with RSV or ATRA on the expression of relevant modifiers of DNA methylation machinery, including DNA Methyltransferase 1 (DNMT1) and Cyclin dependent kinase inhibitor 1A (CDKN1A) in CML cells. Subsequently, the combinatorial effects on promoter methylation and transcript levels of methylation­silenced tumor suppressor genes (TSGs), including phosphatase and tensin homologue (PTEN) and retinoic acid receptor beta (RARB), were estimated using MSRA and qPCR, respectively. The tested TSGs were chosen according to bioinformatical analysis of publicly available clinical data of human DNA methylation and gene expression arrays in leukemia patients. The K562 cell line was used as an experimental CML in vitro model. Following a period of 72 h exposure of K562 cells, the tested combinations led to significant cell growth inhibition and induction of caspase­3­dependent apoptosis. These observations were accompanied by DNMT1 downregulation and CDKN1A upregulation, with a concomitant enhanced decrease in DNMT1 protein level, especially after ATRA treatment with CIF. Concurrent methylation­mediated RARB and PTEN reactivation was detected. The results of the current study demonstrated that CIF that was used in combination with the tested phytochemicals, RSV or ATRA, exhibited a greater ability to remodel DNA methylation marks and promote cell death in CML cells. These results may support the application of CIF combinations with natural bioactive agents in anti­leukemic epigenetic therapy.

Clofarabine Can Replace Anthracyclines and Etoposide in Remission Induction Therapy for Childhood Acute Myeloid Leukemia: The AML08 Multicenter, Randomized Phase III Trial.

PURPOSE: To identify effective and less toxic therapy for children with acute myeloid leukemia, we introduced clofarabine into the first course of remission induction to reduce exposure to daunorubicin and etoposide. PATIENTS AND METHODS: From 2008 through 2017, 285 patients were enrolled at eight centers; 262 were randomly assigned to receive clofarabine and cytarabine (Clo+AraC, n = 129) or high-dose cytarabine, daunorubicin, and etoposide (HD-ADE, n = 133) as induction I. Induction II consisted of low-dose ADE given alone or combined with sorafenib or vorinostat. Consolidation therapy comprised two or three additional courses of chemotherapy or hematopoietic cell transplantation. Genetic abnormalities and the level of minimal residual disease (MRD) at day 22 of initial remission induction determined final risk classification. The primary end point was MRD at day 22. RESULTS: Complete remission was induced after two courses of therapy in 263 (92.3%) of the 285 patients; induction failures included four early deaths and 15 cases of resistant leukemia. Day 22 MRD was positive in 57 of 121 randomly assigned evaluable patients (47%) who received Clo+AraC and 42 of 121 patients (35%) who received HD-ADE (odds ratio, 1.86; 95% CI, 1.03 to 3.41; P = .04). Despite this result, the 3-year event-free survival rate (52.9% [44.6% to 62.8%] for Clo+AraC v 52.4% [44.0% to 62.4%] for HD-ADE, P = .94) and overall survival rate (74.8% [67.1% to 83.3%] for Clo+AraC v 64.6% [56.2% to 74.2%] for HD-ADE, P = .1) did not differ significantly across the two arms. CONCLUSION: Our findings suggest that the use of clofarabine with cytarabine during remission induction might reduce the need for anthracycline and etoposide in pediatric patients with acute myeloid leukemia and may reduce rates of cardiomyopathy and treatment-related cancer.

Evaluation of Pharmacodynamic Responses to Cancer Therapeutic Agents Using DNA Damage Markers.

PURPOSE: We sought to examine the pharmacodynamic activation of the DNA damage response (DDR) pathway in tumors following anticancer treatment for confirmation of target engagement. EXPERIMENTAL DESIGN: We evaluated the time course and spatial activation of 3 protein biomarkers of DNA damage recognition and repair (γH2AX, pS343-Nbs1, and Rad51) simultaneously in a quantitative multiplex immunofluorescence assay (IFA) to assess DDR pathway activation in tumor tissues following exposure to DNA-damaging agents. RESULTS: Because of inherent biological variability, baseline DDR biomarker levels were evaluated in a colorectal cancer microarray to establish clinically relevant thresholds for pharmacodynamic activation. Xenograft-bearing mice and clinical colorectal tumor biopsies obtained from subjects exposed to DNA-damaging therapeutic regimens demonstrated marked intratumor heterogeneity in the timing and extent of DDR biomarker activation due, in part, to the cell-cycle dependency of DNA damage biomarker expression. CONCLUSIONS: We have demonstrated the clinical utility of this DDR multiplex IFA in preclinical models and clinical specimens following exposure to multiple classes of cytotoxic agents, DNA repair protein inhibitors, and molecularly targeted agents, in both homologous recombination-proficient and -deficient contexts. Levels exceeding 4% nuclear area positive (NAP) γH2AX, 4% NAP pS343-Nbs1, and 5% cells with ≥5 Rad51 nuclear foci indicate a DDR activation response to treatment in human colorectal cancer tissue. Determination of effect-level cutoffs allows for robust interpretation of biomarkers with significant interpatient and intratumor heterogeneity; simultaneous assessment of biomarkers induced at different phases of the DDR guards against the risk of false negatives due to an ill-timed biopsy.

Novel Clofarabine-Based Combinations with Polyphenols Epigenetically Reactivate Retinoic Acid Receptor Beta, Inhibit Cell Growth, and Induce Apoptosis of Breast Cancer Cells.

An epigenetic component, especially aberrant DNA methylation pattern, has been shown to be frequently involved in sporadic breast cancer development. A growing body of literature demonstrates that combination of agents, i.e. nucleoside analogues with dietary phytochemicals, may provide enhanced therapeutic effects in epigenetic reprogramming of cancer cells. Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF), a second-generation 2'-deoxyadenosine analogue, has numerous anti-cancer effects, including potential capacity to regulate epigenetic processes. Our present study is the first to investigate the combinatorial effects of ClF (used at IC50 concentration) with epigallocatechin-3-gallate (EGCG, tea catechin) or genistein (soy phytoestrogen), at physiological concentrations, on breast cancer cell growth, apoptosis, and epigenetic regulation of retinoic acid receptor beta (RARB) transcriptional activity. In MCF7 and MDA-MB-231 cells, RARB promoter methylation and expression of RARB, modifiers of DNA methylation reaction (DNMT1, CDKN1A, TP53), and potential regulator of RARB transcription, PTEN, were estimated using methylation-sensitive restriction analysis (MSRA) and quantitative real-time polymerase chain reaction (qPCR), respectively. The combinatorial exposures synergistically or additively inhibited the growth and induced apoptosis of breast cancer cells, followed by RARB hypomethylation with concomitant multiple increase in RARB, PTEN, and CDKN1A transcript levels. Taken together, our results demonstrate the ability of ClF-based combinations with polyphenols to promote cancer cell death and reactivate DNA methylation-silenced tumor suppressor genes in breast cancer cells with different invasive potential.

Clofarabine exerts antileukemic activity against cytarabine-resistant B-cell precursor acute lymphoblastic leukemia with low deoxycytidine kinase expression.

Cytosine arabinoside (Ara-C) is one of the key drugs for the treatment of acute myeloid leukemia. It is also used for consolidation therapy of acute lymphoblastic leukemia (ALL). Ara-C is a deoxyadenosine analog and is phosphorylated to form cytosine arabinoside triphosphate (Ara-CTP) as an active form. In the first step of the metabolic pathway, Ara-C is phosphorylated to Ara-CMP by deoxycytidine kinase (DCK). However, the current cumulative evidence in the association of the Ara-C sensitivity in ALL appears inconclusive. We analyzed various cell lines for the possible involvement of DCK in the sensitivities of B-cell precursor ALL (BCP-ALL) to Ara-C. Higher DCK expression was associated with higher Ara-C sensitivity. DCK knockout by genome editing with a CRISPR-Cas9 system in an Ara-C-sensitive-ALL cell line induced marked resistance to Ara-C, but not to vincristine and daunorubicin, indicating the involvement of DCK expression in the Ara-C sensitivity of BCP-ALL. DCK gene silencing due to the hypermethylation of a CpG island and reduced DCK activity due to a nonsynonymous variant allele were not associated with Ara-C sensitivity. Clofarabine is a second-generation deoxyadenosine analog rationally synthesized to improve stability and reduce toxicity. The IC50 of clofarabine in 79 BCP-ALL cell lines was approximately 20 times lower than that of Ara-C. In contrast to Ara-C, although the knockout of DCK induced marked resistance to clofarabine, sensitivity to clofarabine was only marginally associated with DCK gene expression level, suggesting a possible efficacy of clofarabine for BCP-ALL that shows relative Ara-C resistance due to low DCK expression.