Ara-C Cytotoxicity Research Articles

Acceleration of slow autophagy flux induced by arabinofuranosyl cytidine improves its antileukemic effectiveness in M-NFS-60 cells.

Arabinofuranosyl cytidine (AraC) is an analog of deoxycytidine used as an anticancer drug for leukemic patients. The effective dose always produces severe complications. The present study investigated the modulation of autophagy and its impact on the cytotoxicity of AraC toward murine myelogenous leukemia cells (M-NFS-60). Autophagy was inhibited by NH4Cl or Bafilomycin A1 or enhanced by amino acid starvation, glucose starvation, mild hyperthermia (41 °C), or rapamycin (Rap). Cells were treated with different concentrations, 0 to 2 μM, of AraC in the presence or absence of autophagy modulators. AraC-induced apoptosis is combined with autophagy, especially at lower concentrations. This autophagy is characterized by a slow flux, as indicated by levels of LC3B II and P62 proteins. Inhibition of autophagy did not alter cleaved caspase 3 levels (c-casp.3) or cell viability measured by MTT assays. Conversely, acceleration of AraC-induced autophagy by co-treatment with autophagy inducers reduced cell viability and increased c-casp.3 and c-PARP levels. Further, c-PARP levels were reduced in the presence of caspase inhibitor, Z-VAD-FMK. Enhancement of slow autophagic flux induced by low concentrations of AraC significantly increased the cytotoxicity of AraC toward M-NFS-60 cells. Such coadministration of autophagy inducers might improve the efficacy of AraC treatment and reduce effective doses.

Prolyl oligopeptidase participates in the cytosine arabinoside-induced nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase in a human neuroblastoma cell line

Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a binding partner of prolyl oligopeptidase (POP) in neuroblastoma NB-1 cells and that the POP inhibitor, SUAM-14746, inhibits cytosine arabinoside (Ara-C)-induced nuclear translocation of GAPDH and protects against Ara-C cytotoxicity. To carry out a more in-depth analysis of the interaction between POP and GAPDH, we generated POP-KO NB-1 cells and compared the nuclear translocation of GAPDH after Ara-C with or without SUAM-14746 treatment to wild-type NB-1 cells by western blotting and fluorescence immunostaining. Ara-C did not induce the nuclear translocation of GAPDH and SUAM-14746 did not protect against Ara-C cytotoxicity in POP-KO cells. These results indicate that the anticancer effects of Ara-C not only include the commonly known antimetabolic effects, but also the induction of cell death by nuclear transfer of GAPDH through interaction with POP.

The High NRF2 Expression Confers Chemotherapy Resistance through Upregulated DUSP1 in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a diverse group of clonal marrow stem cell disorders characterized by ineffective hematopoiesis and dysplasia of the cellular elements. Although low-dose cytarabine (Ara-c) has been considered as one of the frontline therapies for high-risk MDS, the overall response rate of single Ara-c treatment was only 30%~40%. Nuclear factor erythroid 2-related factor 2 (NRF2), a basic leucine zipper transcription factor, has been reported to play a pivotal role in chemical detoxification. It is not yet known whether NRF2 has utility as a prognostic biomarker, or whether an elevated NRF2 level is associated with Ara-c resistance in MDS patients.To investigate the expression and effects of NRF2 in MDS patients, gene set enrichment analysis (GSEA) and immunohistochemistry (IHC) were performed. GSEA in a published MDS cohort (n=183) revealed that expressions of NRF2 target genes was significantly enriched in high risk MDS (REAB-1/2) compared to low-risks (RARS and RA). We further detected NRF2 expression levels using IHC staining in the bone marrow (BM) biopsies from a cohort of MDS patients (n=111). Consistent with GSEA results, our IHC data indicated that the NRF2 expression levels of BM from high-risks exceeded low-risk MDS patients by Revised International Prognosis Scoring System (IPSS-R). Importantly, MDS patients with higher NRF2 levels (IHC scores, 4~6) displayed worse overall survival (OS) than patients with lower NRF2 levels (IHC scores, 0~3) (median, 15.7 vs. 22.7 months, P=0.007). Subgroup analysis showed that higher NRF2 levels correlated with poor OS only in chemotherapy group (P=0.04) but not in decitabine (P=0.38) or supportive care group (P=0.19).To recapitulate our findings regarding the function of NRF2 in chemo-resistance, we performed in-vitro and in-vivo experiments. All experiments were performed both in human MDS-derived cell line SKM-1 and MDS mouse model cell line RUNX1 mutant-transduced MllPTD/WT BM cells (MllPTD/WT/RUNX1-S291fs). The cytotoxicity of Ara-c was determined by calculating the half maximal inhibitory concentration (IC50). NRF2 downregulation in MDS cell lines, by inhibitor luteolin, increases IC50 of Ara-c. On the contrary, upregulation of NRF2, mediated by agonist sulforaphane, induces resistance of MDS cells to Ara-C. To better define how suppression of NRF2 sensitized MDS cells to Ara-c, we used lentivirus-mediated shRNA for knockdown of NRF2. Knockdown of NRF2 markedly enhanced early and late apoptosis in MDS cell lines treated with Ara-c. We also established xenograft mouse models through intravenously injecting NRF2 shRNA or scramble shRNA SKM-1 cells into NOD/SCID-IL2Rγnull-SGM3 (NSGS) mice. Knockdown of NRF2 significantly sensitized tumors to Ara-c in our SKM-1 transplanted mouse model. To further investigate the mechanisms involved in NRF2-mediated Ara-c resistance, we analyzed published gene-expression profiles of a large cohort of MDS patients (n=183) and Ara-C sensitive (IC50 <6 μM AraC) and resistant (IC50 >80 μM) AML patient samples (n=10). The data indicated that a group of NRF2 target genes might be responsible for Ara-C resistence. Interestedly, DUSP1 was one of the genes upregulated in both high-risk MDS patients and Ara-c resistant AML patients. Our chromatin immunoprecipitation (ChIP) and qPCR results validated that DUSP1 was a NRF2 direct target gene. Importantly, downregulation of DUSP1 by inhibitor or lentivirus shRNA could abrogate Ara-c resistance in NRF2 elevated MDS cell lines, indicating that NRF2 confers Ara-c resistance partly through its downstream target gene DUSP1 in MDS cells.In conclusion, our clinical and experimental results reveal that 1) NRF2 expression levels are elevated in high-risk MDS patients and serve as a statistically significant prognostic variable for OS in patients with MDS, especially for MDS patients who have received chemotherapy. 2) Pharmacological inhibition of NRF2 re-sensitizes MDS cells to Ara-c treatment while activation of NRF2 by agonist resulted in the reduced sensitivity to Ara-c. 3) NRF2 mediates Ara-c resistance through its direct target gene DUSP1 . Taken together, our findings suggest that silencing NRF2 sensitizes MDS cells to Ara-c treatment; targeting NRF2 in combination with conventional chemotherapy could pave the way for high risk MDS therapy. DisclosuresNo relevant conflicts of interest to declare.

HOTAIRM1 knockdown enhances cytarabine-induced cytotoxicity by suppression of glycolysis through the Wnt/β-catenin/PFKP pathway in acute myeloid leukemia cells

HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) is a long non-coding RNA (lncRNA) that is highly specific for maturing myeloid cells. Dysregulation of HOTAIRM1 has been found to be implicated in the development of acute myeloid leukemia (AML). However, the role of HOTAIRM1 in the drug resistance in AML remains unknown. The present study aimed to investigate the effect of HOTAIRM1 on the cytarabine (Ara-C) resistance in leukemia cell lines and to explore the underlying mechanism. The leukemia cell lines, HL60 and THP-1, were transfected with HOTAIRM1 specific siRNA (si-HOTAIRM1) or control siRNA (si-ctrl), and then treated with Ara-C for 48 h. The mRNA levels of HOTAIRM1 and platelet-type phosphofructokinase (PFKP) were measured using RT-PCR. Cell viability was evaluated by MTT assay. Apoptosis was determined using flow cytometry and caspase-3/7 activity assay. Glycolysis was evaluated by determining the glucose consumption and lactate production. To activate the Wnt/β-catenin signaling pathway, HL60 and THP-1 cells were transfected with β-catenin overexpressing plasmid (pcDNA-β-catenin). Protein levels of PFKP, β-catenin, and c-Myc were examined using western blot analysis. The results showed that knockdown of HOTAIRM1 enhanced Ara-C-induced reduction of cell viability and increase of cell apoptosis. HOTAIRM1 knockdown suppressed the glucose consumption and lactate production, as well as the expression of PFKP in AML cells. Besides, HOTAIRM1 knockdown resulted in a significant inhibitory effect on the Wnt/β-catenin pathway. Furthermore, activating Wnt/β-catenin pathway mitigated the effects of HOTAIRM1 knockdown on glycolysis and Ara-C cytotoxicity in AML cells. In conclusion, knockdown of HOTAIRM1 enhanced Ara-C cytotoxicity through regulating the Wnt/β-catenin/PFKP signaling pathway. These findings suggested that HOTAIRM1 might be a therapeutic target for overcoming the Ara-C resistance in AML.

Chidamide Enhances the Cytotoxicity of Cytarabine and Sorafenib in Acute Myeloid Leukemia Cells by Modulating H3K9me3 and Autophagy Levels.

Previous studies showed that chidamide enhances the cytotoxicity of drugs in acute myeloid leukemia (AML) cells. Therefore, we examined whether chidamide enhanced the cytotoxicity of drugs in AML cells by affecting H3K9me3 and autophagy levels. AML cells (THP-1 and MV4-11 cells) were treated with chidamide, cytarabine (Ara-c), or sorafenib alone or in combination. Cell proliferation and survival rates were analyzed by MTT, flow cytometry, and Western blotting assays. The results showed that a low dose of chidamide enhanced the cytotoxicity of Ara-c or sorafenib in AML cells, decreasing proliferation and increasing apoptosis. H3K9me3 levels as assessed by Western blotting were upregulated by chidamide treatment. Chromatin immunoprecipitation sequencing, which was used to investigate potential signaling pathways, indicated that the autophagy pathway might play a role in the effects of chidamide. The level of autophagy induced in AML cells upon treatment with Ara-c or sorafenib was inhibited by chidamide, and autophagy markers (LC3, P62) were tested by Western blotting. SIRT1 messenger RNA (mRNA) and protein levels were lower in AML cells treated with Ara-c or sorafenib in combination with chidamide than those in cells treated with these drugs alone. Additionally, the Integrative Genomics Viewer results indicate that the H3K9me3 changes were related to SIRT1-binding sites. Together, these results show that chidamide enhances the cytotoxicity of two chemotherapy drugs in AML cells by increasing the H3K9me3 level and inhibiting autophagy via decreasing the expression of SIRT1. Chidamide may be a potential treatment strategy for AML in the future, especially for refractory AML patients.

281P - Selective depletion of tumour-associated SAMHD1 by HSP90 inhibitors enhances the anti-AML effect of cytarabine

BackgroundAcute myeloid leukemia (AML) is the most common acute type of leukemia in adults, with a disappointing prognosis and high death rate. Cytarabine (ara-C) is a primary first-line chemotherapy drug used in the treatment of AML, and resistance to it represents a major source of treatment failure. Sterile alpha motif and HD domain-containing protein 1 (SAMHD1), the only known human dNTPase, is known to attenuate ara-C cytotoxicity in AML cells, resulting in treatment failure and a poor prognosis. Therefore, selective inhibition of SAMHD1 in AML should prove a promising strategy for increasing the efficacy of ara-C. Nevertheless, no effective strategy to target SAMHD1 is available yet. MethodsCell culture, Monocyte isolation and differentiation, Cytotoxicity assays, Protein extraction and Western blot analysis, Immunoprecipitation, Immunofluorescent staining, Tissue staining, Mass spectrum system, mRNA analysis, RNA silencing, Flow cytometry, Orthotopic AML animal model, Heterotopic AML animal model. ResultsHere, we report that SAMHD1 is a novel HSP90-dependent protein in various tumor types. Pharmacological inhibition of HSP90 led to SAMHD1 depletion in a wide range of tumor cell types but had no or minimally effect on SAMHD1 of peripheral blood mononuclear cells (PBMCs) or macrophages from healthy donors due to the phosphorylation status on Thr 592. The combination of ara-C and HSP90 inhibitors showed a synergistic effect in killing AML cells. The HSP90 inhibitor also significantly enhanced the therapeutic efficacy of ara-C in both orthotopic and heterotopic models, alleviating tumor burden and prolonging survival. ConclusionsOverall, these data suggest that the addition of an HSP90 inhibitor to ara-C-based chemotherapy represents a potential new treatment strategy for AML patients. Legal entity responsible for the studyXiaofang Yu. FundingNational Natural Science Foundation of China (81772169). DisclosureAll authors have declared no conflicts of interest.

Commonly Used Chemotherapy Drugs Differentially Determine Microenvironment-Mediated Protection, Via Mitochondrial Transfer, to B-Precursor Acute Lymphoblastic Leukaemia Cells

Murine models of Acute Lymphoblastic Leukaemia (ALL) suggest relapse arises not from intrinsic chemoresistance by genetically distinct cells, but from a subset of cells protected within a specific niche.To confirm the existence of such a niche in patients with ALL and elucidate the mechanism by which stromal cells (MSC) protect ALL cells we isolated MSC from bone marrow of 70 B-ALL patients enrolled on the UKALL14 trial. Immunostaining for f-actin, gene expression profiling and cytokine/chemokine quantification showed that a significant proportion of bone marrow specimens, especially after treatment with a cytarabine (AraC)-containing block, contained MSC with an activated phenotype, analagous to cancer associated fibroblasts (ALL-CAF).We demonstrated that primary ALL cells, ALL cell lines, AraC and daunorubicin (but not vincristine (VCR) or dexamethasone (dex)) generated ALL-CAF de novo from both healthy donor MSC and the MSC cell line HS27a. Notably, the chemotherapy drugs induced distinct morphological changes and differential alpha-smooth muscle expression (figure 1a).Control of oxidative stress via modification of reactive oxygen species (ROS) presented a potential unifying explanation for ALL-CAF generation by both ALL cells and chemotherapy. Using flow cytometry we demonstrated that AraC significantly increased ROS in the B-ALL cell line, SEM, in monoculture but in co-culture with HS27a, ROS was significantly lowered and was not impacted by AraC. The MSC co-culture-mediated reduction in ROS in co-culture corresponded to a significant reduction in cell death (10.5% vs 36%, p = 0.0001). By contrast, VCR did not impact ROS significantly and Dex reduced it. Both were significantly more effective than AraC at inducing SEM cell death in co-culture (VCR 20.2% vs AraC 10.5%, p = 0.0003; Dex 39.1% vs AraC 10.5%, p = 0.0007), despite inducing the same degree of cell death in monoculture.We hypothesised that mitochondrial transfer between ALL-CAF and B-ALL cells could provide a generalised mechanism to overcome the deleterious impact of cell-intrinsic and chemotherapy-driven ROS in B-ALL cells. A ‘mitotracker’ flow cytometry assay showed differential mitochondrial transfer from HS27a to B-ALL cells, in proportion to the baseline ROS levels. We confirmed that mitochondria could also be transferred from healthy donor MSC co-cultured with primary patient ALL cells. Furthermore, AraC, but not VCR or Dex, significantly enhanced mitochondrial transfer, and did so in a dose-dependent manner. To rule out passive transfer of dye, we used the murine stromal line MS5 as an alternative mitochondria donor to SEM cells. Murine mitochondrial, but not nuclear, DNA was clearly seen in flow-sorted SEM cells after co-culture with MS5, at baseline and at higher levels after AraC therapy. We also directly visualised the transfer of mitochondria along tunnelling nanotubes (TNT) by time-lapse confocal imaging (figure 1b). To confirm that mitochondrial transfer was essential in MSC ‘rescue’ of ALL cells, we generated HS27a cells deficient in mitochondria following prolonged culture with low dose ethidium bromide. The mitochondrially-deficient cells retained viability as well as the ability to become ALL-CAF but were clearly defective in their ability to rescue SEM ALL cells from AraC induced cell death.To confirm the functional impact of mitochondrial transfer via TNT, we used actin inhibitor latrunculin B (LatB) and the microtubule damaging agent nocodazole which both significantly blocked the phenomenon. Both LatB and nocodazole significantly restored AraC-related cytotoxicity. Colchicine, another microtubule damaging agent had a similar impact to nocodazole. VCR completely overcame the protective impact of HS27a on AraC cytotoxicity and was additive with AraC in the co-culture system.We have shown that CAF-like MSC provide support to ALL cells under oxidative stress by mitochondrial transfer via TNT. This is disrupted by microtubule damaging agents and conditions provoking their formation are mitigated by Dex, both mainstays of ALL therapy. Our data may explain the ineffectiveness of ROS-inducing chemotherapy at eradicating disease at the niche and provides an explanation of why low dose, microtubule damaging agents such as VCR used in maintenance therapy are effective in ALL. Our findings have immediate implications for the design and scheduling of current combination chemotherapies for ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Extracellular ATP and CD39 Regulates Mitochondrial Function and Cytarabine Resistance through Intrinsic PKA-ATF-PGC1a Pathway in Acute Myeloid Leukemia

Relapses in acute myeloid leukemia (AML) are caused by chemoresistant leukemic populations and new therapeutic approaches that specifically target these cells are urgently needed. Based on transcriptomic analyses of relevant PDX preclinical model of the resistance to cytarabine (AraC) and of the residual disease in patients, we identified ecto-nucleoside triphosphate diphosphohydrolase-1 CD39 (ENTPD1) overexpressed in the residual leukemic cells in vivo after chemotherapy. By flow cytometry, we confirmed that AraC increased cell surface CD39 expression in AML cell lines in vitro and in vivo as well as in 24 diverse patient-derived xenograft models. We further observed this increase in 100 patients at 35-days post-intensive chemotherapy compared to their respective diagnosis. Interestingly, high CD39 expression on AML patients was associated with a worse response to AraC in vivo. Furthermore, we showed that FACS-sorted CD39high AML cells had increased mitochondrial mass and activity, and were resistant to AraC in vitro and in vivo. We demonstrated that CD39 downstream signaling pathway was dependent on cAMP-PKA-PGC1a-TFAM axis and its inhibition by H89 sensitized AML cells to AraC through the inhibition of mitochondrial OxPHOS biogenesis and function. Finally, pharmacological inhibition of CD39 ATP hydrolase activity or genetic invalidation of CD39 protein using two inhibitors or shRNA markedly enhanced AraC cytotoxicity in AML cell lines and primary patient samples in vitro and in vivo. Together, these results indicate CD39 as a new player of the intrinsic chemoresistance pathway and a new therapeutic target to specifically overcome AraC resistance and eradicate these leukemic cells responsible for relapses in AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 315: Cell death of AML blasts induced by cytarabine and enhanced by the Vitamin D2/Carnosic acid cell differentiating combination involves apoptosis signal-regulating kinase 1 (ASK1) activation

Abstract Acute Myeloid Leukemia (AML) is a disease with a grim prognosis and limited curative treatment options. The principal agent used to induce disease remissions is Cytarabine (AraC), typically combined with an anthracycline, though multiple other combinations have been explored. We have previously reported that the exposure of AML blasts, in which the DNA is already damaged by AraC, to the Vitamin D2 analog Doxercalciferol combined with Carnosic Acid (D2/CA) enhances AraC cytotoxicity, resulting in an increased cell kill of the blasts. To obtain an understanding of the underlying mechanisms, and thus facilitate the translation of this finding to the clinic, we conducted in vitro studies on HL60, U937 cell lines and on AML blasts in primary culture. The initial findings were that the kinases BRAF and JNK1 and the pro-apoptotic protein BIM participate in the enhancement of AraC action. We now report that the kinase activity of ASK1 (MAP3K5) is upregulated by both AraC and D2/CA, and this appears to be additive thus increasing the apoptosis by this sequential combination regimen. Interestingly, we also noted that BRAF directly binds to ASK1, and since it is a kinase, this raises the possibility that it activates ASK1 in this experimental system. This possibility is strengthened by the report that JNK activation which leads to apoptosis is dependent on BRAF. The ongoing investigation of additional details of the molecular events responsible for enhanced cell death may provide clues for the improvement of the therapy of AML. (Supported by NIH grant R01CA044722-26 from NCI). Citation Format: Xuening Wang, Jonathan S. Harrison, George P. Studzinski. Cell death of AML blasts induced by cytarabine and enhanced by the Vitamin D2/Carnosic acid cell differentiating combination involves apoptosis signal-regulating kinase 1 (ASK1) activation [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 315.

Randomized phase II trial of cytosine arabinoside with and without the CHK1 inhibitor MK-8776 in relapsed and refractory acute myeloid leukemia

PurposeCytosine arabinoside (AraC) remains the backbone of most treatment regimens for acute myeloid leukemia (AML). Incorporation of AraC into DNA activates checkpoint kinase 1 (Chk1), leading to cell-cycle arrest and diminished AraC cytotoxicity, which can be reversed by the selective Chk1 inhibitor MK-8776. Building on a Phase I trial, we conducted a phase II trial comparing timed sequential AraC with or without MK-8776. MethodsPatients with relapsed or primary refractory AML were randomized 1:1 to receive either AraC with MK-8776 (Arm A); or AraC alone (Arm B). Results32 patients were treated: 14 assigned to Arm A and 18 to Arm B. There were 5 (36%) complete responses (CR/CRi) and 1 (7%) partial response (PR) in Arm A, and 8 (44%) CR/CRis and 1 (6%) PR in Arm B. Median survival did not differ significantly between the two groups (5.9months in Arm A vs. 4.5 months in Arm B). MK-8776 led to a robust increase in DNA damage in circulating leukemic blasts as measured by increased γ-H2AX (16.9%±6.1% prior and 36.4%±6.8% at one hour after MK-8776 infusion, p=0.016). ConclusionResponse rates and survival were similar between the two groups in spite of evidence that MK-8776 augmented DNA damage in circulating leukemic blasts. Better than expected results in the control arm using timed sequential AraC and truncated patient enrollment may have limited the ability to detect clinical benefit from the combination.

Optimal AraC-Cytotoxicity to AML Cells Requires ERK5 Activity.

Cytarabine (AraC) has been the primary treatment agent for acute myeloid leukemia (AML) in the past 30 years, but the precise mechanism of its action is not completely known. Here we assessed the role of ERK5 in AraC-induced cell death in AML cell lines HL60 and U937 using ERK5 inhibitors BIX02189 and XMD8-92. We report that inhibition of MEK5/ERK5 activity reduces AraC-induced cell death, DNA damage, the upregulated DNA damage biomarkers, and produced G2 phase cell cycle arrest. In addition, the pro-survival protein P-Bcl2 Ser70 was found to be associated with decreased AraC-induced cell death following XMD8-92 treatment, suggesting a regulatory role of ERK5 on Bcl2 phosphorylation. Our study shows that the full potency of AraC cytotoxicity requires optimal ERK5 activity, suggesting a novel role of ERK5 in cancer chemotherapy. J. Cell. Biochem. 118: 1583-1589, 2017. © 2016 Wiley Periodicals, Inc.

Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies.

The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP), which causes DNA damage through perturbation of DNA synthesis. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.

SAMHD1 is a biomarker for cytarabine response and a therapeutic target in acute myeloid leukemia

The nucleoside analog cytarabine (Ara-C) is an essential component of primary and salvage chemotherapy regimens for acute myeloid leukemia (AML). After cellular uptake, Ara-C is converted into its therapeutically active triphosphate metabolite, Ara-CTP, which exerts antileukemic effects, primarily by inhibiting DNA synthesis in proliferating cells. Currently, a substantial fraction of patients with AML fail to respond effectively to Ara-C therapy, and reliable biomarkers for predicting the therapeutic response to Ara-C are lacking. SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate. Although it has been postulated that SAMHD1 sensitizes cancer cells to nucleoside-analog derivatives through the depletion of competing dNTPs, we show here that SAMHD1 reduces Ara-C cytotoxicity in AML cells. Mechanistically, dGTP-activated SAMHD1 hydrolyzes Ara-CTP, which results in a drastic reduction of Ara-CTP in leukemic cells. Loss of SAMHD1 activity-through genetic depletion, mutational inactivation of its triphosphohydrolase activity or proteasomal degradation using specialized, virus-like particles-potentiates the cytotoxicity of Ara-C in AML cells. In mouse models of retroviral AML transplantation, as well as in retrospective analyses of adult patients with AML, the response to Ara-C-containing therapy was inversely correlated with SAMHD1 expression. These results identify SAMHD1 as a potential biomarker for the stratification of patients with AML who might best respond to Ara-C-based therapy and as a target for treating Ara-C-refractory AML.

BRAF signals to pro-apoptotic BIM to enhance AraC cytotoxicity induced in AML cells by Vitamin D-based differentiation agents

Vitamin D has so far not fulfilled its early promise as an antineoplastic agent, in spite of compelling in vitro data. With the aim of bringing vitamin D or its derivatives (VDDs) effectively to the clinic, we developed a two-pronged approach. First, by adding the plant-derived Carnosic Acid (CA) to a vitamin D2 derivative Doxercalciferol we increased its differentiation potency without increasing it hypercalcemic properties. Second, we added these two agents together to AML cells already treated with Cytarabine (AraC), the standard drug for the treatment of patients with AML. We now report that BRAF, a part of the MAPK signaling pathway, is required for the optimally increased cell death in this system and acts upstream of BIM, the regulator of the caspase cascade that leads to cell death by apoptosis. It is proposed that this therapeutic regimen should be tested in a clinical trial.

The role of VDR and BIM in potentiation of cytarabine-induced cell death in human AML blasts.

Acute Myeloid Leukemia (AML) has grave prognosis due to aggressive nature of the disease, the toxicity of standard treatment, and overall low cure rates. We recently showed that AML cells in established culture treated with cytarabine (AraC) and a differentiation agent combination show enhancement of AraC cytotoxicity. Here we elucidate molecular changes which underlie this observation with focus on AML blasts in primary culture. The cells were treated with AraC at concentrations achievable in clinical settings, and followed by the addition of Doxercalciferol, a vitamin D2 derivative (D2), together with Carnosic acid (CA), a plant-derived antioxidant. Importantly, although AraC is also toxic to normal bone marrow cell population, the enhanced cell kill by D2/CA was limited to malignant blasts. This enhancement of cell death was associated with activation of the monocytic differentiation program as shown by molecular markers, and the increased expression of vitamin D receptor (VDR). Apoptosis elicited by this treatment is caspase-dependent, and the optimal blast killing required the increased expression of the apoptosis regulator Bim. These data suggest that testing of this regimen in the clinic is warranted.

Panobinostat-Augmented Cytarabine and Daunorubicin (7+3) Induction for Older Patients with AML or High-Risk MDS Is Well Tolerated and Results in Favorable Clinical Outcomes: The Panda Trial

IntroductionPatients aged >60 years (y) with acute myeloid leukemia (AML) have inferior outcomes compared to those <60y. Even those who tolerate induction chemotherapy achieve lower response rates with standard approaches and experience significant toxicity with more intensive combinations. The histone deacetylase (HDAC) inhibitor panobinostat (pano) potentiates anthracycline and ara-C cytotoxicity, likely through enhancement of DNA double-strand breaks leading to increased apoptosis (Maiso et al. 2009; Xie et al. 2013). We hypothesized that addition of short-course HDAC inhibition with pano prior to and during 7+3 induction therapy would be well-tolerated and lead to favorable outcomes in this patient population.MethodsWe conducted a phase I study in patients >60y with newly diagnosed AML or high-risk MDS combining pano at doses ranging from 20 mg to 60 mg given orally on days 1, 3, 5, and 8 of induction with daunorubicin (dauno) 60 mg/m2 on days 3-5 and ara-C 100mg/m2 continuously on days 3-10 (p+7+3). Patients were allowed retreatment on p+5+2, with pano on days 1, 3, and 5 at the same doses, if the nadir BM biopsy had residual leukemia. Upon attainment of CR/CRi, patients were offered a second p+7+3 or alternative consolidation including allogeneic stem cell transplantation (alloHCT). Dose-limiting toxicity was defined based on unexpected toxicities from 7+3 induction and a standard 3+3 design was utilized for dose escalation. Peripheral blood mononuclear cells were isolated on days 1-5 and HDAC expression and histone acetylation were evaluated by western blot and quantified by densitometry.ResultsOverall, 22 patients were treated on the PanDA protocol. Treatment was well-tolerated in all dose-cohorts and the MTD was not reached. We treated 6 patients each at the 60 mg and 50 mg dose in the dose expansion phase due to recurrent grade 1 bradycardia. The median age was 67y-60-69y (13), 70-79y (5), and >80y (4). Eleven patients had de novo AML, 7 had AML with myelodysplasia related changes, 2 had 2ary AML from MPD, 1 treatment-associated myeloid neoplasm, and 1 had RAEB-2. Two patients had progressed on hypomethylating agents. Thirteen (62%) had intermediate-risk cytogenetics (cyto). Amongst 8 euploid patients, 2 had FLT3-ITDmut, 1 had FLT3-TKDmut and 2 had NPM1mut as the sole abnormality. Seven (33%) patients had complex/monosomic cyto. There were no patients with favorable cyto.There were no protocol-defined DLTs. The most common grade 2-4 TEAEs were febrile neutropenia (77%), electrolyte abnormalities (64%), diarrhea (55%), rash (55%), LFT abnormalities (32%), nausea (27%) and anorexia (27%). Of note, 36% experienced asymptomatic sinus bradycardia peaking on day 6 following pano, dauno, and 5HT3 inhibitor anti-emetics. Additionally, 9% experienced new onset grade 2 atrial fibrillation. There was no evidence of myocardial dysfunction on echocardiogram following induction. SAEs reported on study were sepsis (9%), typhlitis (5%), DIC (5%), pneumonitis (5%), and bowel perforation due to mucorales (5%). There were 2 deaths (9%) during induction, one related to typhlitis and the other to sepsis.Of 20 evaluable patients, 8 (40%) achieved CR/CRi . One patient received study treatment as consolidation, 3 received intermediate/high-dose ara-C, 1 received a hypomethylating agent, 2 underwent alloHCT and 1 had no further therapy. Median overall survival was 10 mos (ITT) and 16 mos for those who achieved a CR/CRi (p=0.005). Median relapse-free survival was 10 mos, range 3-27 mos (Figure 1).Baseline expression of HDAC class 1, 2, 3 and 6 was evaluated and was not found to be associated with response to p+7+3 arguing against a direct role in p+7+3-induced cytotoxicity. However, global histone acetylation change after pano exposure (days 1-3) was significantly associated with eventual chemo-ablation at nadir (Figure 2). Ongoing studies are evaluating markers of DNA damage response and apoptosis in patients treated with PanDA vs. concurrent 7+3 controls.ConclusionsThe addition of pano to 7+3 induction for older patients with AML is well-tolerated and results in clinical outcomes that are favorable in our very high-risk population. The recommended dose of pano is 50 mg daily on days 1, 3, 5, and 8 when combined with 7+3 induction in future studies. Importantly, histone acetylation change following exposure to pano but not baseline HDAC expression predicts sensitivity to combined therapy. [Display omitted] [Display omitted] DisclosuresWieduwilt:Sigma-Tau: Research Funding; Alexion: Honoraria. Off Label Use: Panobinostat is approved for multiple myeloma. This paper reports it's use in patients with acute myeloid leukemia.. Olin:Daiichi-Sankyo: Research Funding. Logan:Pharmacyclics: Consultancy; Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy. Damon:Atara: Consultancy; Sunesis: Research Funding; McGraw-Hill: Patents & Royalties: Book Chapter; Sigma-Tau: Research Funding. Boyer:Onyx: Speakers Bureau. Andreadis:genentech: Employment; novartis oncology: Research Funding; cellerant: Research Funding; karyopharm: Research Funding; McGraw-Hill: Patents & Royalties: Book Chapter; Pfizer: Honoraria; Pharmacyclics: Honoraria.

C-Jun Regulates ER Stress Signaling to Promote Chemotherapy Resistance in Acute Myeloid Leukemia

The first-line treatment for acute myeloid leukemia (AML) patients is a combination of cytarabine (Ara-C) and an antracycline, such as daunorubicin (Dnr). However, one third of AML patients are refractory to first-line therapy and 35-60% of those who do initially respond to chemotherapy relapse with resistant disease. Therefore, identifying and deciphering the molecular abnormalities that contribute to chemotherapy resistance in AML is the critical first step for developing new therapies that overcome chemotherapy resistance. In our study, we found that expression of the proto-oncogene c-JUN is elevated in patient-derived AML samples compared with normal hematopoietic progenitor cells. Additionally, expression and phosphorylation of c-JUN further increased in human AML cells exposed to Ara-C or Dnr. Confirming functional functional importance of c-JUN in human AML cells, we found that short hairpin RNAs (shRNAs) that specifically target c-JUN suppress cell growth and induce apoptosis in several genetically distinct human AML cell lines (Kasumi-1, MOLM-14, MV4-11, NOMO-1, OCI-AML3, SKM-1, THP-1 and U937). Furthermore, we have observed that shRNA-mediated inhibition of c-JUN also significantly potentiated the cytotoxic effects of Ara-C (p<0.05), suggesting that c-JUN is required for AML cell survival and may play a critical role in chemotherapy resistance.To elucidate the molecular mechanism by which c-JUN promotes AML cell survival and chemotherapy resistance, we performed global gene expression analyses (RNA-seq) of THP-1 cells expressing either non-targeting control or c-JUN shRNAs. From these analyses, we found that c-JUN inhibition results in decreased expression of numerous genes that are activated in response to endoplasmic reticulum (ER) stress, including a significant reduction in the expression of genes regulated by the ER stress-activated transcription factors ATF4 and DDIT3 (p=2.44x10-20).We therefore hypothesized that c-JUN may control ER stress signaling pathways to promote cell survival and chemotherapy resistance in AML. In support of this hypothesis, we have found that c-JUN inhibition blunts the activation of ER-stress related pathways stimulated by chemical inducers of ER stress, thapsigargin and tunicamycin. Additionally, we found that Ara-C treatment activates ER stress-related signaling pathways and that this activation can be reversed by c-JUN inhibition, which correlates with the increased death of c-JUN shRNA-expressing AML cells exposed to Ara-C.To further elucidate the role of ER-stress related pathways in AML, we focused on the serine/threonine kinase, PERK (encoded by EIF2AK3), which activates signaling pathways that aim to ameliorate ER stress and promote cell survival. Similar to c-JUN inhibition, shRNA-mediated inhibition of PERK not only increased cell death but also enhanced the cytotoxicity of Ara-C in AML cells, indicating that this ER stress pathway is a potential therapeutic target to increase the effectiveness of standard chemotherapy.To investigate the therapeutic potential of targeting the PERK pathway, we treated human AML cells with a chemical inhibitor of PERK, GSK2656157, in combination with Ara-C and found that certain concentrations of the two drugs synergistically increased AML cell killing versus each drug alone. Using leukemia cells derived from a mouse model of AML driven by the leukemogenic fusion allele MLL-AF9 alone or in combination with a FLT3 internal tandem duplication, we observed that the combination of GSK2656157 and Ara-C significantly reduced colony growth in methylcellulose by 80% compared with each drug alone (p<0.05).These findings suggest that c-JUN plays a vital role in mitigating ER stress in AML cells and that targeting c-JUN and/or ER stress signaling pathways is a novel potential therapeutic approach for improving the effectiveness of current first-line AML therapies. DisclosuresNo relevant conflicts of interest to declare.

Randomized Phase II Trial of Timed Sequential Cytosine Arabinoside with and without the CHK1 Inhibitor MK-8876 in Adults with Relapsed and Refractory Acute Myelogenous Leukemia

Background: Cytosine arabinoside (ara-C) remains the backbone of most regimens for the treatment of AML. The incorporation of ara-C into DNA activates checkpoint kinase 1 (Chk1) causing replication fork slowing, the accumulation of cells in S phase, and diminished ara-C cytotoxicity. The selective Chk1 inhibitor MK-8776 abrogates ara-C-induced S-phase arrest, thereby enhancing ara-C-induced apoptosis in vitro.1 A phase I trial demonstrated the safety of MK-8876 in combination with infusional ara-C and correlative studies demonstrated increased ara-C-induced H2Ax phosphorylation in AML cells, consistent with unrepaired DNA damage.2 Herein, we report data from a phase II trial comparing ara-C plus MK8876 with ara-C alone.Methods: Patients with relapsed or refractory AML were randomized to receive either ara-C 2 gm/m2 via continuous infusion (CIV) over 72 hours on days 1 and 10 with MK-8776 100 mg flat dose infused over 30 minutes on days 2, 3, 11, and 12 (combo); or ara-C alone on the same schedule (control). The primary end point was response rate (CR+CRi). Adverse events (AEs) for all patients were reported according to the NCI-CTCAE version 4.0. Correlative studies were performed on blood and marrow samples collected prior to treatment on days 1, 2 and 3 and at one hour after MK-8776 infusion (PB only). Marrow and circulating blasts were analyzed using semi-quantitative flow cytometric determinations of blast cell gH2AX expression, a marker of DNA damage.Results: Thirty-two patients were treated at 4 sites with 14 assigned to the combo arm and 18 to the control arm. The study was stopped early due to lack of drug availability. The median age was 60 (range 29-72) and 17 (53%) were male. Nine (28%) had refractory AML, 16 (50%) were in first relapse, and 7 (22%) were in second relapse. Eleven (34%) had undergone a prior bone marrow transplant (BMT). Risk stratification classified 1 (3%) patient as favorable risk disease, 11 (34%) as intermediate-1, 8 (25%) as intermediate-2, and 11 (34%) as adverse risk.There were 5 (36%) responses (CR/CRi) and 1 (7%) partial response (PR) in the combo arm, and 8 (44%) responses and 1 (6%) PR in the control arm. Median survival did not differ significantly between the two groups (5.9 months in the combo arm vs. 4.2 months in the control arm). Sub-group analysis showed increased responses in the combo arm for the patients in first relapse (50% vs. 40%) and those with a prior BMT (50% vs. 29%). Most non-hematologic grade 3/4 treatment-related AEs were similar in the two groups including febrile neutropenia (64% vs. 61%), infectious complications (50% vs. 50%), and sepsis (21% vs. 17%). Patients in the combo arm experienced more QT prolongation (21% vs. 0%), hypertension (21% vs. 0%), and GI toxicities (21% vs. 0%); while those in the control arm had increased renal (0% vs. 11%) and liver toxicity (7% vs. 17%). Two deaths were attributed to treatment, and both were in the control arm.Eight combo patients had serial PB samples and 2 had serial marrow samples studied by flow cytometry. There was an increase in the percentage of gH2AX+ CD34+ cells on Day 2 directly after MK-8876 infusion (16.9% prior and 36.4% at one hour), which occurred to a lesser extent on Day 3 after MK-8876 infusion (12.7% prior and 15.6% at one hour). Three of eight patients demonstrated a sustained increase in gH2AX+ CD34+ from Day 2 to Day 3, and all three achieved either a PR or CR.Conclusion: This is the first phase II study building on prior work suggesting that inhibition of Chk1 would increase response to ara-C in patients with relapsed or refractory AML. The study confirmed that treatment with the combination is feasible with similar toxicities between the treatment and control groups. The response rates were similar between the two groups with improved responses in patients receiving the combo in first relapse and following BMT. Correlative studies support the finding that responding patients show a sustained increase in DNA damage and this may serve as a biomarker in future studies. Chk1 remains a potential target to improve treatment outcomes with ara-C.

RNA expression of genes involved in cytarabine metabolism and transport predicts cytarabine response in acute myeloid leukemia.

Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69-11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015.

Cytarabine preferentially induces mutation at specific sequences in the genome which are identifiable in relapsed acute myeloid leukaemia

Cytarabine (1-β-D-arabinofuranosylcytosine; Ara-C) is an effective remission induction treatment for acute myeloid leukemia (AML).1,2 However, the majority of patients relapse within 3 years, often with aggressive AML2 characterised by the gain of novel base substitution mutations in genes including IDH2 and TET2.3,4 Chemotherapy used to treat AML is predicted to induce mutations detectable in relapsed disease, although direct evidence for this is lacking. The cytotoxicity of Ara-C is mediated in part by incorporation into replicating DNA leading to inhibition of chain extension. However, even at concentrations that induce significant cell death, arabinofuranosylcytosine triphosphate, the reactive metabolite of Ara-C, is primarily incorporated into DNA at internucleotide positions,5,6 suggesting that this nucleoside analogue is likely mutagenic and could contribute to the aetiology of novel somatic mutations acquired during AML relapse.