Brett Ewald

Nucleoside analogs: molecular mechanisms signaling cell death.

Nucleoside analogs are structurally similar antimetabolites that have a broad range of action and are clinically active in both solid tumors and hematological malignancies. Many of these agents are incorporated into DNA by polymerases during normal DNA synthesis, an action that blocks further extension of the nascent strand and causes stalling of replication forks. The molecular mechanisms that sense stalled replication forks activate cell cycle checkpoints and DNA repair processes, which may contribute to drug resistance. When replication forks are not stabilized by these molecules or when subsequent DNA repair processes are overwhelmed, apoptosis is initiated either by these same DNA damage sensors or by alternative mechanisms. Recently, strategies aimed at targeting DNA damage checkpoints or DNA repair processes have demonstrated effectiveness in sensitizing cells to nucleoside analogs, thus offering a means to elude drug resistance. In addition to their DNA synthesis-directed actions many nucleoside analogs trigger apoptosis by unique mechanisms, such as causing epigenetic modifications or by direct activation of the apoptosome. A review of the cellular and molecular responses to clinically relevant agents provides an understanding of the mechanisms that cause apoptosis and may provide rationale for the development of novel therapeutic strategies.

H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation

Gemcitabine is a nucleoside analogue that is incorporated into replicating DNA, resulting in partial chain termination and stalling of replication forks. The histone variant H2AX is phosphorylated on Ser139 (γ-H2AX) and forms nuclear foci at sites of DNA damage. Here, we characterize the concentration- and time-dependent phosphorylation of H2AX in response to gemcitabine-induced stalled replication forks. The number of γ-H2AX foci increased with time up to 2 to 6 h after exposure to gemcitabine, whereas longer exposures did not cause greater phosphorylation or increase cell death. The percentage of γ-H2AX–positive cells increased with concentrations of gemcitabine up to 0.1 μmol/L, and γ-H2AX was most evident in the S-phase fraction. Phosphorylation of ataxia-telangiectasia mutated (ATM) on Ser1981 was also associated with S-phase cells and colocalized in the nucleus with phosphorylated H2AX foci after gemcitabine exposure. Chemical inhibition of ATM, ATM- and Rad3-related, and DNA-dependent protein kinase blocked H2AX phosphorylation. H2AX and ATM phosphorylation were associated with inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1/Cdc25A/cyclin-dependent kinase 2). Exposure of previously gemcitabine-treated cultures to the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) caused a 10-fold increase in H2AX phosphorylation, which was displayed as an even pan-nuclear staining. This increased phosphorylation was not due to apoptosis-induced DNA fragmentation and was associated with the S-phase fraction and decreased reproductive viability. Thus, H2AX becomes phosphorylated and forms nuclear foci in response to gemcitabine-induced stalled replication forks, and this is greatly increased upon checkpoint abrogation. [Mol Cancer Ther 2007;6(4):1239–48]

Specific activation of microRNA106b enables the p73 apoptotic response in chronic lymphocytic leukemia by targeting the ubiquitin ligase Itch for degradation

Chronic lymphocytic leukemia (CLL) is characterized by cells that exhibit dysfunctional apoptosis. Here, we show that deacetylase inhibition led to the E2F1- and myc-mediated transcriptional activation of the microRNA miR106b in primary CLL cells. Induction of miR106b was associated with a down-regulation in the levels of the E3-ubiquitin ligase Itch. Decreases in Itch protein levels were associated with a reciprocal accumulation of its proapoptotic substrate, TAp73 (p73), and induction of p53 up-regulated modulator of apoptosis (PUMA) mRNA and protein. This event was accompanied by mitochondrial dysfunction, processing of caspase-9, and apoptosis of CLL cells. Ectopic expression of miR106b in CLL cells demonstrated that Itch was a direct target of miR106b such that miR106b-induced decreases in Itch resulted in an accumulation of p73. Thus, our results identify a novel regulatory mechanism wherein microRNA regulate cell survival by mediating the posttranscriptional down-regulation of an ubiquitin ligase, leading to the induction of a proapoptotic regulator in malignant cells. Silencing of miRNA expression in CLL may selectively suppress proapoptotic pathways, providing such tumors with a survival advantage. Consequently, chemotherapeutic drugs that activate miR106b could initiate a p53-independent mechanism that targets CLL cells.

Phase I Study of DNX-2401 (Delta-24-RGD) Oncolytic Adenovirus: Replication and Immunotherapeutic Effects in Recurrent Malignant Glioma

Purpose DNX-2401 (Delta-24-RGD; tasadenoturev) is a tumor-selective, replication-competent oncolytic adenovirus. Preclinical studies demonstrated antiglioma efficacy, but the effects and mechanisms of action have not been evaluated in patients. Methods A phase I, dose-escalation, biologic-end-point clinical trial of DNX-2401 was conducted in 37 patients with recurrent malignant glioma. Patients received a single intratumoral injection of DNX-2401 into biopsy-confirmed recurrent tumor to evaluate safety and response across eight dose levels (group A). To investigate the mechanism of action, a second group of patients (group B) underwent intratumoral injection through a permanently implanted catheter, followed 14 days later by en bloc resection to acquire post-treatment specimens. Results In group A (n = 25), 20% of patients survived > 3 years from treatment, and three patients had a ≥ 95% reduction in the enhancing tumor (12%), with all three of these dramatic responses resulting in > 3 years of progression-free survival from the time of treatment. Analyses of post-treatment surgical specimens (group B, n = 12) showed that DNX-2401 replicates and spreads within the tumor, documenting direct virus-induced oncolysis in patients. In addition to radiographic signs of inflammation, histopathologic examination of immune markers in post-treatment specimens showed tumor infiltration by CD8+ and T-bet+ cells, and transmembrane immunoglobulin mucin-3 downregulation after treatment. Analyses of patient-derived cell lines for damage-associated molecular patterns revealed induction of immunogenic cell death in tumor cells after DNX-2401 administration. Conclusion Treatment with DNX-2401 resulted in dramatic responses with long-term survival in recurrent high-grade gliomas that are probably due to direct oncolytic effects of the virus followed by elicitation of an immune-mediated antiglioma response.

HDAC Inhibition Induces MicroRNA-182, which Targets RAD51 and Impairs HR Repair to Sensitize Cells to Sapacitabine in Acute Myelogenous Leukemia.

Purpose: The double-strand breaks elicited by sapacitabine, a clinically active nucleoside analogue prodrug, are repaired by RAD51 and the homologous recombination repair (HR) pathway, which could potentially limit its toxicity. We investigated the mechanism by which histone deacetylase (HDAC) inhibitors targeted RAD51 and HR to sensitize acute myelogenous leukemia (AML) cells to sapacitabine. Experimental Design: Chromatin immunoprecipitation identified the role of HDACs in silencing miR-182 in AML. Immunoblotting, gene expression, overexpression, or inhibition of miR-182 and luciferase assays established that miR-182 directly targeted RAD51. HR reporter assays, apoptotic assays, and colony-forming assays established that the miR-182, as well as the HDAC inhibition–mediated decreases in RAD51 inhibited HR repair and sensitized cells to sapacitabine. Results: The gene repressors, HDAC1 and HDAC2, became recruited to the promoter of miR-182 to silence its expression in AML. HDAC inhibition induced miR-182 in AML cell lines and primary AML blasts. miR-182 targeted RAD51 protein both in luciferase assays and in AML cells. Overexpression of miR-182, as well as HDAC inhibition–mediated induction of miR-182 were linked to time- and dose-dependent decreases in the levels of RAD51, an inhibition of HR, increased levels of residual damage, and decreased survival after exposure to double-strand damage-inducing agents. Conclusions: Our findings define the mechanism by which HDAC inhibition induces miR-182 to target RAD51 and highlights a novel pharmacologic strategy that compromises the ability of AML cells to conduct HR, thereby sensitizing AML cells to DNA-damaging agents that activate HR as a repair and potential resistance mechanism. Clin Cancer Res; 22(14); 3537–49. ©2016 AACR.

Killing of chronic lymphocytic leukemia by the combination of fludarabine and oxaliplatin is dependent on the activity of XPF endonuclease

Purpose: Chronic lymphocytic leukemia (CLL) resistant to fludarabine-containing treatments responds to oxaliplatin-based therapy that contains fludarabine. We postulated that a mechanism for this activity is the incorporation of fludarabine into DNA during nucleotide excision repair (NER) stimulated by oxaliplatin adducts. Experimental Design: We analyzed CLL cell viability, DNA damage, and signaling pathways in response to treatment by fludarabine, oxaliplatin, or the combination. The dependency of the combination on oxaliplatin-induced DNA repair was investigated using siRNA in CLL cells or cell line models of NER deficiency. Results: Synergistic apoptotic killing was observed in CLL cells after exposure to the combination in vitro. Oxaliplatin induced DNA synthesis in CLL cells, which was inhibited by fludarabine and was eliminated by knockdown of XPF, the NER 5′-endonuclease. Wild-type Chinese hamster ovarian cells showed synergistic killing after combination treatment, whereas only additive killing was observed in cells lacking XPF. Inhibition of repair by fludarabine in CLL cells was accompanied by DNA single-strand break formation. CLL cells initiated both intrinsic and extrinsic apoptotic pathways as evidenced by the loss of mitochondrial outer membrane potential and partial inhibition of cell death upon incubation with FasL antibody. Conclusions: The synergistic cell killing is caused by a mechanistic interaction that requires the initiation of XPF-dependent excision repair in response to oxaliplatin adducts, and the inhibition of that process by fludarabine incorporation into the repair patch. This combination strategy may be useful against other malignancies. Clin Cancer Res; 17(14); 4731–41. ©2011 AACR.

Abstract LB-235: Delta-24-RGD oncolytic adenovirus treatment downmodulates the key regulator of T-cell exhaustion TIM3 in malignant gliomas

T cell exhaustion is a key mechanism of tumor immune suppression and is commonly observed in several cancers. The exhausted T cells in the tumor microenvironment overexpress inhibitory receptors, show decreased effector cytokine production, and exhibit diminished cytolytic capabilities, resulting in the failure to eliminate cancer cells. Restoring the activity of exhausted T cells is a desirable strategy to improve immune-based anti-cancer therapies, and has yielded encouraging results. Exhausted T cells are characterized by the expression of high levels of PD-1 and TIM3, which cooperate to induce T-cell hyporesponsiveness. In this study, we sought to determine whether the infection of human gliomas with oncolytic viruses modifies the expression of T cell markers. As part of a phase 1, dose-escalation, biological-endpoint clinical study (NCT00805376) of Delta-24-RGD, a Rb-targeted, tumor-selective, replication-competent oncolytic adenovirus, conducted in 37 patients with recurrent malignant glioma, 12 patients received an intratumoral injection through an implanted catheter, followed 14-days later by en bloc resection and Delta-24-RGD injections into the post-resection tumor bed. 10 surgical post-treatment specimens and 5 pre-treated tumors were examined for markers of immune response. From all selected cases, formalin fixed, paraffin embedded tissue blocks were examined. IHC was performed using a Leica Bond Max automated stainer as previously described. The slides were scanned in an Aperio AT2 scanner (Leica Biosystems). The IHC scores for CD3, CD4, CD8, CD45Ro, FOXP3, TIM3, LAG-3, VISTA, CD20, CD57, CD68, and OX40 were expressed as cell density in the inflammatory cell population (number of positive cells per mm square of tumor area), except PD-L1, PD-L2, B7-H3, B7-H4, IDO-1, which were expressed using percentage of positive tumor cells, and also as H-Score of the tumor cells (which integrates percentage of tumor cells and intensity of staining, with a final H-score ranging from 0 to 300). Correlations between immunostaining marker values and survival and dose level were assessed using Spearman rank correlation analysis. Differences in immunostaining marker values before and after treatment were assessed using the Wilcoxon rank sum test. These analyses demonstrated that all the target proteins are expressed in gliomas in different patterns and tumor regions. Although, we did not observe significant treatment-related changes in the expression of PD-1 in lymphocytes and PD-L1 in glioma cells, TIM3, a marker for T cell exhaustion, was downregulated after Delta-24-RGD treatment. In addition, expression of B7-H3, a marker of poor prognosis in other tumors, correlated with poorer survival. Because PD-1 and TIM3 cooperate to maintain T-cell exhaustion our data provide a rationale for the combination of Delta-24-RGD and anti-PD-1 antibodies for the treatment of malignant gliomas. Importantly, a multicenter clinical trial to test this strategy has already been open (NCT02798406). Citation Format: Juan Fueyo, Candelaria Gomez-Manzano, Pamela Villalobos, Jaime Rodriguez-Canales, Barbara Mino, Ignacio Wistuba, Kenneth Hess, Marta Alonso, Sonia Tejada, Ricardo Diez-Valle, Brett Ewald, Frank Tufaro, Frederick Lang. Delta-24-RGD oncolytic adenovirus treatment downmodulates the key regulator of T-cell exhaustion TIM3 in malignant gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-235. doi:10.1158/1538-7445.AM2017-LB-235

Abstract 5379: Mechanism of fludarabine and oxaliplatin combined activity depends on the activity of XPF endonuclease

We set out to determine the mechanism by which fludarabine and oxaliplatin exert more than additive cell killing in chronic lymphocytic leukemia (CLL). We postulate that enhanced activity of oxaliplatin and fludarabine combination is based on termination by fludarabine of DNA re-synthesis associated with nucleotide excision repair (NER) following removal of oxaliplatin adducts. The blocked repair would result in single-strand and double-strand DNA breaks which would activate cell DNA damage signaling and apoptosis. More than additive apoptotic killing was observed in 49% (±22) of CLL cells after exposure to oxaliplatin and fludarabine in vitro, although single agents showed minimal increase in cell death (3% ±9 for oxaliplatin, 13% ±14 for fludarabine), as demonstrated by Annexin V and PI staining at 24 hr (expected vs. observed p=0.0003; n=10). Oxaliplatin induced a 2-fold increase in DNA synthesis in CLL cells, which was inhibited by fludarabine, as determined by thymidine incorporation (n=5). Knockdown of XPF, the NER 5′ endonuclease, in CLL patient samples (n=3) eliminated oxaliplatin induced DNA synthesis (3-fold increase in thymidine incorporation with scramble siRNA compared to 1.4-fold increase with XPF siRNA). Wild type CHO (AA8) cells showed more than additive killing after combination treatment similar to the observation with CLL patient samples (predicted survival 50%; observed 21%). In contrast, only additive killing was observed in CHO cells lacking XPF (UV41) with equitoxic doses of oxaliplatin and fludarabine (predicted survival 56%; observed 54%) suggesting that XPF function is essential for the cooperative interaction of the combination. Alkaline assays demonstrated that inhibition of repair by fludarabine was accompanied by DNA strand break formation within 12 hr (n=4) in CLL patients samples. The increase in Olive tail moment ranged from 2- to 5-fold, confirming induction of single-strand breaks. The double-strand break repair proteins ATM, SMC1, H2AX and DNA-PKcs were phosphorylated as early as 2 hr post-treatment with the combination and persisted up to 24 hr (3-fold, 24-fold, 10-fold and 10-fold increase, respectively; n=4). This response suggests that DNA damage generated by fludarabine and oxaliplatin exposure was processed to double-strand DNA breaks in CLL cells. Our work provides evidence that the combination of oxaliplatin and fludarabine results in the formation of toxic DNA damage and that their combined activity depends on a functional NER pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5379.