Vitamin C Enhances PARPi Efficacy for the Treatment of AML

Abstract Poly-ADP-ribose polymerase inhibitors (PARPi) are currently in clinical trial to determine their therapeutic efficacy for the treatment of acute myeloid leukemia (AML). We have shown that vitamin C (VitC), an essential micronutrient and co-factor of Ten-Eleven translocation (TET) proteins, enhances AML sensitivity to PARPi, potentially due to an increased dependency on base-excision repair (BER) enzymes needed to remove TET-catalyzed oxidized methylcytosine bases via active DNA demethylation. TET2 is the most frequently mutated TET gene in patients with AML, and vitamin C treatment can mimic genetic restoration of TET2 function, leading to DNA demethylation, differentiation, and leukemia cell death. Whether vitamin C efficacy in combination with PARPi depends on the level of TET2 functional alleles is not yet known and may stratify whether TET2 wild-type or mutant patients should be targeted by vitamin C adjuvant therapy. We have generated primary murine AML-ETO9a+ and MLL-AF9+ leukemia models with Tet2 +/+, Tet2 +/- and Tet2 -/- alleles to determine the Tet2-dependent efficacy of PARPi treatment when combined with vitamin C. Furthermore, we have performed CRISPR gene knockout and drug library screening in human AML cell lines in combination with vitamin C treatment, and tested a panel of 10 AML cell lines with titrating concentrations of PARPi (Olaparib, Talazoparib, Veliparib and Rucaparib) alone or in combination with vitamin C (L-ascorbic acid) mimicking physiological to pharmacological in vivo doses. Primary murine AML cells and human cell lines were assayed for colony-forming capacity, differentiation, cell cycling, viability and effects on DNA methylation, levels of oxidized 5-mC and gene expression upon combination treatment in vitro and in vivo. TET2 mutant PDX and primary murine AMLs treated in vivo with L-ascorbate (4g/kg) and Olaparib (50mg/kg) by daily IP injection were also monitored for disease burden, cellular differentiation and survival. Vitamin C is known to drive the TET-catalyzed iterative oxidation of 5-methylcytosine (5-mC) leading to the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). We show that VitC-PARPi combination treatment causes an accumulation of oxidized 5-mC intermediates in the AML genome that correlates with increased yH2AX formation in mid-S phase and cell cycle stalling. Vitamin C reduces the IC 50 of Olaparib and Talazoparib by greater than 10-fold in human AML cells lines and primary murine leukemia cells, and treatment in combination promotes myeloid differentiation and blocks colony-forming capacity greater than either alone. In both our in vitro and in vivo studies, Tet2 +/- AML cells exhibit increased sensitivity to vitamin C treatment alone or in combination with PARPi compared to either Tet2 +/+ or Tet2 -/- cells, suggesting that patients with TET2 haploinsufficiency, which represents the majority of TET2 mutant cases, could benefit the most from combined treatment. Our findings confirm that vitamin C can act synergistically with PARPi to block AML cell viability, reduce colony-forming capacity, and decrease leukemia burden in PDX and primary murine leukemia models in a TET2 allelic dose-dependent manner. The combinatorial effect works at clinically relevant concentrations of PARPi, and low-pharmacological doses of vitamin C. These studies suggest that vitamin C can be used as a non-toxic therapeutic adjuvant to PARPi therapy for the treatment of AML. Disclosures Neel: Northern Biologics, LTD: Current equity holder in publicly-traded company, Other: Co- Founder; SAB: Other: Co-Founder; Navire Pharma: Consultancy, Current equity holder in publicly-traded company; Jengu Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Co-Founder; Arvinas, Inc: Consultancy, Current equity holder in publicly-traded company; Recursion Pharma: Current equity holder in publicly-traded company

Genomic Profiling of Uterine Aspirates and cfDNA as an Integrative Liquid Biopsy Strategy in Endometrial Cancer

The incidence and mortality of endometrial cancer (EC) have risen in recent years, hence more precise management is needed. Therefore, we combined different types of liquid biopsies to better characterize the genetic landscape of EC in a non-invasive and dynamic manner. Uterine aspirates (UAs) from 60 patients with EC were obtained during surgery and analyzed by next-generation sequencing (NGS). Blood samples, collected at surgery, were used for cell-free DNA (cfDNA) and circulating tumor cell (CTC) analyses. Finally, personalized therapies were tested in patient-derived xenografts (PDXs) generated from the UAs. NGS analyses revealed the presence of genetic alterations in 93% of the tumors. Circulating tumor DNA (ctDNA) was present in 41.2% of cases, mainly in patients with high-risk tumors, thus indicating a clear association with a more aggressive disease. Accordingly, the results obtained during the post-surgery follow-up indicated the presence of ctDNA in three patients with progressive disease. Moreover, 38.9% of patients were positive for CTCs at surgery. Finally, the efficacy of targeted therapies based on the UA-specific mutational landscape was demonstrated in PDX models. Our study indicates the potential clinical applicability of a personalized strategy based on a combination of different liquid biopsies to characterize and monitor tumor evolution, and to identify targeted therapies

Oxidized mC modulates synthetic lethality to PARP inhibitors for the treatment of leukemia

TET2 haploinsufficiency is a driving event in myeloid cancers and is associated with a worse prognosis in patients with acute myeloid leukemia (AML). Enhancing residual TET2 activity using vitamin C increases oxidized 5-methylcytosine (mC) formation and promotes active DNA demethylation via base excision repair (BER), which slows leukemia progression. We utilize genetic and compound library screening approaches to identify rational combination treatment strategies to improve use of vitamin C as an adjuvant therapy for AML. In addition to increasing the efficacy of several US Food and Drug Administration (FDA)-approved drugs, vitamin C treatment with poly-ADP-ribosyl polymerase inhibitors (PARPis) elicits a strong synergistic effect to block AML self-renewal in murine and human AML models. Vitamin-C-mediated TET activation combined with PARPis causes enrichment of chromatin-bound PARP1 at oxidized mCs and γH2AX accumulation during mid-S phase, leading to cell cycle stalling and differentiation. Given that most AML subtypes maintain residual TET2 expression, vitamin C could elicit broad efficacy as a PARPi therapeutic adjuvant. [Display omitted] •Vitamin C synergizes with PARPis, causing S phase stalling and AML differentiation•Combination treatment efficacy is dependent on TET2 expression in AML•PARPi treatment with vitamin C causes 5fC accumulation in the genome of AML cells•PARPi treatment with vitamin C enriches for PARP1 binding and γH2AX at 5fC sites Vitamin C treatment can slow the progression of AML by enhancing TET2 activity but is not curative as a single agent therapy. Using genetic and compound screening approaches, Brabson et al. identify a rational combination treatment strategy where vitamin C enhances the therapeutic efficacy of PARPis for AML treatment

Transplantation of human neural progenitor cells secreting GDNF into the spinal cord of patients with ALS: a phase 1/2a trial.

Amyotrophic lateral sclerosis (ALS) involves progressive motor neuron loss, leading to paralysis and death typically within 3-5 years of diagnosis. Dysfunctional astrocytes may contribute to disease and glial cell line-derived neurotrophic factor (GDNF) can be protective. Here we show that human neural progenitor cells transduced with GDNF (CNS10-NPC-GDNF) differentiated to astrocytes protected spinal motor neurons and were safe in animal models. CNS10-NPC-GDNF were transplanted unilaterally into the lumbar spinal cord of 18 ALS participants in a phase 1/2a study (NCT02943850). The primary endpoint of safety at 1 year was met, with no negative effect of the transplant on motor function in the treated leg compared with the untreated leg. Tissue analysis of 13 participants who died of disease progression showed graft survival and GDNF production. Benign neuromas near delivery sites were common incidental findings at post-mortem. This study shows that one administration of engineered neural progenitors can provide new support cells and GDNF delivery to the ALS patient spinal cord for up to 42 months post-transplantation

Grape Processing by High Hydrostatic Pressure: Effect on Microbial Populations, Phenol Extraction and Wine Quality

© 2014, Springer Science+Business Media New York. Vitis vinifera (variety Tempranillo) grapes were subjected to high hydrostatic pressure (HHP) treatments of 200, 400 and 550 MPa for 10 min, and its effect on microbial populations, phenol extraction and wine quality was examined. At ≥400 MPa, the wild yeast population was strongly reduced from 104 to <10 cfu/ml. Bacteria showed greater resistance, and a residual load remained even after the treatment at 550 MPa. The extraction of phenolic compounds from the HHP-treated grapes was improved, with higher concentrations of total phenols obtained compared to crushing alone. Anthocyanin extraction was also increased, producing wines with better colour intensity. These wines also had higher methanol and ethanol contents and returned higher aromatic quality and colour scores. The HHP treatment of grapes may assist in the use of yeast starters, increase phenol extraction from grape skins and improve wine quality.This work was funded by the Ministerio de Economía y Competitividad (AGL2013-40503-R).Peer Reviewe

Epigenetic modulation of adult hippocampal neurogenesis by extremely low-frequency electromagnetic fields.

Throughout life adult neurogenesis generates new neurons in the dentate gyrus of hippocampus that have a critical role in memory formation. Strategies able to stimulate this endogenous process have raised considerable interest because of their potential use to treat neurological disorders entailing cognitive impairment. We previously reported that mice exposed to extremely low-frequency electromagnetic fields (ELFEFs) showed increased hippocampal neurogenesis. Here we demonstrate that the ELFEF-dependent enhancement of hippocampal neurogenesis improves spatial learning and memory. To gain insights on the molecular mechanisms underlying ELFEFs\u2019 effects we extended our studies to an in vitro model of neural stem cells (NSCs) isolated from the hippocampi of newborn mice. We found that ELFEFs enhanced proliferation and neuronal differentiation of hippocampal NSCs by regulation of epigenetic mechanisms leading to pro-neuronal gene expression. Upon ELFEF stimulation of NSCs, we observed a significant enhancement of expression of the pro-proliferative gene Hes1 and the neuronal determination genes NeuroD1 and Neurogenin1. These events were preceded by increased acetylation of H3K9 and binding of the phosphorylated transcription factor cAMP response element-binding protein (CREB) on the regulatory sequence of these genes. Such ELFEF-dependent epigenetic modifications were prevented by the Cav1-channel blocker nifedipine, and were associated with increased occupancy of CREB binding protein (CBP) to the same loci within the analyzed promoters. Our results unravel the molecular mechanisms underlying the ELFEFs\u2019 ability to improve endogenous neurogenesis, pointing to histone acetylation\u2013related chromatin remodeling as a critical determinant. These findings could pave the way to the development of novel therapeutic approaches in regenerative medicin