A Randomized Phase II Trial of Epigenetic Priming with Guadecitabine and Carboplatin in Platinum-resistant, Recurrent Ovarian Cancer.

PURPOSE: Platinum resistance in ovarian cancer is associated with epigenetic modifications. Hypomethylating agents (HMA) have been studied as carboplatin resensitizing agents in ovarian cancer. This randomized phase II trial compared guadecitabine, a second-generation HMA, and carboplatin (G+C) against second-line chemotherapy in women with measurable or detectable platinum-resistant ovarian cancer. PATIENTS AND METHODS: Patients received either G+C (guadecitabine 30 mg/m2 s.c. once-daily for 5 days and carboplatin) or treatment of choice (TC; topotecan, pegylated liposomal doxorubicin, paclitaxel, or gemcitabine) in 28-day cycles until progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS); secondary endpoints were RECIST v1.1 and CA-125 response rate, 6-month PFS, and overall survival (OS). RESULTS: Of 100 patients treated, 51 received G+C and 49 received TC, of which 27 crossed over to G+C. The study did not meet its primary endpoint as the median PFS was not statistically different between arms (16.3 weeks vs. 9.1 weeks in the G+C and TC groups, respectively; P = 0.07). However, the 6-month PFS rate was significantly higher in the G+C group (37% vs. 11% in TC group; P = 0.003). The incidence of grade 3 or higher toxicity was similar in G+C and TC groups (51% and 49%, respectively), with neutropenia and leukopenia being more frequent in the G+C group. CONCLUSIONS: Although this trial did not show superiority for PFS of G+C versus TC, the 6-month PFS increased in G+C treated patients. Further refinement of this strategy should focus on identification of predictive markers for patient selection

First-in-Human Study of AT13148, a Dual ROCK-AKT Inhibitor in Patients with Solid Tumors

Purpose: AT13148 is an oral AGC kinase inhibitor, which potently inhibits ROCK and AKT kinases. In preclinical models, AT13148 has been shown to have antimetastatic and antiproliferative activity. Patients and Methods: The trial followed a rolling six design during dose escalation. An intrapatient dose escalation arm to evaluate tolerability and a biopsy cohort to study pharmacodynamic effects were later added. AT13148 was administered orally three days a week (Mon–Wed–Fri) in 28-day cycles. Pharmacokinetic profiles were assessed using mass spectrometry and pharmacodynamic studies included quantifying p-GSK3β levels in platelet-rich plasma (PRP) and p-cofilin and p-MLC2 levels in tumor biopsies. Results: Fifty-one patients were treated on study. The safety of 5–300 mg of AT13148 was studied. Further, the doses of 120–180–240 mg were studied in an intrapatient dose escalation cohort. The dose-limiting toxicities included hypotension (300 mg), pneumonitis, and elevated liver enzymes (240 mg), and skin rash (180 mg). The most common side effects were fatigue, nausea, headaches, and hypotension. On the basis of tolerability, 180 mg was considered the maximally tolerated dose. At 180 mg, mean Cmax and AUC were 400 nmol/L and 13,000 nmol/L/hour, respectively. At 180 mg, ≥50% reduction of p-cofilin was observed in 3 of 8 posttreatment biopsies. Conclusions: AT13148 was the first dual potent ROCK-AKT inhibitor to be investigated for the treatment of solid tumors. The narrow therapeutic index and the pharmacokinetic profile led to recommend not developing this compound further. There are significant lessons learned in designing and testing agents that simultaneously inhibit multiple kinases including AGC kinases in cancer

Decitabine treatment of GL261 glioma increases CTL-mediated killing and enhances CTL reactivation.

<p>(A) 4h killing induced by OT1 or Pmel-1 CTLs on GL261-OVA GDCs (GDCs) and GL261-OVA GICs (GICs). Target cells were plated for 48h with DAC (10μM) and then labeled with CFSE. When target cells were incubated with Pmel-1 CTLs, they were pulsed 1h with 1μg/ml gp100 peptide before the assay. The Effector:Target (E:T) ratio used was 10:1. Live cells were discriminated by LIVE/DEAD fixable yellow dead cell stain. Error bars represent SD. *p<0.05;**p<0.01;***p<0.001, paired t-test, n = 6. (B) 20h killing induced by Pmel-1 CTLs on GL261-OVA GDCs (GDCs) and GL261-OVA GICs (GICs). Target cells were pulsed 1h with 1μg/ml gp100 peptide before the assay. The protocol is described in (A). Error bars represent SD. *p<0.05;**p<0.01;***p<0.001, Paired t-test performed between untreated and treated groups; unpaired t-test performed between GICs and GDCs, n = 3 (C) OT1 and Pmel-1 CTL increase in cell division number after incubation with irradiated GL261-OVA GDCs (GDCs) or GL261-OVA GICS (GICs) calculated as difference in cell division number compared with CTLs incubated alone. CTLs were labeled with CFSE and co-cultured with irradiated GDCs or GICs that were previously treated with Decitabine (10μM, 48h). The number of T cell divisions was assessed by cytofluorometry (CFSE fluorescent peaks of gated events). CTLs were identified by CD8 staining. As positive control, anti-CD3/CD28-coated Dynabeads (Beads αCD3/CD28) were used. Error bars represent SD.*p<0.05;**p<0.01;***p<0.001, paired t-test, n = 3 (D) IFN-γ secretion by OT1 CTLs after incubation with GL261-OVA GDCs (GDCs) or GL261-OVA GICS (GICs). Values were normalised to positive controls (181 ng/ml +/- 84.9). Error bars represent SD.*p<0.05;**p<0.01;***p<0.001, paired t-test, n = 3.</p

GL261 MHCI, ICAM-1 and PDL1 expression is impacted by decitabine.

<p>In the left panels, a representative histogram of GL261-OVA GDC (GDCs) and GL261-OVA GIC (GICs) expression of MHCI (H-2Kb) (A), ICAM-1 (B), and PDL1 (C) is shown (DAC used at 10μM, 48h incubation, both isotype control and antibody staining are shown). Live cells were gated. In the right panels, expression of each molecule (calculated as MFIR) by GL261-OVA GDCs (GDCs) and GL261-OVA GICs (GICs) after 48h DAC treatment is shown. Error bars represent SD. One-way Anova test (Dunnet’s multiple comparison test) was performed to compare the expression of MHCI, ICAM-1 or PDL1 by the same cell type at different DAC concentrations; unpaired t-test was performed to compare the expression of MHCI, ICAM-1 or PDL1 between the two cell types; p<0.05;**p<0.01;***p<0.001, n = 3.</p

Decitabine Treatment of Glioma-Initiating Cells Enhances Immune Recognition and Killing

Malignant gliomas are aggressive brain tumours with very poor prognosis. The majority of glioma cells are differentiated (glioma-differentiated cells: GDCs), whereas the smaller population (glioma-initiating cells, GICs) is undifferentiated and resistant to conventional therapies. Therefore, to better target this pool of heterogeneous cells, a combination of diverse therapeutic approaches is envisaged. Here we investigated whether the immunosensitising properties of the hypomethylating agent decitabine can be extended to GICs. Using the murine GL261 cell line, we demonstrate that decitabine augments the expression of the death receptor FAS both on GDCs and GICs. Interestingly, it had a higher impact on GICs and correlated with an enhanced sensitivity to FASL-mediated cell death. Moreover, the expression of other critical molecules involved in cognate recognition by cytotoxic T lymphocytes, MHCI and ICAM-1, was upregulated by decitabine treatment. Consequently, T-cell mediated killing of both GDCs and GICs was enhanced, as was T cell proliferation after reactivation. Overall, although GICs are described to resist classical therapies, our study shows that hypomethylating agents have the potential to enhance glioma cell recognition and subsequent destruction by immune cells, regardless of their differentiation status. These results support the development of combinatorial treatment modalities including epigenetic modulation together with immunotherapy in order to treat heterogenous malignancies such as glioblastoma