Targeting epigenetics for treatment of BRAF mutated metastatic melanoma with decitabine in combination with vemurafenib: A phase lb study

Abstract

Introduction: Epigenetic modifications play an important role in progression and development of resistance in ^V600EBRAF positive metastatic melanoma. Therefore, we hypothesized that the action of vemurafenib (BRAF inhibitor) can be made more effective by combining with low dose decitabine (a DNA methyltransferase inhibitor). The primary objective of this phase lb study was to determine the dose limiting toxicity and maximum tolerated dose of combination of subcutaneous decitabine with oral vemurafenib in patients with ^V600EBRAF positive metastatic melanoma with or without any prior treatment.

Experimental design: The study employed 3+3 dose escalation combining subcutaneous decitabine at different doses and schedules (4 cohorts) with the standard oral dose of vemurafenib 960 mg twice daily. Preclinical assessment and further analysis were also performed in A375 melanoma cell line.

Results: Fourteen patients received study treatment. No dose limiting toxicity was encountered and maximum tolerated dose was not reached. Important toxicities included fatigue, increased creatinine, neutropenia, leucopenia, hypophosphatemia, rash and hyperuricemia. Three patients achieved complete response, three had partial response and five had stable disease. Preclinical assessment demonstrated action of the combination which delayed the development of acquired resistance and improved duration of treatment sensitivity.

Conclusions: The combination of oral vemurafenib with subcutaneous decitabine is safe and showed activity in ^V600EBRAF positive metastatic melanoma. Since most responses were seen in cohort 1, which utilized low-dose, long-term decitabine, future studies of this combination treatment should utilize longer duration of decitabine, at the lowest dose of 0.1 mg/kg.

Keywords: BRAF; decitabine; epigenetics; melanoma; vemurafenib.

Figure 1. Patient responses to vemurafenib and decitabine across various cohorts

Figure 2. Individual patient responses with vemurafenib and decitabine

Each line represents a single subject.

Figure 3. Best percentage reduction in target lesions from baseline on treatment with vemurafenib and decitabine

Figure 4

(A) dose-response of A375 to increasing concentrations of decitabine. (B) DNMT1 depletion measured at 72 h at subcytotoxic concentrations of decitabine. (C) H2A.X expression observed at 72 h subcytotoxic concentrations of decitabine and at 24 h exposure to doxorubicin. Expression of both DNMT1 and H2A.X is represented relative to the DMSO treated cells. (D) Population doubling level of A375 cells treated with Vemurafenib (3 uM) and/or Decitabine (10 nM) was measured every 4 days for 113 days. (E, F). Every 4 or 8 days, cell viability in response to increasing concentrations of Vemurafenib was measured for the different branches to evaluate their sensitivity to Vemurafenib. IC50 (E) and cell viability at 1 uM (F) were calculated for each branches. ** = p < 0.01, *** = p < 0.001.

Figure 5. Dose response curve to vemurafenib

At different time point of the experiment (expressed in days), sensitivity to vemurafenib was evaluated in the different A375 cell lines. Cells were treated with 10-fold dilution series (1 nM to 10 uM) of Vemurafenib and cell viability was assessed after 72 h by Crystal violet staining. Cell viability results are expressed in fold vs. the untreated cells. The IC50 were calculated with GraphPadPrism software (GraphPad Software, Inc.).

Figure 6. Overall Study Design (Treatment Schema) A subcutaneous dose of decitabine was administered three times/week at 0.1, 0.2 or 0.3 mg/kg for 2 weeks in cohort 1, 2 and 4 respectively while at 0.3 mg/kg for 1 week in cohort 3

Duration of one cycle was 28 days. Decitabine was given during the first 2 cycles only, while vemurafenib was continued indefinitely until disease progression.