it is possible to access the histogram in the file from within a define. I wrote this simple example to demonstrate it:
import ROOT
# this file contains a histo called "hpx"
f = ROOT.TFile("hsimple.root")
# We use a feature called "dynamic scopes" to create a variable holding hpx
ROOT.gInterpreter.ProcessLine("auto histo = hpx;")
df = ROOT.ROOT.RDataFrame(3)
df2 = df.Define("hpx_bin", "histo->GetBinContent(rdfentry_)")
m = df2.Mean("hpx_bin")
print(m.GetValue())
Here I used the hsimple.root file in the tutorials directory of ROOT, the hpx histogram contained in it as well the deafult variable rdfentry_.
Let us know how it goes for your example.