USEOFFLOWCYTOMETRYTOSTUDY DRUG TARGET INHIBITION IN LABORATORY ANIMALS AND IN EARLY-PHASE CLINICAL TRIALS - Flow Cytometry in Drug Discovery and Development - page 152

(a)

Untreated Sorafenib GM-CSF

P-STAT5 PE

P atient 1 P atient 2 P atient 3

(b)

Control

U0126

Rapamycin

U0126 +
rapamycin

SCF

P-ERK

FIGURE 7.1 (a) Constitutive STAT5 phosphorylation in peripheral blasts of a patient with
acute myeloid leukemia carrying a Flt-3 internal tandem duplication. Aliquots of the sample
were incubated with the Flt-3 inhibitor sorafenib, or activated using GM-CSF, and then fixed
and processed as described in the text. Blast cells were identified using anti-CD34 and color
gated. The blast cells have a side scatter that is intermediate between lymphocytes and
granulocytes, and their P-STAT5 fluorescence is relatively high in the untreated control sample.
The intensity decreases to that of the granulocytes in the presence of sorafenib, consistent with
the ITD mutation activating P-STAT5, whereas GM-CSF activates STAT5 in the granulocytes
via JAK, so that their intensity is similar to that of the blast cells. This effect can be used as a
positive control. (b) Constitutive activation of S6 ribosomal protein in peripheral blast cells
(color gated) from three AML patients. Note the considerable intercellular heterogeneity
of P-S6, which ranges over two decades in all three cases. The MEK inhibitor U0126 decreases
P-S6 in patients 2 and 3 and P-ERK in all three cases. The mTOR inhibitor rapamycin decreases
P-S6 in patient 1, but not the other cases, whereas the combination of U0126 and rapamycin
reduces P-S6 to baseline in all three cases. A treatment protocol incorporating a rapamycin
analogue might therefore be more effective in patient 1, and is predicted to cause a decrease
in the constitutive P-S6 levels that could be monitored using this assay.

There is currently interest in agents that inhibit the PI3 kinase/Akt pathway, since
this is aberrantly activated in many cancers, and a need for effective pharmacody-
namic monitoring of these agents, given the importance of this pathway in the
maintenance of normal tissues. Having been unsuccessful in attempts to develop a
surrogate blood marker for PI3 kinase inhibition based on the use of phosphospecific