(A) mRNA expression analysis of MDMX in human cancers
using The Cancer Genome Atlas (TCGA) data sets and (B) MDMX and
MDM2 mRNA expression in hematopoietic stem (HSC) and granulocyte-monocytic
progenitor-enriched (GMP) cells from acute myeloid leukemia (AML) patients (n
= 14) and healthy controls (n = 6) (data shown as mean ± SD, where
**P < 0.01, ***P < 0.001; n.s., not
significant). (C) Fluorescence polarization (mP) of a fixed concentration of
ALRN-3618 was measured in the presence of varying concentrations (log M) of mouse double minute
2 homolog (MDM2) and MDMX (top panels) (n = 2). Graphs display varying concentrations of
ALRN-6924 used to displace ALRN-3618 at its EC80 from each target protein (bottom
panels). (D) IC50 and Kl values (n = 2) for each target
protein, as determined from a nonlinear four-parameter curve obtained from plotting
fluorescence polarization versus drug concentration in (C) (bottom). (E)
Sensorgrams for the binding of ALRN-6924 (blue) and RG7388 (red) to surfaces with immobilized
MDM2 (top) and MDMX (bottom) (n = 2). (F) Enzyme-linked immunosorbent assay
demonstrating the expression of human p21 protein (left) (21 hours after treatment with
ALRN-6924) and cellular caspase activity (right) (48 hours after treatment with ALRN-6924)
determined using Caspase-Glo 3/7 assay reagent in SJASA-1 cells (n = 2; data
shown as mean ± SD). ACC, adrenocortical carcinoma; DLBC, diffuse large B cell lymphoma;
PCPG, pheochromocytoma and paraganglioma; RCC, renal cell carcinoma.