32D

rhTPO Promoted Proliferative Potential of 32D Cells with TPO Receptor Overexpression via Regulating STAT3/STAT5 Expression

Aplastic anemia (AA) serum suppresses proliferation and increases apoptosis of myeloid progenitor 32D cells. Qian's team asked whether recombinant human thrombopoietin (rhTPO) can rescue these defects and clarify the underlying mechanism.

32D cells were infected with lentivirus to overexpress the TPO receptor; Western blot confirmed robust up-regulation (Fig. 1A). CCK-8 assays showed that rhTPO promoted proliferation in a concentration-dependent manner, with maximal effect at 100 U/ml, which was chosen for subsequent experiments. To compare the influence of normal versus aplastic anaemia (AA) serum, four groups were established: (1) normal serum, (2) AA serum, (3) normal serum + 100 U/ml rhTPO, and (4) AA serum + 100 U/ml rhTPO. Trypan-blue exclusion revealed that AA serum almost abolished proliferation at 24, 48 and 72 h, whereas rhTPO increased cell numbers but failed to restore them to the normal-serum level (Fig. 2A). CCK-8 gave the same picture: AA serum strongly inhibited proliferation, and rhTPO only partially reversed this inhibition without reaching normal-serum values (Fig. 2B). They previously observed that AA serum raises p-STAT3 and lowers p-STAT5 during 32D apoptosis. Here, Western blot showed that rhTPO decreased the elevated p-STAT3 and restored the reduced p-STAT5 in AA serum-treated cells (Fig. 2C, D). Thus, rhTPO's ability to rescue proliferation correlates closely with its modulation of STAT3/5 phosphorylation.

Discovery of Novel Heteroaryl Alkynes for Highly Potent KIT^D816V Cells Inhibition to Treat Gastrointestinal Stromal Tumors

80% GIST carry KIT exon 9/11 mutations; imatinib and later TKIs treat them, but secondary ATP-pocket or A-loop mutations-especially D816V-drive pan-resistance. Existing multikinase inhibitors show modest D816V potency and off-target toxicity, leaving a clinical void. Xie's team aims to develop a highly potent, selective agent that robustly suppresses KIT^D816V-driven GIST with minimal wild-type KIT interference.

They previously identified type II o-aminopyridyl alkyne CSF-1R inhibitors (e.g., 13, Fig. 3A). Switch-control theory predicts they can flip KIT D816V back to its inactive state. Because KIT's A-loop is phosphate-independent and type II inhibition is hard to reproduce biochemically, they screened directly on KIT^D816V-transformed 32D cells. Compound 13 showed modest activity (GI₅₀ = 1010 nM). Iterative optimization of linker, tail, central ring and hinge binder then delivered heteroaryl alkynes 54 and 57 with greatly improved potency (Fig. 3A). In 32D-KIT^D816V cells (Fig. 3B), 54 and 57 outperformed ripretinib by abolishing KIT, STAT3 and ERK phosphorylation. Across a panel of clinically relevant KIT mutants (Fig. 3C), both compounds maintained sub-nanomolar to picomolar GI₅₀ values; 54 was 16-19-fold more potent than ripretinib against V559D, V559D/V654A and V559D/N822K. In KIT-wt-amplified lines (NCI-H526, Mo7e, HMC-1) 54/57 showed markedly reduced activity, yielding wide therapeutic windows, and exhibited ≥1428-fold selectivity over EGFRwt or PDGFRαwt-driven cells. Ripretinib, by contrast, retained strong inhibition of KITwt (GI₅₀ ≈ 260 nM) and PDGFRαwt cells, displaying narrower margins. Thus 54/57 promise potent mutant-KIT suppression with diminished on-target wild-type toxicity.