Ponatinib (AP24534)

Cell lines

SW26 (ALT-positive) and SW39 (telomerase-positive) isogenic cell lines derived from IMR90; osteosarcoma (OS) cell lines (SAOS-2, U2OS, T1000, CAL72); well-differentiated liposarcoma (LPS) cell lines; telomerase-positive cell lines (HT161, HOS); normal lung-derived IMR90 fibroblasts

Preparation Method

The cells were used in a comparative anti-cancer compound library screen and subsequent validation assays. ALT cell lines used in these experiments were ATRX-deficient. ALT cell lines were treated with Ponatinib at 125-250nM.

Reaction Conditions

125-250nM; 24-72h

Applications

Ponatinib exhibited enhanced killing of ALT-positive cells compared to telomerase-positive cells. Ponatinib induced an increase in extrachromosomal telomeric C-circles (a marker of ALT activity) in all tested ALT cell lines after 72 hours. Ponatinib provoked telomeric dysfunction, increased levels of telomere dysfunction-induced foci (TIFs), and caused specific telomere damage and replicative stress (evidenced by colocalization of pS33 RPA and telomeric DNA). Ponatinib interfered with telomeric replication and synthesis, decreasing newly BrdU-labelled telomeres and reducing ALT telomere DNA synthesis in APBs (ATSA assay). Ponatinib treatment also induced DNA damage (increased γH2AX), promoted the formation of large telomere foci, and triggered senescence in ALT cell lines. Ponatinib inhibited an ABL1-JNK-JUN signaling circuit, reducing phosphorylation of JUN at Serine 63 and Threonine 91/93 and decreasing total JUN protein levels.

Animal models

Female C57BL/6J mice (for B16-F10 melanoma and GL261 glioblastoma models) and BALB/c mice (for 4T1 breast carcinoma model), 6-8 weeks old

Preparation Method

B16-F10 melanoma tumour model was established by injecting 2×10⁵ cells with matrigel subcutaneously into the right flank of C57BL/6J mice. 4T1 breast carcinoma model was established by injecting 8×10⁵ cells in the mammary pad of BALB/c female mice. GL261 glioblastoma model was established by implanting 1×10⁶ cells in the right flank of C57BL/6 mice. Once the tumour volume reached 50–100mm³, mice were randomly divided into groups and the Ponatinib (15mg/kg) was administered daily for 10 days.

Dosage form

15mg/kg; i.p.; daily for 10day

Applications

Ponatinib treatment significantly delayed the growth of B16-F10 melanoma and 4T1 breast tumours and reduced tumour weights. Ponatinib enhanced CD8⁺ T cell infiltration in the tumour microenvironment (TME), downregulated PD-L1 expression and downstream signalling molecules (p-P38, p-Erk, p-JNK), increased cleaved caspase-3 expression, regulated the Th1/Th2 balance (increased IFN-γ, decreased IL-4, increased Th1/Th2 ratio), increased IL-2 levels, lowered FoxP3 expression, and depleted tumour-associated macrophages (TAMs). Ponatinib also induced a favourable systemic antitumour immunity by enhancing CD8⁺ T cell population, tumour-specific CTL activity, balancing the Th1/Th2 ratio, and lowering PD-L1 expression.

References:
[1] Kusuma FK, Prabhu A, Tieo G, et al. Signalling inhibition by ponatinib disrupts productive alternative lengthening of telomeres (ALT). Nat Commun. 2023 Apr 6;14(1):1919.
[2] Barnwal A, Tamang R, Sanjeev Das, et al. Ponatinib delays the growth of solid tumours by remodelling immunosuppressive tumour microenvironment through the inhibition of induced PD-L1 expression. Br J Cancer. 2023 Oct;129(6):1007-1021.