The First-in-Class PTPN2/1 Inhibitor Abbv-CLS-484 Disrupts Mitochondrial Renewal and Blocks Tfrc-Mediated PINK1-Prkn-Dependent Mitophagy to Exert Anti-Tumor Activities in ALK-Positive Anaplastic Large Cell Lymphoma
Authors: Liang Jinhua , Wang Weiting , Duan Ziwen , Xing Tong-Yao , Shen Hao-Rui , Yin Hua , Wang Li , Li Jianyong , Wei Xu 
Published: December 2024

Background: Anaplastic large cell lymphoma (ALCL) is a heterogeneous subtype of T-cell lymphoma usually driven by genetic alterations affecting the anaplastic lymphoma kinase (ALK) gene. Although the prognosis is generally favorable, up to 30-40% of ALK+ ALCL patients still suffer from relapses or disease progression. Notably, protein tyrosine phosphatase non-receptor type 2 Gene (PTPN2) has been reported as a cancer target in vivo CRISPR screening (Manguso RT, Nature), and the first-in-class potent PTPN2/1 active-site inhibitor ABBV-CLS-484 (AC484) has shown promising prospects for cancer treatments in solid tumors (NCT04777994). Therefore, the study aims to explore the role and underlying mechanism of PTPN2 and the efficacy of AC484 in ALK+ ALCL.

Methods : Paraffin-embedded tumor sections of ALK+ ALCL patients were collected from the First Affiliated Hospital of Nanjing Medical University with written informed consent. SUDHL1 and Karpas299 are ALK+ ALCL cell lines. Gene knockout was achieved by CRISPR/Cas9 system. RNA sequencing was performed following PTPN2 knockout in Karpas299. All animal studies were approved by the Institutional Animal Care and Use Committee. Wild-type and PTPN2 knockout cells were injected subcutaneously into NOD/SCID male mice. Ten days after injection, AC484 was administered once a day at 3 mg kg-1 for 15 days, by gavage. Transmission electron microscopy (TEM) was used to observe the ultrastructure of the mitochondria. Single-cell RNA sequencing was performed on Karpas299 treated with 5,10,15 μM AC484 for 72 h.

Results:PTPN2 was identified as the essential gene for the growth and survival of ALK+ ALCL based on the results of the whole-genome loss-of-function screens in ALK+ ALCL cells (SUDHL1, Karpas299, KIJK, SUPM2 and DEL). High PTPN2 expression was observed in ALK+ ALCL cell lines and patients. Functional experiments in vitro and in vivo indicated that PTPN2 depletion could suppress tumor cell proliferation, promote apoptosis, and provoke cell cycle arrest. For further investigation, KEGG analysis showed significant enrichment in the Mitophagy pathway following PTPN2 knockout. High magnification of electron micrographs showed the accumulation of damaged swelling mitochondria in PTPN2-deficient tumor cells. LC3 II expression was significantly downregulated in mitochondria by PTPN2 knockout in the presence of Bafilomycin A1 (BafA1), suggesting the inhibition of mitophagy induced by PTPN2 silencing. Since TFRC was reported as a mitochondrial regulator (Senyilmaz D, Nature), we identified that PTPN2 negatively regulated TFRC expression by transcription factor HIF1A and PTPN2 deficiency resulted in PINK1-PRKN-mediated mitophagy inhibition and damaged mitochondria clearance through disinhibiting TFRC expression, leading to cell death in ALK+ ALCL. The process was ferroptosis-independent. As previously reported (Karaca AE, Blood), PTPN1 has no effect on tumor growth and survival in ALK+ ALCL.

AC484 was introduced due to the good application potential and prospects in tumor treatment. The IC50 for 72h was estimated to be 6.285 μM on SUDHL1 and 5.838 μM on Karpas299. The mice in the AC484-treated group showed significant tumor growth rate and tumor regression. To further explore the therapeutic potential of AC484, we found the negative effects of AC484 in the mitochondria. The Mitophagy activity was scored using AUCell analysis of single-cell RNA sequencing following AC484 treatment and cells in AC484 treat groups had lower scores, indicating poor activation in mitophagy. TEM analysis observed damaged swelling mitochondria aggregated in tumor cells treated with AC484 in mouse xenograft models. AC484 also reduced the aggregation of LC3 caused by BafA1 and inhibited the PINK1-PRKN pathway with an increase of TFRC expression which indicated AC484 exerted anti-tumor activities by TFRC-mediated PINK1-PRKN-dependent mitophagy.

Conclusion :In this study, we demonstrated that PTPN2 played cancer-promoting effects by TFRC-mediated PINK1-PRKN-dependent mitophagy in vitro and in vivo, independent of ferroptosis. Furthermore, the first-in-class PTPN2/1 inhibitor AC484 showed a great anti-tumor effect against ALK+ ALCL by disrupting mitochondrial function and mitophagy. Thus, we provide insight into the selection of treatment options for ALK+ ALCL with poor prognosis and provide the basis for the conduct of clinical trials on AC484.