Oncogenic fusions of the anaplastic lymphoma kinase (ALK) gene drive a subtype of anaplastic large cell lymphoma (ALCL), leading to the constitutive activation of ALK that triggers downstream oncogenic pathways such as MAPK/ERK, JAK/STAT, PI3K/AKT to promote cellular proliferation and survival. We recently discovered PTPN2 and PTPN1 phosphatases as two direct regulators of ALK phosphorylation, that is crucial to provide an optimal signaling in lymphoma cells: any alteration of ALK activation may reduce the cellular fitness. In this context, PTPN2 and PTPN1 are dependance genes in ALK+ ALCL with a mechanism that is not fully elucidated yet.

ALK+ and ALK- ALCL cell lines were treated in vitro with the ALK inhibitor crizotinib (TKI) or the PTPN2/PTPN1 phosphatase inhibitor ABBV-CLS-484 (AC484) with increasing concentrations at various time points. Viability and cell cycle were analyzed by flow cytometry and CellTiter-Glo. ALK and downstream oncogenic signaling activation were assessed by Western Blotting. In vivo studies were performed by injecting murine ALK+ ALCL cells into syngeneic BALB/c mice or by engrafting human ALK+ ALCL cell lines or patient-derived xenografts (PDXs) in NSG immunodeficient mice. Mice were treated with TKI or AC484. Tumor growth was evaluated by caliper or imaging, and residual tumor cells were assessed by histology and immunohistochemistry. Anti-ALK CD8+ T cell immune responses were measured by flow cytometry.

AC484 induced a rapid hyperphosphorylation of ALK and its downstream mediators in all murine or human ALK+ ALCL cell lines, an effect that is opposite to the blockade of ALK and downstream signaling obtained with ALK TKIs. AC484 promoted apoptotic and DNA damage responses, although with kinetics delayed compared to TKI. ALK- ALCL and PTPN1/PTPN2 knock-out ALK+ lymphoma cells were insensitive to AC484, highlighting the specificity of AC484 for ALK and PTPN1/PTPN2. AC484 induced apoptosis in ALK+ ALCL cells that became resistant to TKI, suggesting its use as a therapeutic option for untreatable TKI-resistant ALCL. In vivo, AC484 promoted a marked reduction of tumor growth in human ALK+ ALCL grafted into NSG mice. Remarkably, AC484 induced a complete eradication of ALK+ lymphoma cells injected s.c. or i.v. in immunocompetent mice, concomitant to a potent increase of the anti-ALK CD8+ T cell immune response.

We propose AC484 as one of the first drugs that exploits oncogenic signaling amplification, instead of blockade, as a novel therapeutic approach. AC484 markedly amplifies ALK signaling resulting in oncogenic stress and anti-tumor activity selectively in ALK+ ALCL even if resistant to ALK TKI. In addition, AC484 strongly boosts anti-ALK immune responses suggesting that the immune modulating activity of AC484 could contribute to its curative potential.