Pharmacologic overactivation of ALK activity by PTPN2/PTPN1 inhibition induces a combination of tumor-intrinsic oncogenic stress and immune responses to promote tumor eradication in ALK-positive lymphoma

Abstract

Abstract Introduction Anaplastic Lymphoma Kinase (ALK)-positive Anaplastic Large Cell Lymphoma (ALCL) is characterized by the presence of ALK oncogenic fusions, most frequently the nucleophosmin 1 (NPM1)-ALK fusion. Treatment with standard chemotherapy regimens is curative in about 70% of ALK+ ALCL, while the ALK tyrosine kinase inhibitor (TKI) crizotinib is approved for use in children and young adults with relapsed or refractory disease. Despite the potent activity of crizotinib that strongly inhibitor lymphoma growth and induces complete remission in most patients, ALK+ ALCL is not eradicated by ALK TKIs resulting in development of resistance or relapses in almost half of the patients. Importantly, patients with ALK+ ALCL develop spontaneous anti-ALK immune responses, including ALK-specific CD+ and CD8+ T cells responses, that correlate with prognosis but are not sufficient to achieve tumor eradication. Recently, we discovered that PTPN1 and PTPN2 phosphatases directly regulate the phosphorylation of ALK and genetic knock-out of PTPN1 or PTPN2 is not tolerated by ALK+ ALCL cells due to a toxicity induced by ALK over-signaling. In this work, we evaluated the effects of the pharmacologic inhibition of PTPN2/1 in ALK+ ALCL. We tested in vitro and in vivo in mouse models the inhibitor ABBV-CLS-484 which is currently evaluated in a Phase 1 trial in subjects with locally advanced or metastatic tumors (NCT04777994). Materials and Methods In vitro treatment with the ALK inhibitor crizotinib (TKI) or the PTPN2/PTPN1 phosphatase inhibitor ABBV-CLS-484 (AC484) with increasing concentrations at various time points was performed on ALK+ and ALK- ALCL cell lines. Viability and cell cycle were analyzed by flow cytometry and CellTiter-Glo. Western Blot was performed to characterize ALK and downstream oncogenic signaling activation. Murine ALK+ ALCL cells were injected into syngeneic BALB/c mice and human ALK+ ALCL cell lines or patient-derived xenograft (PDXs) were engrafted in NSG immunodeficient mice. Mice were treated with TKIs or AC484: tumor growth was evaluated by caliper or imaging, and residual tumor cells were assessed by histology and immunohistochemistry. To analyze the ALK-specific immune response, anti-ALK CD8+ T-cells were measured by flow cytometry. Results and Discussion While treatment of human and murine ALK+ ALCL cell lines with ALK TKIs induced the expected blockade of ALK phosphorylation and downstream oncogenic signaling, a completely opposite effect was obtained when treating ALK+ ALCL cell lines with AC484, consistent in a rapid hyperphosphorylation of ALK and its downstream mediators was demonstrated by Western Blotting. AC484 promoted apoptosis and reduction of cell cycle, although with kinetics delayed compared to TKI. ALK-negative ALCL and PTPN1/PTPN2 knock-out ALK+ lymphoma cells were insensitive to AC484, highlighting the specificity of AC484 for ALK and PTPN1/PTPN2. Moreover, ALK+ ALCL cells that become resistant to an ALK TKI were markedly sensible to AC484 treatment, suggesting its use as a therapeutic option for untreatable TKI-resistant ALCL. In vivo, AC484 promoted a significant reduction of tumor growth in human ALK+ ALCL grafted into NSG mice. Remarkably, a complete eradication of syngeneic ALK+ lymphoma cells injected subcutis or systemically in immunocompetent mice was obtained in mice treated with AC484, while crizotinib induced only a complete remission followed by lymphoma relapse. Interestingly, this was concomitant to a significant increase of the spontaneous anti-ALK CD8+ T-cell immune response achieved by AC484 compared to crizotinib. We demonstrated that the enhanced anti-ALK immune response elicited by AC484 treatment contributed to lymphoma eradication because cure was not achieved when the same tumors were injected in immunodeficient mice. Conclusion AC484 has the potential to shift the paradigm of treatment for relapsed/refractory ALK+ ALCL that do not respond to chemotherapy or ALK TKI by exploiting an amplification of oncogenic signaling rather than its inhibition. Remarkably, AC484 amplifies ALK signaling resulting in oncogenic stress and anti-tumor activity selectively in ALK+ ALCL cells, even if resistant to ALK TKI. The anti-tumor activity of AC484 was demonstrated also in vivo, promoting the complete eradication of ALK+ lymphoma in immunocompetent but not in immunodeficient mice, demonstrating the importance of inducing a more potent anti-ALK immune response to achieve cure.