Anaplastic large cell lymphomas (ALCLs) frequently carry oncogenic fusions involving the anaplastic lymphoma kinase (ALK) gene. Targeting ALK using tyrosine kinase inhibitors (TKIs) is a therapeutic option in cases relapsed after chemotherapy, but TKI resistance may develop. By applying genomic loss-of-function screens, we identified PTPN1 and PTPN2 phosphatases as consistent top hits driving resistance to ALK TKIs in ALK+ ALCL. Loss of either PTPN1 or PTPN2 induced resistance to ALK TKIs in vitro and in vivo. Mechanistically, we demonstrated that PTPN1 and PTPN2 are phosphatases that bind to and regulate ALK phosphorylation and activity. In turn, oncogenic ALK and STAT3 repress PTPN1 transcription. We found that PTPN1 is also a phosphatase for SHP2, a key mediator of oncogenic ALK signaling. Downstream signaling analysis showed that deletion of PTPN1 or PTPN2 induces resistance to crizotinib by hyperactivating SHP2, the MAPK, and JAK/STAT pathways. RNA sequencing of patient samples that developed resistance to ALK TKIs showed downregulation of PTPN1 and PTPN2 associated with upregulation of SHP2 expression. Combination of crizotinib with a SHP2 inhibitor synergistically inhibited the growth of wild-type or PTPN1/PTPN2 knock-out ALCL, where it reverted TKI resistance. Thus, we identified PTPN1 and PTPN2 as ALK phosphatases that control sensitivity to ALK TKIs in ALCL and demonstrated that a combined blockade of SHP2 potentiates the efficacy of ALK inhibition in TKI-sensitive and -resistant ALK+ ALCL.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non–small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

Anaplastic Large Cell Lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma frequently driven by the chimeric tyrosine kinase NPM-ALK, generated by the t (2,5)(p23;q35) translocation. While ALK+ ALCL belongs to mature T cell lymphomas, loss of T cell identity is observed in the majority of ALCL secondary to a transcriptional and epigenetic repressive program induced by oncogenic NPM-ALK. While inhibiting the expression of T cell molecules, NPM-ALK activates surrogate TCR signaling by directly inducing pathways downstream the TCR. CD45 is a tyrosine phosphatase that plays a central role in T cell activation by controlling the TCR signaling and regulating the cytokine responses through the JAK/STAT pathway and exists in different isoforms depending on the stage of T-cell maturation, activation and differentiation. ALK+ ALCL cells mainly express the isoform CD45RO in keeping with their mature/memory T cell phenotype. Because of its regulatory effect on the JAK/STAT pathway that is essential for ALK+ ALCL, we investigated whether CD45 expression was affected by oncogenic ALK. We found that most ALK+ ALCL cell lines express the CD45RO isoform with modest CD45RA expression and that NPM-ALK regulated the expression of these CD45 isoforms. Regulation of CD45 expression was dependent on ALK kinase activity as CD45RO expression was increased when NPM-ALK kinase activity was inhibited by treatment with ALK tyrosine kinase inhibitors (TKIs). Silencing ALK expression through shRNA or degradation of ALK by the PROTAC TL13-112 caused upregulation of CD45RO both at mRNA and protein levels with minimal changes on CD45RA, overall indicating that oncogenic ALK downregulates the expression of CD45. CD45 repression was mediated by STAT3 as demonstrated by ChIP-seq data on ALCL cells treated with the ALK-TKI crizotinib or cells treated with a STAT3 degrader. Next, we found that knocking-out CD45 with the CRISPR/Cas9 system resulted in increased resistance to ALK TKI treatment and CD45 was down-regulated in ALCL cells that developed resistance in vitro to ALK TKIs. Overall, these data suggest that CD45 expression is regulated by ALK via STAT3 and acts as a rheostat of ALK oncogenic signaling and resistance to TKI treatment in ALCL.