Abstract LT018: The perivascular niche protects ALK+ lymphoma cells from ALK inhibition through the CCL19/21-CCR7 axis

The ALK inhibitor crizotinib showed promising therapeutic efficacy for relapsed/refractory Anaplastic Large Cell Lymphoma (R/R ALCL). However, a fraction of ALK+ R/R ALCL patients do not achieve complete remission due to crizotinib resistance that develops within the first 3 months of therapy. In patients that achieve complete remission, crizotinib discontinuation causes rapid disease relapses due to the expansion of persister lymphoma cells never completely eradicated by the ALK inhibitor. There is, in fact, growing evidence that ALK+ ALCL can persist for years in patients being undetectable. ALCL grows around blood and lymphatic vessels in the lymph node. We hypothesize that this perivascular niche provides pro-survival signals contributing to ALK+ ALCL persistence and TKI resistance. By RNA-seq analysis on ALK+ ALCL cells and scRNA-seq analysis in one ALK+ ALCL primary sample, we found that ALK+ cells expressed the C-C chemokine receptor type 7 (CCR7), while endothelial cells and fibroblasts expressed the unique CCR7 ligands, the chemokine (C-C motif) ligand 19 (CCL19) and 21 (CCL21). Therefore, we explored whether the CCL19/21-CCR7 chemokine-receptor signaling axis could be involved in the persistence of ALK+ ALCL cells during ALK inhibitor treatment. We show that treatment with crizotinib caused upregulation of CCR7 in ALK+ ALCL cells via STAT3, as demonstrated by ChIP-seq data. Besides, stimulation of ALK+ ALCL cells with both CCL19/21 potently activated the MAPK signaling and sustained MAPK activation during ALK inhibition by crizotinib. Mechanistically, we demonstrate that this MAPK activation was mediated by PI3Kγ-dependent CCR7 signaling. This effect was more marked in human ALK+ ALCL cells that express high levels of PI3Kγ, while it was strongly reduced in murine lymphoma PI3KγKO cells, generated from NPM-ALK transgenic mice crossed with PI3KγKO mice. Treatment with the PI3Kγ/δ dual inhibitor duvelisib abrogated the MAPK phosphorylation induced by CCL19/21. When we knocked-out the CCR7 gene via CRISPR/Cas9, human ALK+ ALCL showed markedly reduced activation of the MAPK pathway upon stimulation with CCL19/21. Next, we developed a microphysiological model of ALCL cells in the perivascular niche with a 3D vasculature using a microfluidic chip. In this model, ALCL cells circulate inside the chip in continuous contact with a perfusable vasculature. We, then, demonstrated that the presence of endothelial cells conferred resistance to crizotinib and sustained cell viability of CCR7WT cells, whereas the protective effect was lost in CCR7KO cells. In in vivo experiments, CCR7 was required for lymphoma cell survival and diffusion to the brain during crizotinib treatment. Overall, our results suggest that the perivascular niche could promote the survival of ALK+ ALCL persister cells and protect them from the effect of ALK TKIs via the CCL19/21-CCR7 axis. The disruption of this survival axis could contribute to eradicating minimal residual disease in combination with ALK TKI. Citation Format: Cristina Mastini, Marco Campisi, Carlotta Costa, Chiara Ambrogio, Giulia Germena, Silvia Peola, Cinzia Martinengo, Enrico Patrucco, Ines Mota, Maddalena Arrigoni, Martina Olivero, Raffaele Calogero, Valeria Chiono, Roger D. Kamm, Emilio Hirsch, Jon C. Aster, Claudia Voena, Roberto Chiarle. The perivascular niche protects ALK+ lymphoma cells from ALK inhibition through the CCL19/21-CCR7 axis [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT018.