Abstract 552: Stromal facilitated multifactorial resistance to tumor cells against targeted therapies in ALK+ NSCLC
Authors: Desai Bina , Miti Tatiana , Miroshnychenko Daria , Marusyk Viktoriya , Gatenbee Chandler , Henry Menkara , Rix Uwe , Anderson Alexander , Haura Eric , Basanta David , Marusyk Andriy 
Published: June 2023

Abstract
Despite inducing strong and durable remissions, inhibitors of mutant ALK (ALKi) are not curative for advanced ALK+ lung cancers, as residual tumors eventually develop resistance and relapse. Apart from tumor cells’ intrinsic ability to escape from therapy, there is a growing body of evidence suggesting the contribution of extrinsic factors, produced by cancer-associated fibroblasts (CAFs). Despite the multitude of studies that demonstrated the ability of multiple CAF-produced factors to reduce the sensitivity of tumor cells to ALKi, the contribution of these effects toward responses in vivo remains unresolved. To study the impact of stroma on the ability of tumor cells to survive and develop resistance to ALKi, we derived multiple isolates of fibroblasts from clinical samples. Consistent with previous reports, we found that co-culture with CAFs or CAF-conditioned media protects tumor cells against ALKi. We observed that the degree of protection varies between different CAF isolates. This variability in the extent of protection could be attributed to variability in the levels of secreted hepatocyte growth factor (HGF) a known paracrine mediator of environmental resistance. Moreover, exogenous HGF phenocopied the effect of CAFs while blocking HGF-cMET signaling with neutralizing antibodies or pharmacologically abrogated the protective effect. To test the relevance of these findings in vivo, we took advantage of the inability of murine HGF to activate human cMET by comparing the response of ALK+ xenograft tumors to front-line ALKi alectinib between NSG mice and NSG-derivative strain with humanized HGF. In contrast to the in vitro data, HGF status had only a minimal impact on the remission-relapse dynamics of xenograft tumors. This lack of differences reflected a strong HGF-independent sheltering effect of the stromal niche. Our histological analyses of samples at different points of remission-relapse response revealed that while alectinib potently suppressed tumor cell proliferation, proximity to stroma reduced this cytostatic effect, without impacting cell proliferation in the absence of therapy. To gain insights into the mechanistic underpinning of this HGF-independent effect, we used spatial transcriptomics, comparing stroma-proximal and stroma-distant tumor regions. These analyses, as well as functional validation studies, indicate that stroma-sheltering effects are mediated by multiple mechanisms, acting in an additive fashion. In summary, our studies indicate that therapy resistance of tumor cells reflects the combined action of both intrinsic and microenvironmental factors. Our findings indicate that focusing on a single resistance mechanism at a time is unlikely to induce strong, durable responses. Instead, tackling the issue of therapy resistance necessitates the consideration of multiple resistance mechanisms as well as the moving target nature of tumors under therapy.
Citation Format: Bina Desai, Tatiana Miti, Daria Miroshnychenko, Viktoriya Marusyk, Chandler Gatenbee, Menkara Henry, Uwe Rix, Alexander Anderson, Eric Haura, David Basanta, Andriy Marusyk. Stromal facilitated multifactorial resistance to tumor cells against targeted therapies in ALK+ NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 552.