Organoid Acquired Resistance

PROJECT SUMMARY
The overall goal of Project 3 of this U54 application is the organoid-based definition of mechanisms of acquired
resistance (AR) to targeted therapy in non-small cell lung cancer (NSCLC). The treatment of NSCLC employs
numerous therapies directed against the EGFR, ALK, KRAS G12C, ROS1 and PD-1/PD-L1 pathways. However,
despite initial responses, these molecularly-targeted therapies are not curative, and virtually all patients
eventually succumb to progressive disease. Acquired resistance features prominently in relapse, such as with
EGFR T790M or C797S mutations following gefitinib or osimertinib treatment; however, known AR mechanisms
cannot be identified for a significant proportion of patients receiving targeted therapy. Further compounding this
dilemma are the distinct contributions of tumor-intrinsic versus tumor microenvironmental (TME) influences. The
TME in particular contains extracellular matrix (ECM), fibroblast, vascular and immune components that can
vitally impact the development and manifestations of AR. The study of TME-dependent and -independent
mechanisms of AR have been further hindered by a distinct lack of in vitro human experimental systems allowing
holistic recapitulation of the TME in patient biopsies.
Our application leverages the synergistic strengths of the Sourav Bandyopadhyay and Calvin Kuo groups
in organoid and proteomics technology to address mechanisms of EGFR and KRAS G12C AR in NSCLC. We
conduct advanced proteomic approaches and organoid culture both from primary tumor biopsies at acquired
resistance (from Project 1) as well as from patient-derived xenograft (PDX) tumors grown in human immune
reconstituted mice (from Project 2). Our organoids are either “epithelial-only” or novel air-liquid interface (ALI)
organoid cultures that co-preserve tumor epithelium en bloc with tumor-infiltrating fibroblasts and immune
subsets (T, B, NK, myeloid). Aim 1 leverages the results of a NSCLC organoid CRISPR screen to determine if
osimertinib-sensitizing loci can overcome, delay or alter acquired resistance in clinical biopsy/PDX AR organoids,
emphasizing hits with TME mechanisms. Aim 2 uses our mass spectrometry platforms to characterize total
proteome and phospho-proteomic changes in treatment-naive, residual and acquired resistance organoids and
PDX, while pursuing lead candidate TROP2 and new target nominations by genetic, mechanistic and
pharmacologic studies. Lastly, Project 1 has identified MIF/CD74/CD44 and CD47/SIRPa tumor-fibroblast-
macrophage pathways upregulated during AR which are then functionally explored in Aim 3 using ALI organoids
from AR clinical biopsies and PDX tumors. Overall, we propose a comprehensive, translational approach that
exploits complementary organoid and proteomics expertise to directly study biopsies and PDX from NSCLC
patients with acquired resistance to targeted therapy, towards development of improved therapeutic approaches.

Grant Number: 5U54CA224081-08
NIH Institute/Center: NIH
Principal Investigator: Sourav Bandyopadhyay