Receptor Tyrosine Kinase Signaling Networks Define Sensitivity to ERBB Inhibition and Stratify Kras-Mutant Lung Cancers

Abstract

Most non-small cell lung cancers (NSCLCs) contain non-targetable mutations, including KRAS, TP53 or STK11/LKB1 alterations. By coupling ex vivo drug sensitivity profiling with in vivo drug response studies, we aimed to identify drug vulnerabilities for these NSCLC subtypes. Primary adenosquamous carcinoma (ASC) or adenocarcinoma (AC) cultures were established from Kras G12D ;Lkb1 fl/fl (KL) tumors or AC cultures from Kras G12D ;p53 fl/fl (KP) tumors. While p53 null cells readily propagated as conventional cultures, Lkb1 null cells required conditional reprograming for establishment. Drug response profiling revealed short-term response to MEK inhibition, yet, long-term clonogenic assays demonstrated resistance, associated with sustained or adaptive activation of receptor tyrosine kinases (RTKs): activation of ERBBs in KL cultures, or FGFR in AC cultures. Furthermore, pan-ERBB inhibition reduced the clonogenicity of KL cultures, which was exacerbated by combinatorial MEK inhibition, while combinatorial MEK and FGFR inhibition suppressed clonogenicity of AC cultures. Importantly, in vivo studies confirmed KL-selective sensitivity to pan-ERBB inhibition, which correlated with high ERBB ligand expression and activation of ERBB receptors, implying that ERBB network activity may serve as a predictive biomarker of drug response. Interestingly, in human NSCLCs, phosphorylation of EGFR or ERBB3 was frequently detected in ASCs and squamous cell carcinomas. We conclude that analysis of in situ ERBB signaling networks in conjunction with ex vivo drug response profiling and biochemical dissection of adaptive RTK activities may serve as valid diagnostic approach to identify tumors sensitive to ERBB network inhibition.

Functional diagnostics using fresh uncultured lung tumor cells to guide personalized treatments

“…1E-F), matching previous findings in CR cultures. 19 In further agreement with published data, 22,23 a synergistic interaction was detected between MEK and ERBB receptor family inhibition in KL, but not in KP FUTCs ( Fig. 2A).…”

“…1D and F), corroborating findings from cultured lung cancer models. [19][20][21] Lastly, we confirmed that gemcitabine, an approved chemotherapeutic agent for NSCLC treatment, potently inhibited the viability of both KL and KP cells ( Fig. 1E-F), matching previous findings in CR cultures.…”

“…1A), and (iii) we have gained understanding on their tumor subtype-selective drug sensitivities. 19 First, we confirmed that FUTCs survive and grow during culture by measuring cellular ATP levels using CellTiter-Glo (CTG). FUTCs from the different tumor groups exhibited significantly higher CTG readouts after three days of culture (Fig.…”

“…18 For FUTC isolation, individual lung tumors were manually minced using a sterile scalpel and then enzymatically digested in HBSS containing 2 mg/ml collagenase, 0.3 mg/ml dispase and 10 mg/ml bovine serum albumin (BSA), using a Miltenyi gentleMACS dissociator, as described. 19…”

“…Murine lung tumor-derived FUTCs or CR cultures at passage 4 were used for DSRT following published procedures, 19 with minor modifications. Briefly, 2500 FUTCs/well or 1500 CR cells/well were seeded in 384-well plates and were exposed to drugs on the following day.…”