Robert C. Doebele

TrkB (NTRK2) overexpression in neuroblastoma is reported to be associated with poor prognostic features, including MYCN amplification and advanced tumor stages. In this study, TrkB expression in available cell lines was characterized, and allosteric inhibitors of ligand-activated TrkB were designed, characterized, and tested. The designed compounds commonly bear a triazole group at their center, which bridges three aromatic components. Based on the chemical design, 25 compounds were synthesized for the cell-based assay to examine the neuroblastoma cell growth inhibition. Several cell lines were used in the assay based on TrkB expression levels. TrkB non-enhanced and TrkA overexpressing cell cultures were also tested for comparison. Many of the synthesized compounds suppressed the phosphorylation of the TrkB (cytoplasmic side) induced by brain-derived neurotrophic factor (BDNF) binding to the extracellular domain of TrkB. Several compounds showed effective suppression of phosphorylation to a similar degree as larotrectinib, an approved TrkB inhibitor targeting the cytoplasmic tyrosine kinase domain. The compounds’ potency to suppress neuroblastoma cell growth was evaluated using a basal high TrkB-expressing culture. Select compounds showed strong cell growth suppression, with 50% inhibitory activities below 30 nM. Two potent compounds were also tested to determine their inhibitory activities on other cell lines. Cell growth inhibition was observed for three cell cultures, but moderate for one. Neither the approved TrkB inhibitor, larotrectinib, nor our allosteric compounds could completely inhibit the growth of all neuroblastoma cell lines natively expressing TrkB.

Cancer-associated fibroblasts (CAFs) are associated with tumor progression and drug resistance. Here, we investigated the mechanisms underlying the cross-talk between CAFs and tumor cells in non–small cell lung cancer (NSCLC). In NSCLC cell lines with EML4-ALK fusions, we observed substantial CAF-mediated drug resistance to clinically used inhibitors of the tyrosine kinase ALK. Array-based cytokine profiling of CAF-derived conditioned medium indicated that a major contributor to the phenomenon was the secreted growth factor HGF, and blocking its receptor MET overcame paracrine resistance to ALK inhibition. However, cell-selective labeling of the proteome in cocultures also revealed an equally important contribution by the fibronectin-integrin pathway, specifically integrin β ₁ , which was confirmed through pharmacological inhibition and cell-specific silencing or knockout. Concurrent targeting of MET and integrin signaling effectively abrogated ALK inhibitor resistance in EML4-ALK ⁺ NSCLC cells cocultured with CAFs. Moreover, the combination of the ALK inhibitor alectinib with the MET inhibitor capmatinib and/or the integrin inhibitor cilengitide was more effective than single-agent treatment in suppressing tumor growth in allografted mice. The findings illustrate a previously unappreciated complex nature of concurrent paracrine and juxtacrine mechanisms of CAF-driven resistance that may inform the development of more effective therapeutic approaches.

Purpose: Mouse double minute 2 (MDM2) is an E3 ubiquitin ligase that degrades the tumor suppressor p53. In cancers, MDM2 amplification (MDM2amp) leads to overexpression of MDM2, inducing p53 degradation and a p53-null phenotype even in the absence of TP53 mutations. We report here the preclinical and clinical activities of milademetan, a potent and selective oral small-molecule inhibitor of the MDM2–p53 interaction, in MDM2amp, TP53 wild-type (WT) solid tumors. Patients and Methods: Milademetan was tested against a variety of cell line and xenograft tumor models. This supported a phase II basket study (MANTRA-2) in patients with advanced MDM2amp, TP53-WT solid tumors. The primary endpoint was the objective response rate, and key secondary endpoints included progression-free survival and adverse events. Results: Milademetan showed potent activity against MDM2amp, TP53-WT laboratory models. In the phase II trial, 40 patients received milademetan, 31 of whom had centrally confirmed molecular testing. The best overall response was 19.4% (6/31) with one confirmed response (3.2%) and five unconfirmed partial responses, including a patient with endometrial stromal sarcoma who achieved a 100% target lesion reduction. The median progression-free survival was 3.5 months (95% confidence interval, 1.8–3.7). Grade 3 or 4 adverse events observed included thrombocytopenia, neutropenia, anemia, leukopenia, and diarrhea. Conclusion: Milademetan had a manageable safety profile and achieved responses against a variety of refractory MDM2amp, TP53-WT solid tumors, but tumor reductions were short-lived. Subsequent efforts should focus on combination strategies, further biomarker refinement, or novel MDM2 targeting approaches to achieve more durable clinical benefit.

Drug resistance remains a challenge for targeted therapy of cancers driven by EML4-ALK and related fusion oncogenes. EML4-ALK forms cytoplasmic protein condensates, which result from networks of interactions between oncogene and adapter protein multimers. While these assemblies are associated with oncogenic signaling, their role in drug response is unclear. Here, we use optogenetics and live-cell imaging to find that EML4-ALK assemblies suppress transmembrane receptor tyrosine kinase (RTK) signaling by sequestering RTK adapter proteins including GRB2 and SOS1. Furthermore, ALK inhibition, while suppressing oncogenic signaling, simultaneously releases the sequestered adapters and thereby resensitizes RTK signaling. Resensitized RTKs promote rapid and pulsatile ERK reactivation that originates from paracrine ligands shed by dying cells. Reactivated ERK signaling promotes cell survival, which can be counteracted by combination therapies that block paracrine signaling. Our results identify a regulatory role for RTK fusion assemblies and uncover a mechanism of tolerance to targeted therapies.

EGFR to MIG6 ratios in relation with crizotinib IC50s in cell lines.

Result of RNA-seq differential expression analysis for H3122-CR vs H3122.

The complete list of identified phosphopeptides after global (pS/T/Y) phosphopeptide enrichment in crizotinib vs DMSO treated CUTO28 cells with fold changes and p-value calculation.

Result of RNA-seq differential expression analysis for CUTO37-ER vs CUTO37.

EGFR to MIG6 ratios in relation with crizotinib IC50s in cell lines.

Supplementary Table S2 from MIG6 Mediates Adaptive and Acquired Resistance to ALK/ROS1 Fusion Kinase Inhibition through EGFR Bypass Signaling