In situ Modeling of Acquired Resistance to RTK/RAS Pathway Targeted Therapies

Sealover, Nancy E.; Theard, Patricia T.; Hughes, Jacob M; Linke, Amanda J; Daley, Brianna R; Kortum, Robert L.

doi:10.2139/ssrn.4510603

Cited by 4 publications

(3 citation statements)

References 40 publications

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“…The doses of cisplatin that inhibited survival were higher in H82 than the other two SCLC-N cell lines, H524 and HCC33, which were more sensitive to the drug. This allowed us to use the same dose in each control and KO cell line to determine the concentration of cisplatin that caused prolonged growth arrest (> 6 weeks) prior to cell outgrowth in a majority of cell populations for each cell line, in a multi-well resistance assay (39) . For the assay, control (NTC) and KSR1 KO cells were plated at low density (3000 cells/well; <10% confluent) in multiple 96-well plates and each plate was either left untreated (to assess the time required for normal cell outgrowth) or treated weekly with a single inhibitory dose of cisplatin (ED85 dose for H82, ED75 for H524 and HCC33) for up to 14-15 weeks.…”

Section: Resultsmentioning

confidence: 99%

“…Clonogenicity is an in vitro index of self-renewal, a key property of TICs (15) . Clonogenicity was tested through single cell colony forming assays (15,41) . Following confirmation of KSR1 knockout by western blotting, control (NTC) and KSR1 KO cells were sorted by FACS and plated as single cells in 96-well plates to be analyzed for colony formation by CellTiter-Glo®.…”

Section: Resultsmentioning

confidence: 99%

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KSR1 regulates small-cell lung carcinoma tumor initiation and cisplatin resistance

Chatterjee,

Svoboda,

Huisman

et al. 2024

Preprint

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Small-cell lung cancer (SCLC) is designated a recalcitrant cancer due to its five-year relative survival rate of less than 7%. First line SCLC treatment has not changed in the last 40 years. The NeuroD1 subtype of SCLC (SCLC-N) commonly harbors MYC amplifications and other hallmarks of aggressive behavior. Finding novel therapeutic options that effectively eliminate residual disease observed after initial response to therapy is essential to improving SCLC patient outcome. Tumor-initiating cells (TICs) are reported as the sanctuary population within the bulk tumor responsible for therapeutic resistance and relapse. In contrast to earlier studies in which ERK activation is reported to be inhibitory to growth and tumor development, we show that KSR1 signaling is conserved in SCLC-N and that it regulates tumor initiation through ERK. Thus, KSR1 function in SCLC-N serves as a novel model for understanding the role of KSR1-dependent signaling in normal and malignant tissues. We further show that KSR1 mediates cisplatin resistance in SCLC-N cells. CRISPR/Cas9-mediated KSR1 knockout causes a dramatic increase in sensitivity to cisplatin and is coincident with a marked decrease in TICs, indicating that targeting KSR1 might be selectively toxic to cells responsible for therapeutic resistance and tumor initiation. Our data show that KSR1, a molecular scaffold for the Raf/MEK/ERK signaling cascade, is critical for tumor initiation and clonogenicity, bothin vitroandin vivoin the highly aggressive, metastatic and therapy resistant NeuroD1 subtype of SCLC. These findings shed light on a key distinct protein responsible for regulation in SCLC-N tumors, and a potential subtype specific therapeutic target.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

KSR1 regulates small-cell lung carcinoma tumor initiation and cisplatin resistance

Chatterjee,

Svoboda,

Huisman

et al. 2024

Preprint

View full text Add to dashboard Cite

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“…Wells are re-fed weekly and wells showing >50% confluence are scored as resistant to that dose of drug(s). We previously showed that while low doses of RTK/RAS pathway targeted inhibitors cause a proliferative delay, acquired resistance to RTK/RAS pathway inhibitors including G12Cis can be modeled using a ≥ EC80 dose of G12Ci (76). For H358 cells, 10 nM adagrasib modeled G12Ci acquired resistance whereas 1, 3, or 6 nM caused a slight delay in proliferation but still showed intrinsic resistance.…”

Section: Sos1 Inhibition Limits G12c Inhibitor Resistance In Kras G12...mentioning

confidence: 99%

SOS1 inhibition enhances the efficacy of and delays resistance to G12C inhibitors in lung adenocarcinoma

Daley,

Sealover,

Sheffels

et al. 2023

Preprint

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SummaryClinical effectiveness of KRAS G12C inhibitors (G12Cis) is limited both by intrinsic and acquired resistance, necessitating the development of combination approaches. We found that targeting proximal receptor tyrosine kinase (RTK) signaling using the SOS1 inhibitor (SOS1i) BI-3406 both enhanced the potency of and delayed resistance to G12Ci treatment, but the extent of SOS1i effectiveness was modulated by both SOS2 expression and the specific mutational landscape. SOS1i enhanced the efficacy of G12Ci and limited rebound RTK/ERK signaling to overcome intrinsic/adaptive resistance, but this effect was modulated by SOS2 protein levels. Survival of drug-tolerant persister (DTP) cells within the heterogeneous tumor population and/or acquired mutations that reactivate RTK/RAS signaling can lead to outgrowth of tumor initiating cells (TICs) that drive therapeutic resistance. G12Ci drug tolerant persister cells showed a 2-3-fold enrichment of TICs, suggesting that these could be a sanctuary population of G12Ci resistant cells. SOS1i re-sensitized DTPs to G12Ci and inhibited G12C-induced TIC enrichment. Co-mutation of the tumor suppressorKEAP1limits the clinical effectiveness of G12Cis, andKEAP1andSTK11deletion increased TIC frequency and accelerated the development of acquired resistance to G12Ciin situ. SOS1i both delayed acquired G12Ci resistance and limited the total number of resistant colonies regardless ofKEAP1andSTK11mutational status. These data suggest that SOS1i could be an effective strategy to both enhance G12Ci efficacy and prevent G12Ci resistance regardless of co-mutations.SignificanceThe SOS1 inhibitor BI-3406 both inhibits intrinsic/adaptive resistance and targets drug tolerant persister cells to limit the development of acquired resistance to clinical KRASG12Cinhibitors in lung adenocarcinoma cells.

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SOS2 modulates the threshold of EGFR signaling to regulate osimertinib efficacy and resistance in lung adenocarcinoma

Theard,

Linke,

Sealover

et al. 2024

Molecular Oncology

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Son of sevenless 1 and 2 (SOS1 and SOS2) are RAS guanine nucleotide exchange factors (RasGEFs) that mediate physiologic and pathologic receptor tyrosine kinase (RTK)‐dependent RAS activation. Here, we show that SOS2 modulates the threshold of epidermal growth factor receptor (EGFR) signaling to regulate the efficacy of and resistance to the EGFR tyrosine kinase inhibitor (EGFR‐TKI) osimertinib in lung adenocarcinoma (LUAD). SOS2 deletion (SOS2KO) sensitized EGFR‐mutated cells to perturbations in EGFR signaling caused by reduced serum and/or osimertinib treatment to inhibit phosphatidylinositol 3‐kinase (PI3K)/AKT pathway activation, oncogenic transformation, and survival. Bypassing RTK reactivation of PI3K/AKT signaling represents a common resistance mechanism to EGFR‐TKIs; SOS2KO reduced PI3K/AKT reactivation to limit osimertinib resistance. In a forced HGF/MET‐driven bypass model, SOS2KO inhibited hepatocyte growth factor (HGF)‐stimulated PI3K signaling to block HGF‐driven osimertinib resistance. Using a long‐term in situ resistance assay, most osimertinib‐resistant cultures exhibited a hybrid epithelial/mesenchymal phenotype associated with reactivated RTK/AKT signaling. In contrast, RTK/AKT‐dependent osimertinib resistance was markedly reduced by SOS2 deletion; the few SOS2KO cultures that became osimertinib resistant primarily underwent non‐RTK‐dependent epithelial–mesenchymal transition (EMT). Since bypassing RTK reactivation and/or tertiary EGFR mutations represent most osimertinib‐resistant cancers, these data suggest that targeting proximal RTK signaling, here exemplified by SOS2 deletion, has the potential to delay the development osimertinib resistance and enhance overall clinical responses for patients with EGFR‐mutated LUAD.

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In situ Modeling of Acquired Resistance to RTK/RAS Pathway Targeted Therapies

Cited by 4 publications

References 40 publications

KSR1 regulates small-cell lung carcinoma tumor initiation and cisplatin resistance

KSR1 regulates small-cell lung carcinoma tumor initiation and cisplatin resistance

SOS1 inhibition enhances the efficacy of and delays resistance to G12C inhibitors in lung adenocarcinoma

SOS2 modulates the threshold of EGFR signaling to regulate osimertinib efficacy and resistance in lung adenocarcinoma

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