Resistance mechanisms to tyrosine kinase inhibitors in solid tumors

Project Information

TKI resistance

Grant agreement ID: 717034

DOI

10.3030/717034

Project closed

EC signature date 28 November 2016

Start date 1 December 2016

End date 30 November 2021

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 500 000,00

EU contribution

€ 1 500 000,00

Coordinated by

INSTITUT GUSTAVE ROUSSY
France

Periodic Reporting for period 4 - TKI resistance (Resistance mechanisms to tyrosine kinase inhibitors in solid tumors)

Reporting period: 2021-06-01 to 2021-11-30

Summary of the context and overall objectives of the project

‘Precision medicine’ is based on the selection of therapeutic agents for each individual patient based on the mutations, translocations and copy number variations identified in their cancer cells. In tumors in which such genetic alterations could be identified (i.e. 20-40% of lung cancers, 30% of urothelial carcinomas), cancer cells are highly dependent on the function of a single oncogene, defined as ‘driver oncogene’ for their proliferation and survival. Currently, more than 25 oncology drugs that target kinases have been approved, and many more are currently being clinically evaluated. From a biological point of view, cancer cells seem to have an intriguingly strong capacity of adaptation to a loss of the oncogenic signal their own survival depends on, thereby limiting the long term benefit of these new targeted therapies. The therapeutic effect of the latest generation inhibitors of ALK (lorlatinib), of FGFR (erdafitinib, pemigatinib, infigratinib and TAS120) and of EGFR (osimertinib), despite greater efficacy and selectivity than previous generations, is still limited by tumor adaptation mechanisms.
My project aims to better understand the mechanisms of resistance to novel tyrosine kinase inhibitors (TKI) in cancer patients with oncogenic addiction. By establishing new experimental models of resistance directly from patient biopsies (cell lines and xenografts), we elucidate the molecular mechanisms by which cancer cells escape targeted therapies and identify a way to overcome resistance.
My project is divided into several aims:
1) Establish unique laboratory models of TKI tumor adaptation from biopsy-derived cancer models (human primary culture and PDX)
2) Characterize the molecular mechanisms of tumor adaptation to oncogenic deaddiction.
3) Decipher the molecular basis of persister cells in order to discover strategies on how to block or postpone patients relapse.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

I have studied tumor samples from 93 patients who progressed on a kinase inhibitor and established 43 PDX and/or primary cell lines new models. We validated the oncogenic driver presence in the models by sequencing and confirmed their resistance to the targeted treatment.
Using tumor sequencing and laboratory studies we successfully identified a resistance mechanism in 70 patients (75%). 41% of the patients acquired a secondary mutation in the oncogenic target itself to block the inhibitor binding and 36% of patients activated another kinase to bypass the inhibition of the oncogenic driver.
More specifically, I studied in depth the resistance mechanisms specific to the last generation ALK TKI lorlatinib. Combining the establishment of patient-derived models (PDX and cell lines), high-throughput sequencing of circulating tumor DNA (ctDNA), gene knockout by CRISPR-Cas9, bioinformatics modeling of the clonal evolution in spatially and temporally spaced biopsies, I presented the first translational evaluation of acquired resistance to this novel and clinically important TKI. The identified resistance mechanisms include an epithelial-mesenchymal transition (EMT) sensitive to the combined inhibition of ALK and SRC kinases, new complex secondary mutations of ALK (G1202R + F1174L and C1156Y + G1269A), as well as a bypassing signal induced by the loss of NF2 and overcome by mTOR inhibitors.
I also fully characterized the resistance mechanisms in 12 FGFR driven patient derived models from urothelial and cholangiocarcinoma cancers with acquired resistance to the new FGFR inhibitors erdafitinib and TAS-120. The resistance mechanisms identified are the acquisition of secondary mutations in FGFR (n = 6) and the activation of a bypass (n = 6). To deepen our knowledge on FGFR mechanisms of activation or of resistance, we have cloned in the laboratory 33 unknown fusions and mutations detected in FGFR. These studies include a functional characterization of the oncogenic properties and inhibitor blockade provided by the genomic alterations.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We perform single cell sequencing on tissues from TKI resistant patients, in which multiple alterations have been identified to decipher the tumor heterogeneity at resistance. Facing potential toxicity profiles clinicians and regulatory agencies favor a sequential administration of pharmacological agents rather than a simultaneous treatment. However if two driver alterations exist within a single cell, a sequential approach would not lead to a major benefit. The clear determination of a dual-driver alteration at the single cell level would therefore have a major impact on the clinical practices. Recent technical advances in term of single cell isolation including from FFPE material and sequencing methods now allow sorting out this crucial question. This data will help understanding mechanisms underlying cell adaptation to oncogene-driven tumor inhibition and could have significant clinical impact by increasing the accuracy of molecular diagnosis and precision of personalized therapy.
We link our studies of TKI resistance to DNA repair deficiencies. Facing the systematic aspect of acquired resistance to targeted therapies, we hypothesize that exposure to kinase inhibitory agents would directly cause a modulation of DNA repair that would favor the appearance of mutations or chromosomal alterations which would lead to the emergence of resistance mechanisms within tumor cells. We recently developed our own double-strand break substrate. The substrate is based on a lentiviral vector containing a specific CRISPR-Cas9 cleavage site and several reporter gene modules expressing different fluorescent patterns depending on the repair pathway employed by the cell. We are currently treating TKI sensitive cell models with the kinase inhibitor after infection with the lentiviral substrate to evaluate the DNA repair capabilities at the single cell level. After the identification of main DNA repair pathways altered by TKI treatment and in order to prevent the acquisition of mutations and the emergence of resistance we will combine a kinase inhibitor with the inhibition of a DNA repair pathway in order to induce a synthetic lethality.
For patients who receive treatment with a targeted therapy, achieving a complete response to therapy is rare. The residual disease contains persisting tumor cells, which might be clonally derived from a small resistant subpopulation present at baseline and/or through the induction of adaptive changes within the tumor cells in response to TKI therapy. These persisting cells have the capability to acquire additional resistance mechanisms in vitro and ultimately give rise to resistant, progressive disease. Systemic therapy targeting these persisting tumor cells might eliminate this reservoir of resistant cells. Elucidating the mechanisms, likely epigenetic rewiring, allowing cancer cell drug tolerance is of major clinical interest and represents the most promising strategy to reach the dream of a cancer cure in the metastatic setting.

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