Mechanisms of acquired resistance to anti-EGFR targeted therapies in metastatic colorectal cancer

Colorectal cancer (CRC), the third most common cancer type in western countries, affects more than 200,000 patients worldwide every year. Screening, surgery and medical therapies are successful in the management of early-stage CRC, but far less efficacious in advanced stages of the disease. A key reason of the limited success of CRC-directed therapies is its intrinsic cancer heterogeneity, which is more prominent in the metastatic setting. Molecular characterization of CRCs revealed that heterogeneity plays an important role especially in the context of resistance to molecular targeted therapy.

The anti Epidermal Growth Factor Receptor (EGFR) monoclonal antibodies cetuximab and panitumumab are in clinical use to treat different malignancies, including metastatic colorectal cancer (CRC). In this setting, response rates to either drug as monotherapy are around 10-15% in an unselected population. This phenomenon has been recognized as primary resistance and it is due to the presence of several genetic alterations in EGFR signaling pathway. The most frequent genetic alterations are KRAS exon 2 mutations account for more than 40% of patients. KRAS exons 3 and 4, NRAS exons 2, 3, 4, BRAF exon 15 mutations and amplification of KRAS, HER2 and MET account for around 20% of mCRC patients which do not benefit from anti-EGFR treatment. RAS wild type population represents the sum of the anti-EGFR therapies responders (10-15%) and the fraction of patients who do not benefit from those treatments even in the absence of known primary resistance genetic alterations.

Clinical data indicate that even the best responses obtained in RAS wild type tumors are transient and even the best responses do not last longer than 12 months on average. The molecular bases of secondary (acquired) resistance to anti-EGFR targeted therapies in CRCs are poorly understood and the difficulties in accessing tumor samples from cetuximab or panitumumab refractory patients are among the reasons that have so far impaired research on this topic. Most importantly, CRC patients with acquired resistance to anti-EGFR targeted therapies have no further therapeutic options, which makes the investigation about molecular mechanisms even more needed.

Development of CRC cell lines with acquired resistance to cetuximab and panitumumab

Due to the lack of preclinical CRC models of secondary resistance to anti-EGFR drugs, we took advantage from CRC cell model to explore molecular mechanisms of acquired resistance. We exploited five CRC cell lines (LIM1215, DiFi, HCA-46, OXCO-2 and NCIH508) which we found to be highly sensitive to anti-EGFR monoclonal antibodies, cetuximab and panitumumab, treatment. To better characterize these models, we analyzed the status of genes involved in primary resistance such as KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4), BRAF (exon 15) and also EGFR, HER2 and MET. The five cell lines did not shown any KRAS and NRAS mutations. BRAF was also wild type at mutational hotspots known to confer primary resistance to EGFR blockade. The DiFi cell line displayed high-level amplification of the EGFR gene. MET and HER2 gene copies were not significantly altered in the cell lines panel.

Overall, this first characterization show that our cell lines faithfully recapitulate the profile of CRC patients which benefits from anti-EGFR treatment, demonstrating the good potential of the panel. The five cell lines, which were initially very sensitive to anti-EGFR monoclonal antibodies, were made resistant through continuous exposition to the drugs cetuximab or panitumumab until the development of secondary resistance. Resistance protocol was repeated for several time in some of the cell models, increasing the number of acquired resistant cell lines (Figure 1).

Figure 1. Anti-EGFR sensitive cell lines and generation of resistant derivatives

When cetuximab- and panitumumab-resistant derivatives were generated we started genetic analysis to determine molecular mechanisms of acquired resistance; therefore, Sanger sequencing of candidates gene involved in primary resistance, was performed on genomic DNA extracted from parental and cetuximab resistant cells (referred as R-cetux) and panitumumab resistant cell (referred as R-panit).

Sequencing analyses of the resistant populations revealed molecular alterations in KRAS, BRAF and NRAS genes, and several resistant derivatives contained concomitant multiple mutations highlighting a high level of heterogeneity.

Mutational analysis of candidate resistance genes was unsuccessful on some of the resistant derivatives, like DiFi resistant cells. Therefore, we performed an unbiased gene copy number analysis and mutational profiling by 454 next-generation sequencing. After deep sequencing analysis, DiFi R-cetux cells differed from their sensitive parental counterpart by two focal molecular alterations: the EGFR gene copy number was reduced, whereas the KRAS gene was amplified. In light of these results, we decided to investigate the presence of KRAS gene amplification as a mechanism of acquired resistance on the entire panel of our resistant derivatives by Real Time PCR. This analysis confirmed amplification of KRAS in DiFi R1-cetux (and DiFi R2-cetux) and revealed the same alteration in HCA-46 R1-cetux and NCIH508 R-cetux and NCIH508 R-panit cell lines.

Altogether, these results suggest that multiple genetic mechanisms can drive resistance to EGFR blockade and several sub-clones often coexist in the population that emerges after selection with anti-EGFR therapies (Figure 2).

Figure 2. Anti-EGFR resistant derivatives and their genetic alterations

Patients which relapse after anti-EGFR treatment harbor heterogeneous RAS genes alterations

Experiments performed in preclinical models indicate that the emergence of KRAS mutations is associated with acquired resistance to EGFR blockade. To determine whether KRAS alterations are clinically relevant mechanisms of acquired cetuximab resistance, we examined tumor biopsies from ten patients with CRC who had become refractory to either cetuximab or panitumumab. We identified one individual whose tumor at progression displayed KRAS amplification that was not present in a matched pre-cetuximab biopsy. In a different patient, Sanger sequencing identified a KRAS p.Q61H mutation in a biopsy obtained after disease progression on cetuximab treatment; the remaining eight tumor samples obtained in patients with acquired resistance to anti-EGFR therapy were wild-type KRAS by this technique. To determine whether the Sanger technology may have been underpowered to detect KRAS mutations in the biopsies obtained after cetuximab or panitumumab progression, these remaining cases were analyzed using deep sequencing technologies (454 or BEAMing). These techniques identified the KRAS p.G13D mutation in four samples, and the simultaneous presence of G12D and G13D mutations in one case.

The cell-based findings suggest that upon EGFR blockade, multiple resistant clones emerge, and that resistance is often driven by genetically distinct mechanisms. We hypothesized that the results obtained by tissue biopsies could underestimate the intrinsic heterogeneity of a metastasis because they can account only of a small portion of the single metastasis. The scenario is further complicated by the fact that patients with metastatic CRC usually have multiple lesions. Therefore, biopsies represent only a snapshot of the overall disease and, accordingly, are not well suited to monitor the emergence of resistant clones, which can be located in distant metastatic lesions. To overcome, at least in part, these limitations, we have implemented a ‘liquid biopsy’ approach to analyze circulating free tumor DNA as it is more likely to capture the overall tumor genetic complexity in patients with advanced disease. We exploited the highly sensitive BEAMing technique to measure tumor derived DNA mutations in the blood of patients. We obtained plasma samples from 4 patients who responded and then became refractory to either cetuximab or panitumumab. BEAMing probes designed to identify the same somatic variants found in cells lines were used to monitor KRAS, BRAF and NRAS mutations in plasma. Notably, in two cases multiple KRAS variants were detected at relapse but not at baseline suggesting the emergence of several independent clones during treatment. In patient #2 the concomitant presence of KRAS and NRAS mutations was observed in the relapse sample. The same occurred at relapse in patient #3. In the last patient (#4) mutations in KRAS, NRAS and BRAF were not found at progression (in patients #3 and 4 the plasma baseline was not available). These results suggest that therapy with anti-EGFR antibodies selects multiple clones carrying heterogeneous patterns of mutations, a situation akin with what we observed in preclinical models (Figure 3).

Figure 3. Tissue and Liquid biopsies of patients with acquired resistance to anti-EGFR

Genetic alterations in distinct members of the EGFR signaling cascade biochemically converge to activate MEK and ERK

As described above, escape from EGFR blockade in CRC cells is associated with the emergence of distinct alterations in several genes. We hypothesized that the plethora of molecular determinants causative of acquired drug resistance would ultimately converge at a limited number of signaling switches which, in turn, could be rationally targeted by further lines of therapy. To test this assumption we performed biochemical analysis of the resistant cell derivatives. For these studies, we elected to use LIM1215 R-panit (KRAS G13D, NRAS G12C), OXCO-2 R1-cetux (KRAS G12D, BRAF V600E), HCA-46 R-panit (KRAS G12C) and NCIH508 R-cetux (KRAS amplified) as they are representative of the distinct resistant mechanisms which emerged upon selection with cetuximab and panitumumab. We assessed the phosphorylation status of EGFR and its downstream effectors MEK and ERK by Western blot analysis. We found that MEK and/or ERK were consistently activated in resistant cells as compared to their parental counterparts. These data lead unequivocally to a common route of pathway dependency that, independently of the genetic mechanism of resistance, converge to the sustained activation of MAPK pathway. However, we found that pharmacological inhibition of MEK was unable to successfully block the growth of resistant cells. The fact that cells that developed acquired resistance to EGFR blockade, display constitutive activation of MEK but are only modestly affected by MEK inhibition is not obvious. To study this further, we performed biochemical analyses in the presence of EGFR and MEK inhibitors (cetuximab and pimasertib, respectively) alone or in combination.

Notably, we found that in the absence of cetuximab the MEK inhibitor pimasertib leads to efficient phospho-ERK suppression, but this is however transient and after few hours ERK became phosphorylated again. Concomitant to ERK reactivation, we observed increased phosphorylation of EGFR. Phosphorylation of the receptor increased over time and was maximal after 6-12 hours depending on the cell model. When the same experiment was performed in the presence of cetuximab together with the MEK inhibitor, EGFR phosphorylation was suppressed and this was accompanied by prolonged abrogation of ERK phosphorylation. Importantly, these results are consistent across multiple cell models of acquired resistance to EGFR blockade and irrespectively of their mutational status and were validated in several pharmacological assays on our cell models in vitro and in vivo.

Combinatorial treatment is effective in a tumor mouse xenograft from a relapsed patient

To corroborate the translational relevance of our work, we generated a mouse xenotransplant from a thin needle biopsy derived from a metastatic CRC patient who responded and subsequently relapsed upon EGFR therapy (cetuximab) due to the acquisition of a KRAS exon 4 mutation (p.A146T). This mutation is identical to one of the KRAS variants we found in LIM1215 cells that developed resistance to cetuximab (R4 derivative). This Patient-Derived Xenograft (PDX) therefore perfectly parallels the cell line models described above. We compared the morphological features of the biopsy obtained from the lung metastasis from which the xenopatient was established and specimen from the xenotransplant grown in NOD-SCID mice and, as shown, xenografted tumors retained the hystopathological characteristics of their original patient counterpart. We therefore expanded the first mouse in four cohorts in order to test the efficacy of the combinatorial treatment. Cetuximab or the MEK inhibitor pimasertib alone had limited effectiveness, while combinatorial treatment prominently impaired tumor growth and even induced shrinkage in fully established xenotumors derived from this relapsed metastatic CRC patient. These results suggest that patients who become resistant to EGFR monoclonal antibodies by the emergence of genetic alterations in the RAS pathway may derive significant clinical benefit from combinatorial treatment with EGFR and MEK inhibitors (Figure 4).

Figure 4. PDX from a relapsed patient is sensitive to combinatorial treatment

Conclusions

In this work, we discovered for the first time different mechanisms can trigger secondary resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer but, most importantly, we highlight the fact that regardless of the single genetic alteration, the net output is invariably sustained activation of MAPK pathway.

This finding prompted us to use this particular characteristic as an Achilles’s Hill of our models of acquired resistance. Only when EGFR was co-targeted together with MEK secondary resistance could be reverted. Notably, we provide evidence that data generated in cell models are clinically relevant by showing that the same results can be obtained with bioptic and plasma analyses and xenografted tumors derived from a patient who responded and then relapsed upon cetuximab therapy.

Overall, our data strongly support the design of clinical trials in which CRC patients at relapse after anti-EGFR treatment can be re-challenged with a MEK inhibitor together with the monoclonal antibody offering a new therapeutic option for these patients.