IGF2 is an actionable target that identifies a distinct subpopulation of colorectal cancer patients with marginal response to anti-EGFR therapies

Response to cetuximab in “quadruple negative” mCRC xenopatients: A population study in 125 cases

Clinical studies have recently demonstrated that, in addition to the validated KRAS mutations in exon 2, additional KRAS mutations in exons 3 and 4, as well as NRAS, BRAF, and concurrent PIK3CA (exon 20) mutations, predict a lack of response in patients who receive anti-EGFR antibodies (5, 8). We obtained similar results in a series of 66 xenopatients wild type for exon 2 of KRAS (10). We decided to expand our investigation of cetuximab activity on a wider population scale, with a specific focus on quadruple negative cases that did not harbor resistance-conferring mutations in KRAS, NRAS, BRAF, or PIK3CA. We therefore assessed anti-EGFR response in an extended suite of xenopatients, for a total of 125 quadruple negative models (77 newly engrafted samples and 48 samples from our former collection) (10). In accordance with previously established procedures (10), we defined a “case” as one (or, more often, two) xenopatient lines derived from an individual patient specimen, with each line consisting of 12 mice (6 treated with placebo and 6 treated with cetuximab). To segregate response classes, we adopted a volume-based designation inspired by clinical standards: (i) regression [objective response (OR)] was classified as a reduction of at least 50% in the mean volume of tumors for each case, taking as reference the baseline (pretreatment) tumor volume; (ii) progressive disease (PD) was categorized as a volume increase of at least 35%; and, finally, (iii) intermediate responses (tumor volume changes between 35% increase and 50% reduction) were considered as stable disease (SD). On the basis of comparative calculations, these three-dimensional cutoffs were the closest approximations for the unidimensional RECIST (Response Evaluation Criteria in Solid Tumors) criteria used in patients, which refer to the longest diameter of target lesions.

Figure 1A depicts a waterfall plot of the effect of cetuximab at 3 weeks after the start of treatment, with xenopatient cases ranked by tumor volume changes relative to baseline: OR was observed in 31 cases (24.8%), SD in 60 cases (48%), and PD in 34 cases (27.2%). Because this set of xenopatients was chosen to include a genetically selected subpopulation free of resistance-conferring mutations in KRAS, NRAS, BRAF, and PIK3CA, the distribution of response rates compared favorably with data obtained in less stringently selected series. Indeed, mCRC patients for whom only KRAS (exon 2) wild-type status was assessed have been shown to experience a 12.8 to 17% frequency of tumor regression and a 34% frequency of SD (3, 4). Similarly, in our original collection of 66 xenopatients—which excluded KRAS (exon 2) mutations, but included all other mutant genotypes—OR occurred in 11 cases (16.7%) and SD in 27 cases (40.9%) (10).

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Although inclusion of only quadruple negative tumors led to a higher frequency of treatment response, cases with SD were still predominant over regressions (Fig. 1A). Moreover, cases exhibiting overt tumor shrinkage [defined as a mass reduction of more than 75%, a cutoff arbitrarily set at a size equidistant between borderline regression (50%) and complete mass obliteration (100%)] remained a small minority (9 of 125, 7.2%) (Fig. 1A). These findings suggest that this refinement of genetic selection may not be sufficient to enrich for full responders. We therefore elected to exploit the merits of xenopatients—namely, their suitability for multiple exploratory therapies and the tumor molecular information associated with each xenopatient model—to test rational combinations as a means to improve the efficacy of single-agent cetuximab in SD cases.

Molecular correlations between expression of EGFR pathway components and response to cetuximab

Retrospective clinical trials have shown a correlation between gene copy number gain (and consequent overexpression) of EGFR or higher expression of two EGFR ligands, amphiregulin and epiregulin, and increased sensitivity to anti-EGFR treatment (16–21). We broadened these observations by extracting the transcript levels of EGFR family receptors with known activity in CRC (EGFR, HER2, and HER3) and EGFR cognate ligands (EGF, epiregulin, TGF-α, β-cellulin, and heparin-binding EGF) out of an Illumina beadchip array data set. Baseline gene expression information was available for 105 of the 125 quadruple negative models analyzed for cetuximab response; for each model, two independent xenografts were subjected to microarray analysis, for a total of 210 profiles (table S1). Because amphiregulin expression proved to be undetectable (likely due to probe failure), the same cases were profiled for amphiregulin expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR). We note that these surveys were performed on tumor samples after the first passage in vivo. In this condition, the human stroma was substituted by murine components, as demonstrated by lack of immunoreactivity for vimentin, a prototypical stromal marker, when using human-specific antibodies (fig. S1); therefore, this analysis covered only receptors and autocrine ligands expressed by cancer cells.

As expected, we observed a positive association between elevated expression of EGFR, epiregulin, and amphiregulin and OR to cetuximab (Fig. 1, B and C, fig. S2, and table S1). Again, in accordance with previous observations (10–12, 22), HER2 was strongly overexpressed in a fraction of PDs, and the expression of TGF-α was increased in a portion of cases displaying resistance to treatment (Fig. 1B, fig. S2, and table S1). Extension of gene expression analysis to the other receptors and ligands revealed that the HER3 receptor, β-cellulin, and heparin-binding EGF were overall more expressed in responders than in resistant cases (Fig. 1B, fig. S2, and table S1).

In addition to baseline profiles, on-treatment microarray data were also available for 14 cetuximab-sensitive models that had received the antibody for 72 hours or 6 weeks (table S2). This longitudinal analysis indicated progressive reduction (with statistical significance after 6 weeks) in the expression levels of EGFR (P = 0.017 relative to baseline expression), epiregulin (P = 1 × 10^–4), and heparin-binding EGF (P = 0.035), and increased expression of HER2 (P = 0.002) and HER3 (P = 0.013) (fig. S3, A and B). This suggests gradual tumor adaptation to EGFR blockade by down-regulation of EGFR-dependent signals and up-regulation of HER2/HER3-based compensatory pathways.

Combination therapies against the EGFR pathway in xenopatients showing partial sensitivity to single-agent cetuximab

When compared with resistant cases, the baseline expression of EGFR, HER3, amphiregulin, epiregulin, β-cellulin, and heparin-binding EGF was higher not only in responders but also in SDs (Fig. 1, B and C, fig. S2, and table S1); heparin-binding EGF was even more expressed in SDs than in ORs (Fig. 1B, fig. S2, and table S1). Increased representation of EGFR, HER3, and EGFR ligands not only in ORs but also in SDs suggests high EGFR pathway activity in this latter subpopulation, which may sustain a certain degree of functional dependency on EGFR signals.

On the basis of these clues, we explored whether potentiated inactivation of EGFR was sufficient to induce massive regression (more than 75%) in those tumors that responded to cetuximab alone with only growth inhibition or limited shrinkage. Specifically, we randomly extracted from our entire collection of quadruple negative xenopatients 21 SD cases (fig. S4) and comparatively tested the effects of cetuximab alone versus a combination of cetuximab plus lapatinib, a dual EGFR/HER2 small-molecule inhibitor (combo1). After 4 weeks of treatment with combo1, five cases showed more than 75% tumor volume reduction (Fig. 2A), which was not achieved by single-agent treatment with cetuximab (table S3). Figure 2B shows the response to individual inhibitors or combo1 over time in three representative cases. Lapatinib alone was overall poorly effective, irrespective of the final outcome of the combination therapy. In cases in which combo1 was advantageous, regression occurred rapidly and proceeded steadily until almost complete eradication of the tumor mass (M079). In other instances, tumor growth kinetics were affected similarly by cetuximab alone and cetuximab plus lapatinib, with cases experiencing either long-lasting (M304) or short-lived (M241) disease stabilization.

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Anti–Ki-67 staining of end-of-treatment tumor sections from a representative responder (M079) revealed proliferation defects consistent with the tumor growth curves: cetuximab, but not lapatinib, decreased the number of proliferating cells, which was further abated by combo1 (Fig. 3A). Similarly, cetuximab considerably reduced the phosphorylation of extracellular signal–regulated kinase (ERK) and S6, two canonical EGFR downstream signals along the RAS and phosphatidylinositol 3-kinase (PI3K) pathways, respectively (Fig. 3A). S6 phosphorylation was incrementally depressed by combo1 relative to cetuximab alone, whereas the plateau of ERK deactivation achieved by cetuximab monotherapy was not further affected by combinatorial treatment (Fig. 3A). Reductions in proliferation index and transducers’ activity were accompanied by a corresponding decrement in tumor cellularity at morphological inspection (fig. S5A). Apoptosis, as assessed by active caspase-3 immunoreactivity, was barely detectable in control tumors, was only modestly triggered by combo1 after acute treatment (24 hours), and was again almost imperceptible after prolonged therapy (6 weeks) (fig. S5B). These results suggest that the contribution of combo1 was to shift the steady-state proliferation kinetics of the tumor mass toward a new equilibrium in which attrition was favored over growth, without appreciable induction of cancer cell death.

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Lapatinib inactivates both EGFR and HER2. Therefore, it could synergize with anti-EGFR antibody therapy either by disrupting the enzymatic potential of the EGFR/HER2 complex or by complementing cetuximab for more drastic and specific inhibition of EGFR. To address this issue, we treated M079 with erlotinib, an EGFR-specific inhibitor free from off-target effects at standard concentrations. Similar to lapatinib, erlotinib monotherapy was unable to induce regression, and the combo of cetuximab and erlotinib was as effective as the lapatinib combo in prompting tumor involution (Fig. 3B). As an alternative proof of concept, we boosted EGFR inactivation by increasing the dosage of cetuximab up to fivefold (from 20 to 100 mg/kg). Again, intensified (and selective) EGFR neutralization resulted in tumor shrinkage (Fig. 3B), further pointing to more pronounced blockade of EGFR as a means to achieve better responses.

Expression analysis of parameters that influence response to combination therapies against the EGFR pathway

Concomitant blockade of EGFR by cetuximab and lapatinib/erlotinib was sufficient to turn cetuximab-induced SDs into major ORs in a subset of cases (5 of 21, 23.8%); however, the majority of tumors did not benefit from the addition of small-molecule EGFR inhibitors (Fig. 2A). To explore whether sensitivity to combo1 was more pronounced in SD cases with particularly high EGFR pathway activity, we compared the expression of EGFR family receptors and ligands in combo1 responders versus resistant cases. Although individual differences were quantitatively modest, a general trend toward higher expression of several family members was noticed in responders (HER3, +34%; EGF, +24%; epiregulin, +16%; EGFR, +10%) (fig. S6 and table S1). This suggests that relatively weak increases in the expression of multiple EGFR pathway components might sustain a stronger dependency on EGFR signals and increase the cells’ responsiveness to potentiated EGFR inhibition.

We also reasoned that the engagement of survival signals redundant to those transduced by the EGFR pathway and mediated by other cytoprotective cues could be responsible for additional mechanisms of resistance to enhanced EGFR inhibition (23). A recent genome-scale survey of CRC tumors reported considerably higher IGF2 gene expression (as much as 100-fold increase) in 15% of samples (24). The encoded product, insulin-like growth factor 2 (IGF2), triggers stereotyped antiapoptotic signals that are conveyed by the high-affinity IGF1 tyrosine kinase receptor (IGF1R) and the low-affinity insulin receptor (IR) (25). We detected strong IGF2 overexpression by RT-qPCR in 6 of the 21 cases (28.6%) that had been treated with combo1 (Fig. 4A and table S1). In accordance with the assumption that high IGF2 levels may protect from EGFR pathway inactivation, these 6 IGF2 overexpressors were among the 16 cases in which the cetuximab plus lapatinib combination was not superior to cetuximab alone (6 of 16, 37.5%) (Fig. 4A and table S1). Consistent with ligand-dependent receptor activation, IGF1R tyrosine phosphorylation was strong in two representative models with IGF2 overexpression (M040 and M044) and almost undetectable in two models with lower IGF2 expression (M280 and M328) (fig. S7 and table S1).

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When we reanalyzed the levels of EGFR family ligands and receptors in cases without IGF2 overexpression, the higher expression of EGFR and epiregulin in sensitive cases became more evident (EGFR, +27%; epiregulin, +30%; HER3, +23%; EGF, +19%) (fig. S8 and table S1). This further credentials high EGFR pathway activity as a determinant of responsiveness to potentiated EGFR blockade in cases without other obvious mechanisms of desensitization.

Overexpression of IGF2 and reduced sensitivity to cetuximab: Correlations in xenopatients

Extension of IGF2 expression analysis to the entire xenopatient collection (table S1) confirmed the presence of a defined subpopulation of IGF2 overexpression outliers (fig. S9). Although the association was not statistically significant by Fisher’s exact test (P = 0.078), a 3.6-fold enrichment of IGF2 outliers was detected in SD cases (14 of 60, 23.2%) compared to cases that responded with tumor shrinkage (OR) (2 of 31, 6.4%) (Fig. 4B). The percentage of tumors with increased IGF2 expression was higher in SDs than in overtly resistant tumors (PD) (6 of 34, 17.6%) (Fig. 4B). These gene expression data were obtained from passaged samples, indicating autocrine production of IGF2 by cancer cells, similar to that described for EGFR family ligands and receptors. Changes in IGF2 expression, consistent with RT-qPCR data, were confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA) in a subset of 25 cases randomly selected from all samples (Fig. 4C). qPCR gene copy number analysis, performed on the same 25 tumors subjected to ELISA assays, revealed that a subset of samples with higher levels of IGF2 also displayed copy number gains of the IGF2 locus (Fig. 4D), in accordance with previous findings (24).

IGF2 is part of a complex network that encompasses the canonical receptor IGF1R, a second growth factor with stimulating activity (IGF1) and additional components with inhibitory functions, including six IGF binding proteins (IGFBPs) that limit ligand bioavailability, as well as one receptor (IGF2R) that targets the ligands for degradation in the absence of signal transduction (25). Expression analysis of all these genes did not reveal substantial differences between IGF2 overexpressors and the rest of the cases (fig. S10 and table S1). Similarly, IGF2 levels were not consistently affected by cetuximab in the 14 sensitive models for which expression data after 72 hours and 6 weeks of antibody treatment were available (fig. S11 and table S2). Hence, IGF2 activity in IGF2 outliers depended on intrinsic expression rather than on modulation by regulatory factors. Moreover, IGF2 expression was not influenced by EGFR blockade.

Overexpression of IGF2 and reduced sensitivity to cetuximab: Correlations in patients

To assess the clinical relevance of these results, we explored whether IGF2 overexpressors were enriched in tumors from patients who exhibited attenuated response to anti-EGFR antibodies. We started by mining gene expression profiles from a publicly available microarray data set comprising 48 KRAS wild-type CRC liver metastases from patients treated with cetuximab monotherapy (GSE5851, table S1) (20). In agreement with results in xenopatients, a distinct subpopulation featuring strong IGF2 overexpression (more than fourfold above the median expression in the entire data set) was detected in many cases categorized as SDs (10 of 17, 58.8%) but not in ORs (0 of 6, P = 0.051 by Fisher’s exact test) (Fig. 5A); IGF2 overexpressors were less represented among PDs (5 of 25, 20%) (Fig. 5A).

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Then, we analyzed IGF2 expression in a retrospective cohort of 35 quadruple negative cases from primary CRCs collected at our institutions and annotated for therapeutic responses to cetuximab and irinotecan in the chemorefractory setting (table S1) (26). Again, samples featuring more than fourfold IGF2 overexpression segregated in the SD category (3 of 14, 21.4%), with no IGF2-high cases among ORs (0 of 19, P = 0.067 by Fisher’s exact test) or PDs (0 of 2) (Fig. 5B). Also in patient material, in agreement with results in xenopatients, IGF1R was highly phosphorylated in one representative IGF2-overexpressing tumor (S0307F111a) and weakly phosphorylated in another case with lower IGF2 expression (S0307F118a) (fig. S12 and table S1).

In aggregate, when combining both data sets, more than fourfold overexpression of IGF2 was observed in 41.9% of patients whose tumors stabilized on anti-EGFR antibodies (13 of 31) and in none of the patients who responded with tumor regression (0 of 25) (P = 0.0002 by Fisher’s exact test) (Fig. 5, A and B, and table S1). Among the 27 patients with fully resistant tumors, IGF2 overexpression was observed in 5 cases (18.5%) (Fig. 5, A and B, and table S1). When coupled with results in xenopatients, these findings strongly support the role of IGF2 as a determinant of reduced sensitivity to EGFR-targeted therapies in mCRC.

Overexpression of IGF2 and reduced sensitivity to cetuximab in cell cultures

We undertook functional studies in cell cultures to validate the possible role of IGF2 in blunting sensitivity to anti-EGFR antibodies. On the basis of gene expression data obtained by Illumina beadchip arrays in a panel of 151 CRC cell lines (table S4), we selected two models that featured different endogenous levels of IGF2 (NCI-H508, low expression; HDC142, high expression). Differences in gene expression were confirmed at the transcript level by RT-qPCR (Fig. 6A) and at the protein level by ELISA (Fig. 6B). Mutational profiling showed that both cell lines are wild type for KRAS (exons 2 to 4), NRAS (exons 2 and 3), HRAS (exons 2 and 3), and BRAF (exon 15) (27, 28). HDC142 is wild type for PIK3CA (exons 9 and 20), whereas NCI-H508 carries a PIK3CA exon 9 mutation (E545K) (27, 28), which has been demonstrated not to influence response to anti-EGFR antibodies (7). Hence, the genetic makeup of these cell lines is congruent with the clinical observations about the mutational status of CRC patients most likely to respond to anti-EGFR antibodies.

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In line with in vivo results, IGF2 content dictated sensitivity to EGFR blockade, with modest growth inhibition in IGF2-overexpressing HDC142 cells (at cetuximab concentrations as high as 20 μg/ml) and more pronounced inhibition in low-IGF2 NCI-H508 (Fig. 6C). Similarly, exogenous transduction of NCI-H508 with IGF2-encoding lentiviral vectors (Fig. 6, A and B) resulted in weakened response to EGFR inactivation compared with controls: even with higher concentrations of cetuximab (up to 40 μg/ml), the inhibition plateau was about 20% in IGF2 ectopic overexpressors versus 70% in mock-transduced cells (Fig. 6D). Collectively, these in vitro results solidify the notion that elevated IGF2 expression decreases CRC responsiveness to anti-EGFR antibodies.

Targeting the IGF2 pathway to improve response to cetuximab

We reasoned that pharmacologic interception of IGF2-driven survival signals may sensitize tumors to EGFR inhibition. Many drugs targeting the IGF1/IGF2 pathway are in late-stage clinical studies, in particular monoclonal antibodies and small-molecule inhibitors of IGF1R (25). Among them, the adenosine triphosphate (ATP)–mimetic IGF1R/IR inhibitor BMS-754807 (29, 30) is one of the most advanced compounds, although the enrolment into trials for this drug has been recently halted because of a lack of efficacy in unselected patients (31). Prompted by the assumption that high-level overexpression of IGF2 could be exploited as a predictive biomarker for molecular stratification of responders, we investigated whether concomitant inactivation of EGFR (by cetuximab) and IGF1R/IR (by BMS-754807) could be beneficial in IGF2-overexpressing CRC tumors.

We tested the efficacy of BMS-754807 in four IGF2-overexpressing xenopatients (M040, M044, M105, and M380) and one control with low IGF2 levels (M053). For each case, the original tumor specimen was passaged in vivo until we had enough mice for concomitant evaluation of four treatment arms: (i) vehicle; (ii) cetuximab alone; (iii) BMS-754807 alone; (iv) cetuximab and BMS-754807 (combo2). BMS-754807 alone slightly retarded tumor growth, regardless of IGF2 expression (Fig. 7A and fig. S13A). Cetuximab monotherapy was confirmed to induce disease stabilization with the exception of case M105, which had experienced SD in our initial screening but proved to be refractory to EGFR inhibition in this new set of experiments (Fig. 7A and fig. S13A). The antitumor activity of combo2 was superior to that of either monotherapy in the four IGF2 overexpressors, with variable induction of tumor shrinkage in M040, M044, and M380, and delay of tumor growth in M105 (Fig. 7A and fig. S13A). Conversely, M053 xenopatients, which had normal IGF2 levels, did not receive any benefit from combo2 compared with cetuximab alone (Fig. 7A). This indicates that BMS-754807 is effective only in the presence of high IGF2 content, supporting compound specificity.

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Anti–Ki-67 staining of a representative IGF2-overexpressing tumor (M040) indicated that single-agent cetuximab or BMS-754807 was sufficient to decrease cell proliferation, which was further diminished by combination therapy (Fig. 7B). Again, similar to combo1, no evident effect on apoptosis was detected (fig. S13B). At the signaling level, the combination of cetuximab and BMS-754807 was more potent than cetuximab alone in lowering the phosphorylation levels of S6 and ERK (Fig. 7B). Histologically, the posttreatment tissue of tumors responsive to combo2 displayed reduced cellularity and pseudoglandular differentiation, with acinar structures lined by pluristratified epithelium and embedded in large necrotic areas (fig. S13B). Together, these results provide strong indication that dual blockade of EGFR and IGF2 signaling might be beneficial to improve response in cases that display incomplete sensitivity to anti-EGFR antibodies and aberrantly high IGF2 expression. The observation that susceptibility to treatment was dictated by the degree of IGF2 expression suggests that evaluation of IGF2 content could be exploited to select for potentially responsive patients.