Luca Lazzari

Blockade of EGFR and MEK Intercepts Heterogeneous Mechanisms of Acquired Resistance to Anti-EGFR Therapies in Colorectal Cancer

Colorectal cancers that become resistant to EGFR inhibitors through a variety of mechanisms can be effectively treated by inhibiting MEK in conjunction with EGFR. Circulating Tumor DNA for Early Detection and Managing Resistance Cancer evolves over time, without any warning signs. Similarly, the development of resistance to therapy generally becomes apparent only when there are obvious signs of tumor growth, at which point the patient may have lost valuable time. Although a repeat biopsy may be able to identify drug-resistant mutations before the tumor has a chance to regrow, it is usually not feasible to do many repeat biopsies. Now, two studies are demonstrating the utility of monitoring the patients’ blood for tumor DNA to detect cancer at the earliest stages of growth or resistance. In one study, Bettegowda and coauthors showed that sampling a patient’s blood may be sufficient to yield information about the tumor’s genetic makeup, even for many early-stage cancers, without a need for an invasive procedure to collect tumor tissue, such as surgery or endoscopy. The authors demonstrated the presence of circulating DNA from many types of tumors that had not yet metastasized or released detectable cells into the circulation. They could detect more than 50% of patients across 14 tumor types at the earliest stages, when these cancers may still be curable, suggesting that a blood draw could be a viable screening approach to detecting most cancers. They also showed that in patients with colorectal cancer, the information derived from circulating tumor DNA could be used to determine the optimal course of treatment and identify resistance to epidermal growth factor receptor (EGFR) blockade. Meanwhile, Misale and colleagues illustrated a way to use this information to overcome treatment resistance. These authors also found that mutations associated with EGFR inhibitor resistance could be detected in the blood of patients with colorectal cancer. In addition, they demonstrated that adding MEK inhibitors, another class of anticancer drugs, can successfully overcome resistance when given in conjunction with the EGFR inhibitors. Thus, the studies from Bettegowda and Misale and their colleagues show the effectiveness of analyzing circulating DNA from a variety of tumors and highlight the potential applications of this technology for early detection, monitoring resistance, and devising treatment plans to overcome resistance. Colorectal cancers (CRCs) that are sensitive to the anti–epidermal growth factor receptor (EGFR) antibodies cetuximab or panitumumab almost always develop resistance within several months of initiating therapy. We report the emergence of polyclonal KRAS, NRAS, and BRAF mutations in CRC cells with acquired resistance to EGFR blockade. Regardless of the genetic alterations, resistant cells consistently displayed mitogen-activated protein kinase kinase (MEK) and extracellular signal–regulated kinase (ERK) activation, which persisted after EGFR blockade. Inhibition of MEK1/2 alone failed to impair the growth of resistant cells in vitro and in vivo. An RNA interference screen demonstrated that suppression of EGFR, together with silencing of MEK1/2, was required to hamper the proliferation of resistant cells. Indeed, concomitant pharmacological blockade of MEK and EGFR induced prolonged ERK inhibition and severely impaired the growth of resistant tumor cells. Heterogeneous and concomitant mutations in KRAS and NRAS were also detected in plasma samples from patients who developed resistance to anti-EGFR antibodies. A mouse xenotransplant from a CRC patient who responded and subsequently relapsed upon EGFR therapy showed exquisite sensitivity to combinatorial treatment with MEK and EGFR inhibitors. Collectively, these results identify genetically distinct mechanisms that mediate secondary resistance to anti-EGFR therapies, all of which reactivate ERK signaling. These observations provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with targeted agents. We propose that MEK inhibitors, in combination with cetuximab or panitumumab, should be tested in CRC patients who become refractory to anti-EGFR therapies.

Tumor heterogeneity and lesion-specific response to targeted therapy in colorectal cancer

UNLABELLED How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent therapy is unknown. We studied EGFR blockade in colorectal cancer to assess whether tissue and liquid biopsies can be integrated with radiologic imaging to monitor the impact of individual oncogenic alterations on lesion-specific responses. Biopsy of a patients progressing liver metastasis following prolonged response to cetuximab revealed a MEK1(K57T) mutation as a novel mechanism of acquired resistance. This lesion regressed upon treatment with panitumumab and the MEK inhibitor trametinib. In circulating tumor DNA (ctDNA), mutant MEK1 levels declined with treatment, but a previously unrecognized KRAS(Q61H) mutation was also identified that increased despite therapy. This same KRAS mutation was later found in a separate nonresponding metastasis. In summary, parallel analyses of tumor biopsies and serial ctDNA monitoring show that lesion-specific radiographic responses to subsequent targeted therapies can be driven by distinct resistance mechanisms arising within separate tumor lesions in the same patient. SIGNIFICANCE Molecular heterogeneity ensuing from acquired resistance drives lesion-specific responses to subsequent targeted therapies. Analysis of a single-lesion biopsy is inadequate to guide selection of subsequent targeted therapies. ctDNA profiles allow the detection of concomitant resistance mechanisms residing in separate metastases and assessment of the effect of therapies designed to overcome resistance.

ROR1 is a pseudokinase that is crucial for MET-driven tumorigenesis

The human kinome includes Ror1, a poorly characterized orphan receptor. Here we report the findings of an investigation of Ror1 contributions to cancer, undertaken through an integrated screening of 43 cancer cell lines where we measured protein expression, tyrosine phosphorylation, and growth response following RNAi-mediated Ror1 suppression. Ror1 was expressed in approximately 75% of the cancer cell lines without apparent histotype distribution. Gastric carcinoma cells (HS746T) and non-small cell lung carcinoma cells (NCI-H1993) exhibited high levels of Ror1 tyrosine phosphorylation, and Ror1 suppression caused growth inhibition. Biochemical assays revealed unexpectedly that Ror1 is a pseudokinase that is devoid of catalytic activity. Intriguingly, the two cell lines featuring tyrosine-phosphorylated Ror1 both exhibited amplification and activation of the Met oncogene. Ror1 phosphorylation was abrogated by Met inhibition, indicating Met-dependent transphosphorylation of Ror1. Conversely, Ror1 was not transphosphorylated by other constitutively active tyrosine kinases, including EGFR and ErbB2. Constitutive silencing of Ror1 in HS746T and NCI-H1993 carcinoma cells impaired proliferation in vitro and induced a dramatic inhibition of tumorigenesis in vivo. Together, our findings suggest a critical role for Ror1 in malignant phenotypes sustained by the Met oncogene.

Emergence of Multiple EGFR Extracellular Mutations during Cetuximab Treatment in Colorectal Cancer

Purpose: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models. Experimental Design: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance. Results: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab. Conclusions: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor–antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies. Clin Cancer Res; 21(9); 2157–66. ©2015 AACR.

Acquired Resistance to the TRK Inhibitor Entrectinib in Colorectal Cancer

UNLABELLED Entrectinib is a first-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patients relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profiling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1, p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confirmed that either mutation renders the TRKA kinase insensitive to entrectinib. These findings can be immediately exploited to design next-generation TRKA inhibitors. SIGNIFICANCE We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identification of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the first time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements.

RAF Suppression Synergizes with MEK Inhibition in KRAS Mutant Cancer Cells

KRAS is the most frequently mutated oncogene in human cancer, yet no therapies are available to treat KRAS mutant cancers. We used two independent reverse genetic approaches to identify components of the RAS-signaling pathways required for growth of KRAS mutant tumors. Small interfering RNA (siRNA) screening of 37 KRAS mutant colorectal cancer cell lines showed that RAF1 suppression was synthetic lethal with MEK inhibition. An unbiased kinome short hairpin RNA (shRNA)-based screen confirmed this synthetic lethal interaction in colorectal as well as in lung cancer cells bearing KRAS mutations. Compounds targeting RAF kinases can reverse resistance to the MEK inhibitor selumetinib. MEK inhibition induces RAS activation and BRAF-RAF1 dimerization and sustains MEK-ERK signaling, which is responsible for intrinsic resistance to selumetinib. Prolonged dual blockade of RAF and MEK leads to persistent ERK suppression and efficiently induces apoptosis. Our data underlie the relevance of developing combinatorial regimens of drugs targeting the RAF-MEK pathway in KRAS mutant tumors.

Ron Kinase Transphosphorylation Sustains MET Oncogene Addiction

Receptors for the scatter factors HGF and MSP that are encoded by the MET and RON oncogenes are key players in invasive growth. Receptor cross-talk between Met and Ron occurs. Amplification of the MET oncogene results in kinase activation, deregulated expression of an invasive growth phenotype, and addiction to MET oncogene signaling (i.e., dependency on sustained Met signaling for survival and proliferation). Here we show that cancer cells addicted to MET also display constitutive activation of the Ron kinase. In human cancer cell lines coexpressing the 2 oncogenes, Ron is specifically transphosphorylated by activated Met. In contrast, Ron phosphorylation is not triggered in cells harboring constitutively active kinase receptors other than Met, including Egfr or Her2. Furthermore, Ron phosphorylation is suppressed by Met-specific kinase inhibitors (PHA-665752 or JNJ-38877605). Last, Ron phosphorylation is quenched by reducing cell surface expression of Met proteins by antibody-induced shedding. In MET-addicted cancer cells, short hairpin RNA-mediated silencing of RON expression resulted in decreased proliferation and clonogenic activity in vitro and tumorigenicity in vivo. Our findings establish that oncogene addiction to MET involves Ron transactivation, pointing to Ron kinase as a target for combinatorial cancer therapy.

MM-151 overcomes acquired resistance to cetuximab and panitumumab in colorectal cancers harboring EGFR extracellular domain mutations

An oligoclonal antibody overcomes drug resistance in colorectal cancers with EGFR mutations. The more the merrier Monoclonal antibodies against the epidermal growth factor receptor (EGFR), which drives cancer growth, are frequently used in colorectal cancer. Unfortunately, the cancers commonly develop drug-resistant mutations, and the monoclonal antibodies become ineffective. To overcome this problem, Arena et al. used an oligoclonal mixture of monoclonal antibodies called MM-151, which binds multiple parts of the EGFR molecule at once, so that the cancer cannot develop resistance by mutating one site at a time. The authors demonstrated that this approach is effective in both preclinical models and patients who were resistant to other anti-EGFR therapies, paving the way for further clinical development of the oligoclonal antibody. The anti–epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab are used to treat RAS wild-type colorectal cancers (CRCs), but their efficacy is limited by the emergence of acquired drug resistance. After EGFR blockade, about 20% of CRCs develop mutations in the EGFR extracellular domain (ECD) that impair antibody binding and are associated with clinical relapse. We hypothesized that EGFR ECD–resistant variants could be targeted by the recently developed oligoclonal antibody MM-151 that binds multiple regions of the EGFR ECD. MM-151 inhibits EGFR signaling and cell growth in preclinical models, including patient-derived cells carrying mutant EGFR. Upon MM-151 treatment, EGFR ECD mutations decline in circulating cell-free tumor DNA (ctDNA) of CRC patients who previously developed resistance to EGFR blockade. These data provide molecular rationale for the clinical use of MM-151 in patients who become resistant to cetuximab or panitumumab as a result of EGFR ECD mutations.

Acquired RAS or EGFR mutations and duration of response to EGFR blockade in colorectal cancer

Blockade of the epidermal growth factor receptor (EGFR) with the monoclonal antibodies cetuximab or panitumumab is effective in a subset of colorectal cancers (CRCs), but the emergence of resistance limits the efficacy of these therapeutic agents. At relapse, the majority of patients develop RAS mutations, while a subset acquires EGFR extracellular domain (ECD) mutations. Here we find that patients who experience greater and longer responses to EGFR blockade preferentially develop EGFR ECD mutations, while RAS mutations emerge more frequently in patients with smaller tumour shrinkage and shorter progression-free survival. In circulating cell-free tumour DNA of patients treated with anti-EGFR antibodies, RAS mutations emerge earlier than EGFR ECD variants. Subclonal RAS but not EGFR ECD mutations are present in CRC samples obtained before exposure to EGFR blockade. These data indicate that clonal evolution of drug-resistant cells is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies.

The ROR1 pseudokinase diversifies signaling outputs in MET-addicted cancer cells.

MET is a master gene controlling a genetic program driving proliferation, apoptosis protection and invasion. The ROR1 pseudokinase acts as a MET substrate. However, its contribution to MET signaling and MET‐dependent biological outcomes remains to be elucidated. By structure‐function analysis of ROR1 mutants, we show that ROR1 encompasses two major substrate regions: one is located in the proline‐rich domain and is directly phosphorylated by MET; the other resides in the pseudokinase domain and is phosphorylated through intermediate activation of SRC. Differential phosphorylation of these two regions dictates the execution of specific responses: phosphorylation of the ROR1 proline‐rich domain by MET—but not phosphorylation of the pseudokinase domain by SRC—is necessary and sufficient to control MET‐driven proliferation and protection from apoptosis. Differently, both the proline‐rich and the pseudokinase domains mediate cell invasion. Consistent with the role of ROR1 in specifying the functional consequences of MET‐dependent signals, ROR1 silencing leads to selective attenuation of only some of the signal transduction pathways sustained by MET. These data enlighten the so far elusive function(s) of pseudokinases and identify a mechanism of biological diversification, based on substrate specificity of oncogenic kinases.