Emily F. Dunn

Understanding resistance to EGFR inhibitors—impact on future treatment strategies

EGFR is a tyrosine kinase that participates in the regulation of cellular homeostasis. Following ligand binding, EGFR stimulates downstream cell signaling cascades that influence cell proliferation, apoptosis, migration, survival and complex processes, including angiogenesis and tumorigenesis. EGFR has been strongly implicated in the biology of human epithelial malignancies, with therapeutic applications in cancers of the colon, head and neck, lung, and pancreas. Accordingly, targeting EGFR has been intensely pursued, with the development of a series of promising molecular inhibitors for use in clinical oncology. As is common in cancer therapy, challenges with respect to treatment resistance emerge over time. This situation is certainly true of EGFR inhibitor therapies, where intrinsic and acquired resistance is now well recognized. In this Review, we provide a brief overview regarding the biology of EGFR, preclinical and clinical development of EGFR inhibitors, and molecular mechanisms that underlie the development of treatment resistance. A greater understanding of the mechanisms that lead to EGFR resistance may provide valuable insights to help design new strategies that will enhance the impact of this promising class of inhibitors for the treatment of cancer.

Nuclear EGFR Contributes to Acquired Resistance to Cetuximab

Epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase involved in the etiology of several human cancers. Cetuximab is an EGFR-blocking antibody that has been approved for the treatment of patients with head and neck squamous cell carcinoma and metastatic colorectal cancer. Previous reports have shown that EGFR translocation to the nucleus is associated with cell proliferation. Here we investigated mechanisms of acquired resistance to cetuximab using a model derived from the non-small cell lung cancer line H226. We demonstrated that cetuximab-resistant cells overexpress HER family ligands including epidermal growth factor (EGF), amphiregulin, heparin-binding EGF and β-cellulin. Overexpression of these ligands is associated with the nuclear translocation of the EGFR and this process was mediated by the Src family kinases (SFK). Treatment of cetuximab-resistant cells with the SFK inhibitor, dasatinib, resulted in loss of nuclear EGFR, increased membrane expression of the EGFR and resensitization to cetuximab. In addition, expression of a nuclear localization sequence-tagged EGFR in cetuximab-sensitive cells increased resistance to cetuximab both in vitro and in mouse xenografts. Collectively, these data suggest that nuclear expression of EGFR may be an important molecular determinant of resistance to cetuximab therapy and provides a rationale for investigating nuclear EGFR as a biomarker for cetuximab response. Further, these data suggest a rationale for the design of clinical trials that examine the value of treating patients with cetuximab-resistant tumors with inhibitors of SFKs in combination with cetuximab.

The Role of Src in Solid Tumors

The proto-oncogene c-Src (Src) encodes a nonreceptor tyrosine kinase whose expression and activity are correlated with advanced malignancy and poor prognosis in a variety of human cancers. Nine additional enzymes with homology to Src have been identified and collectively are referred to as Src family kinases (SFKs). Together, SFKs represent the largest family of nonreceptor tyrosine kinases and interact directly with receptor tyrosine kinases, G-protein-coupled receptors, steroid receptors, signal transducers and activators of transcription, and molecules involved in cell adhesion and migration. These interactions lead to a diverse array of biological functions including proliferation, cell growth, differentiation, cell shape, motility, migration, angiogenesis, and survival. Studies investigating mutational activation of Src in human cancers suggest that this may be a rare event and that wild-type Src is weakly oncogenic. Thus, the role of Src in the development and progression of human cancer remains unclear. Recently, it was suggested that increased SFK protein levels and, more importantly, SFK tyrosine kinase activity are linked to cancer progression and metastatic disease by facilitating the action of other signaling proteins. This accumulating body of evidence indicates that SFKs may represent a promising therapeutic target for the treatment of solid tumors. This review discusses the role of SFKs in solid tumors and the recent therapeutic advances aimed at targeting this family of tyrosine kinases in cancer.

Epidermal Growth Factor Receptor cooperates with Src Family Kinases in acquired resistance to cetuximab

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a major role in oncogenesis. Cetuximab is an EGFR-blocking antibody that is FDA approved for use in patients with metastatic colorectal cancer (mCRC) and head and neck squamous cell carcinoma (HNSCC). Although cetuximab has shown strong clinical benefit for a subset of cancer patients, most become refractory to cetuximab therapy. We reported that cetuximab-resistant NSCLC line NCI-H226 cells have increased steady-state expression and activity of EGFR secondary to altered trafficking/degradation and this increase in EGFR expression and activity lead to hyper-activation of HER3 and down stream signals to survival. We now present data that Src family kinases (SFKs) are highly activated in cetuximab-resistant cells and enhance EGFR activation of HER3 and PI(3)K/Akt. Studies using the Src kinase inhibitor dasatinib decreased HER3 and PI(3)K/Akt activity. In addition, cetuximab-resistant cells were resensitized to cetuximab when treated with dasatinib. These results indicate that SFKs and EGFR cooperate in acquired resistance to cetuximab and suggest a rationale for clinical strategies that investigate combinatorial therapy directed at both the EGFR and SFKs in patients with acquired resistance to cetuximab.

Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, Sprycel), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases (SFKs). We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (EGFR) and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo and HCT116) cell lines and two KRAS wild-type cell lines (SW48 and CaCo2). In vitro, using poly-D-lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab, whereas KRAS wild-type lines showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo. To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments. Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation, as measured by Ki67, and higher rates of apoptosis, as measured by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways. Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting.

Severe Late Toxicities Following Concomitant Chemoradiotherapy Compared to Radiotherapy Alone in Cervical Cancer: An Inter-era Analysis

PURPOSE To compare rates of severe late toxicities following concomitant chemoradiotherapy and radiotherapy alone for cervical cancer. METHODS AND MATERIALS Patients with cervical cancer were treated at a single institution with radiotherapy alone or concomitant chemoradiotherapy for curative intent. Severe late toxicity was defined as grade≥3 vaginal, urologic, or gastrointestinal toxicity or any pelvic fracture, using Common Terminology Criteria for Adverse Events version 4.0 (CTCAE), occurring ≥6 months from treatment completion and predating any salvage therapy. Severe late toxicity rates were compared after adjusting for pertinent covariates. RESULTS At 3 years, probability of vaginal severe late toxicity was 20.2% for radiotherapy alone and 35.1% for concomitant chemoradiotherapy (P=.026). At 3 years, probability of skeletal severe late toxicity was 1.6% for radiotherapy alone and 7.5% for concomitant chemoradiotherapy (P=.010). After adjustment for case mix, concomitant chemoradiotherapy was associated with higher vaginal (hazard ratio [HR] 3.0, 95% confidence interval [CI], 1.7-5.2, P<.001), and skeletal (HR 7.0, 95% CI 1.4-34.1, P=.016) severe late toxicity. Compared to high dilator compliance, moderate (HR 3.6, 95% CI 2.0-6.5, P<.001) and poor (HR 8.5, 95% CI 4.3-16.9, P<.001) dilator compliance was associated with higher vaginal severe late toxicity. Age>50 was associated with higher vaginal (HR 1.8, 95% CI 1.1-3.0, P=.013) and skeletal (HR 5.7, 95% CI 1.2-27.0, P=.028) severe late toxicity. Concomitant chemoradiotherapy was not associated with higher gastrointestinal (P=.886) or urologic (unadjusted, P=.053; adjusted, P=.063) severe late toxicity. CONCLUSION Compared to radiotherapy alone, concomitant chemoradiotherapy is associated with higher rates of severe vaginal and skeletal late toxicities. Other predictive factors include dilator compliance for severe vaginal late toxicity and age for severe vaginal and skeletal late toxicities.

Dasatinib blocks cetuximab- and radiation-induced nuclear translocation of the epidermal growth factor receptor in head and neck squamous cell carcinoma

BACKGROUND AND PURPOSE The aberrant expression of epidermal growth factor receptor (EGFR) has been linked to the etiology of head and neck squamous cell carcinoma (HNSCC). The first major phase III trial combining cetuximab with radiation confirmed a strong survival advantage. However, both cetuximab and radiation can promote EGFR translocation to the nucleus where it enhances resistance to both of these modalities. In this report we sought to determine how to block cetuximab- and radiation-induced translocation of EGFR to the nucleus in HNSCC cell lines. MATERIAL AND METHODS We utilized three established HNSCC cell lines, SCC1, SCC6 and SCC1483 and measured nuclear translocation of EGFR after treatment with cetuximab or radiation. We then utilized dasatinib (BMS-354825), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases, to determine if SFKs blockade could abrogate cetuximab- and radiation-induced nuclear EGFR translocation. RESULTS Cetuximab and radiation treatment of all three HNSCC lines lead to translocation of the EGFR to the nucleus. Blockade of SFKs abrogated cetuximab- and radiation-induced EGFR translocation to the nucleus. CONCLUSIONS The data presented in this report suggest that both cetuximab and radiation can promote EGFR translocation to the nucleus and dasatinib can inhibit this process. Collectively these findings may suggest that dasatinib can limit EGFR translocation to the nucleus and may enhance radiotherapy plus cetuximab in HNSCC.

Involving American Indians and medically underserved rural populations in cancer clinical trials

Purpose To assess cancer clinical trial recruitment and reasons for nonaccrual among a rural, medically underserved population served by a community-based cancer care center. Methods We prospectively tracked clinical trial enrollment incidence among all new patients presenting at the Rapid City Regional Cancer Care Institute. Evaluating physicians completed questionnaires for each patient regarding clinical trial enrollment status and primary reasons for nonenrollment. Patients who identified as American Indian were referred to a program where patients were assisted in navigating the medical system by trained, culturally competent staff. Results Between September 2006 and January 2008, 891 new cancer patients were evaluated. Seventy-eight patients (9%; 95% confidence intervals, 7—11%) were enrolled on a clinical treatment trial. For 73% (95% confidence intervals, 69—75%) of patients (646 of 891) lack of relevant protocol availability or protocol inclusion criteria restrictiveness was the reason for nonenrollment. Only 45 (5%; 95% confidence intervals, 4—7%) patients refused enrollment on a trial. Of the 78 enrolled on a trial, 6 (8%; 95% confidence intervals, 3—16%) were American Indian. Three additional American Indian patients were enrolled under a nontreatment cancer control trial, bringing the total percentage enrolled of the 94 American Indians who presented to the clinic to 10% (95% confidence intervals, 5—17%). Limitations Eligibility rates were unable to be calculated and cross validation of the number in the cohort via registries or ICD-9 codes was not performed. Conclusion Clinical trial participation in this medically underserved population was low overall, but approximately 3-fold higher than reported national accrual rates. Lack of availability of protocols for common cancer sites as well as stringent protocol inclusion criteria were the primary obstacles to clinical trial enrollment. Targeted interventions using a Patient Navigation program were used to engage AI patients and may have resulted in higher clinical trial enrollment among this racial/ethnic group. Clinical Trials 2009; 6: 610—617. http://ctj.sagepub.com

Effects of Treatment Duration During Concomitant Chemoradiation Therapy for Cervical Cancer

PURPOSE To determine whether extended treatment duration (TD) impacts in-field relapse and survival in the setting of concomitant chemoradiation therapy (CRT) for cervical cancer. METHODS AND MATERIALS A total of 480 consecutive cervical cancer patients treated with radiation therapy (RT) alone or concomitant CRT for curative intent were retrospectively analyzed. Relapse was defined as in-field with respect to external beam radiation therapy fields. The effects of TD on in-field relapse, disease-free survival (DFS), and overall survival (OS) rates were assessed continuously and categorically within the separate RT and CRT cohorts. Covariates included age, histology, stage, and cumulative dose to point A. In-field relapse, DFS, and OS rates were estimated with Kaplan-Meier analysis; comparisons used log-rank statistic. Multivariate analysis used the Cox proportional hazards model. RESULTS A total of 372 patients (RT n=206, CRT n=166) were evaluable, with a median follow-up for relapse-free patients of 4.2 years (RT 4.4 years, CRT 4.2 years; P=.807). Treatment duration was longer in the RT cohort (median 55 days; range 35-99 days) versus the CRT cohort (median 51 days; range 35-92 days) (P=.001). In the RT cohort, TD ≥62 days trended to significance for predicting inferior DFS (hazard ratio 1.42, 95% confidence interval 0.86-1.98, P=.086). However, in the CRT cohort, TD assessed continuously or categorically across multiple cutoff thresholds did not predict for in-field relapse, DFS, or OS. CONCLUSION With RT alone, extended TD ≥62 days may adversely impact treatment efficacy. With the addition of concomitant chemotherapy to RT, however, extended TD has no effect on treatment efficacy.

Nuclear Epidermal Growth Factor Receptor Is a Functional Molecular Target in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a subclass of breast cancers (i.e., estrogen receptor–negative, progesterone receptor–negative, and HER2-negative) that have poor prognosis and very few identified molecular targets. Strikingly, a high percentage of TNBCs overexpresses the EGF receptor (EGFR), yet EGFR inhibition has yielded little clinical benefit. Over the last decade, advances in EGFR biology have established that EGFR functions in two distinct signaling pathways: (i) classical membrane-bound signaling and (ii) nuclear signaling. Previous studies have demonstrated that nuclear EGFR (nEGFR) can enhance resistance to anti-EGFR therapies and is correlated with poor overall survival in breast cancer. On the basis of these findings, we hypothesized that nEGFR may promote intrinsic resistance to cetuximab in TNBC. To examine this question, a battery of TNBC cell lines and human tumors were screened and found to express nEGFR. Knockdown of EGFR expression demonstrated that TNBC cell lines retained dependency on EGFR for proliferation, yet all cell lines were resistant to cetuximab. Furthermore, Src Family Kinases (SFKs) influenced nEGFR translocation in TNBC cell lines and in vivo tumor models, where inhibition of SFK activity led to potent reductions in nEGFR expression. Inhibition of nEGFR translocation led to a subsequent accumulation of EGFR on the plasma membrane, which greatly enhanced sensitivity of TNBC cells to cetuximab. Collectively, these data suggest that targeting both the nEGFR signaling pathway, through the inhibition of its nuclear transport, and the classical EGFR signaling pathway with cetuximab may be a viable approach for the treatment of patients with TNBC. Mol Cancer Ther; 13(5); 1356–68. ©2014 AACR.