Targeting the effector domain of the myristoylated alanine rich C-kinase substrate enhances lung cancer radiation sensitivity

Lung cancer is the leading cause of cancer related deaths. Common molecular drivers of lung cancer are mutations in receptor tyrosine kinases (RTKs) leading to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pro-growth, pro-survival signaling pathways. Myristoylated alanine rich C-kinase substrate (MARCKS) is a protein that has the ability to mitigate this signaling cascade by sequestering the target of PI3K, phosphatidylinositol (4,5)-bisphosphate (PIP2). As such, MARCKS has been implicated as a tumor suppressor, though there is some evidence that MARCKS may be tumor promoting in certain cancer types. Since the MARCKS function depends on its phosphorylation status, which impacts its subcellular location, MARCKS role in cancer may depend highly on the signaling context. Currently, the importance of MARCKS in lung cancer biology is limited. Thus, we investigated MARCKS in both clinical specimens and cell culture models. Immunohistochemistry scoring of MARCKS protein expression in a diverse lung tumor tissue array revealed that the majority of squamous cell carcinomas stained positive for MARCKS while other histologies, such as adenocarcinomas, had lower levels. To study the importance of MARCKS in lung cancer biology, we used inducible overexpression of wild-type (WT) and non-phosphorylatable (NP)-MARCKS in A549 lung cancer cells that had a low level of endogenous MARCKS. We found that NP-MARCKS expression, but not WT-MARCKS, enhanced the radiosensitivity of A549 cells in part by inhibiting DNA repair as evidenced by prolonged radiation-induced DNA double strand breaks. We confirmed the importance of MARCKS phosphorylation status by treating several lung cancer cell lines with a peptide mimetic of the phosphorylation domain, the effector domain (ED), which effectively attenuated cell growth as measured by cell index. Thus, the MARCKS ED appears to be an important target for lung cancer therapeutic development

Cetuximab Augments Cytotoxicity with Poly (ADP-Ribose) Polymerase Inhibition in Head and Neck Cancer

Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of head and neck cancers and confers increased resistance and inferior survival rates. Despite targeted agents against EGFR, such as cetuximab (C225), almost half of treated patients fail this therapy, necessitating novel therapeutic strategies. Poly (ADP-Ribose) polymerase (PARP) inhibitors (PARPi) have gained recent attention due to their unique selectivity in killing tumors with defective DNA repair. In this study, we demonstrate that C225 enhances cytotoxicity with the PARPi ABT-888 in UM-SCC1, UM-SCC6, and FaDu head and neck cancer cells. The mechanism of increased susceptibility to C225 and PARPi involves C225-mediated reduction of non-homologous end-joining (NHEJ)- and homologous recombination (HR)-mediated DNA double strand break (DSB) repair, the subsequent persistence of DNA damage, and activation of the intrinsic apoptotic pathway. By generating a DSB repair deficiency, C225 can render head and neck tumor cells susceptible to PARP inhibition. The combination of C225 and the PARPi ABT-888 can thus be an innovative treatment strategy to potentially improve outcomes in head and neck cancer patients. Furthermore, this strategy may also be feasible for other EGFR overexpressing tumors, including lung and brain cancers

Cetuximab (C225) attenuates non-homologous end-joining (NHEJ) repair.

<p>C225 attenuates irradiation (IR)-induced DNA-Pk Thr2609 foci, well established markers of non-homologous end joining (NHEJ)-mediated DNA DSB repair in (A) UM-SCC1, (B) UM-SCC6, and (C) FaDu head and neck cancer cells. Cells were treated with vehicle, 2.5 µg/mL C225, or 5.0 µg/mL C225 for 16 hours and subsequently subjected to mock or 4 Gy IR. At the indicated time following IR, cells were processed for immunofluorescence staining for DNA-Pk Thr2609 foci. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with >10 foci (*p<0.05, **p<0.01 compared to cells not exposed to C225). (D) C225 reduces phospho-Thr2609 DNA-Pk levels in UM-SCC6 head and neck cancer cells. Cells were treated with vehicle or 2.5 µg/mL C225 for 16 hours and subsequently subjected to mock or 4 Gy IR. One hour following IR, cells were processed for Western blot analysis for phospho-Thr2609 DNA Pk levels. Total DNA Pk was also analyzed and tubulin was used as loading control.</p

Cetuximab (C225) increases DNA damage by inhibiting DNA double strand break (DSB) repair in head and neck cancer cells.

<p>(A) C225 increases the number of cells with DSBs as evidenced by γ-H2AX foci, a commonly used marker for DSBs. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with >10 foci (*p<0.05, **p<0.01 compared to vehicle control). (B) C225 increases γ-H2AX protein levels in treated cells. UM-SCC1, UM-SCC6, and FaDu cells were treated with vehicle, 2.5 µg/mL C225, or 5.0 µg/mL C225 for 16 hours. Following the treatment period, cells were processed for (A) immunofluorescence staining for γ-H2AX foci or (B) western blot analysis for γ-H2AX levels. Shown is the representative Western blot of 3 independent experiments.</p

Cetuximab (C225) attenuates homologous recombination (HR) repair.

<p>C225 attenuates IR-induced Rad51 foci, well characterized markers of homologous recombination (HR)-mediated DNA DSB repair in (A) UM-SCC1, (B) UM-SCC6, and (C) FaDu cells. Cells were treated with vehicle, 2.5 µg/mL C225, or 5.0 µg/mL C225 for 16 hours and subsequently subjected to mock or 4 Gy irradiation (IR). At the indicated times following IR, cells were processed for immunofluorescence staining for Rad51 foci. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with >10 foci (*p<0.05, **p<0.01 compared to vehicle at each respective time point). The inset in (A) is a representative image of UM-SCC1 cells exhibiting Rad51 foci following IR.</p

Cetuximab (C225) enhances cytotoxicity with the PARP inhibitor ABT-888 in head and neck cancer cells.

<p>(A) Combination C225 and ABT-888 reduces the viability of UM-SCC1, UM-SCC6, and FaDu head and neck cancer cells. Cells were treated with either vehicle or 2.5 µg/mL C225 for 16 hours and subsequently exposed to vehicle or 10 µM ABT-888. Twenty-four hours following ABT-888, cell viability was assayed with the ATPlite system (Perkin Elmer). Shown is the representative data of at least 3 independent experiments of the cell viability following various treatments as measured by relative ATP levels (mean +/− SEM, *p<0.01, **p<0.001 compared to vehicle control). (B–D) Combination C225 and ABT-888 reduces the colony forming ability of (B) UM-SCC1, (C) UM-SCC6, and (D) FaDu head and neck cancer cells. Cells were treated with either vehicle or 2.5 µg/mL C225 for 16 hours. Following the treatment period, cells were seeded for colony formation assays and subjected to various doses of ABT-888. Shown is the mean survival fraction (+/− SEM) from at least 3 independent colony formation assay experiments following treatment (**p<0.001).</p

Novel EGFR ectodomain mutations associated with ligand-independent activation and cetuximab resistance in head and neck cancer.

Epidermal growth factor receptor (EGFR) is a pro-tumorigenic receptor tyrosine kinase that facilitates growth for cancer cells that overexpress the receptor. Monoclonal anti-EGFR antibody Cetuximab (CTX) provides significant clinical benefit in patients with head and neck squamous cell carcinoma (HNSCC). Missense mutations in the ectodomain (ECD) of EGFR can be acquired under CTX treatment and mimic the effect of large deletions on spontaneous untethering and activation of the receptor. Little is known about the contribution of EGFR ECD mutations to EGFR activation and CTX resistance in HNSCC. We identified two concurrent non-synonymous missense mutations (G33S and N56K) mapping to domain I in or near the EGF binding pocket of the EGFR ECD in patient-derived HNSCC cells that were selected for CTX resistance through repeated exposure to the agent in an effort to mimic what may occur clinically. Structural modeling predicted that the G33S and N56K mutants would restrict adoption of a fully closed (tethered) and inactive EGFR conformation while not permitting association of EGFR with the EGF ligand or CTX. Binding studies confirmed that the mutant, untethered receptor displayed reduced affinity for both EGF and CTX but demonstrated sustained activation and presence at the cell surface with diminished internalization and sorting for endosomal degradation, leading to persistent downstream AKT signaling. Our results demonstrate that HNSCC cells can select for EGFR ECD mutations under CTX exposure that converge to trap the receptor in an open, ligand-independent, constitutively activated state. These mutants impede the receptor's competence to bind CTX possibly explaining certain cases of CTX treatment-induced or de novo resistance to CTX

Combination cetuximab (C225) and ABT-888 induces persistent DNA double strand break damage in head and neck cancer cells.

<p>(A–C) DNA damage 2, 24, and 48 hours following vehicle, C225, PARPi, or both C225+PARPi was assessed by γ-H2AX foci in (A) UM-SCC1, (B) UM-SCC6, and (C) FaDu cells. Cells were treated with vehicle or various doses of C225 for 16 hours and subsequently exposed to vehicle or various doses of ABT-888. At the indicated times following PARP inhibition, cells were processed for immunofluorescence staining for γ-H2AX foci. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with >10 foci (*p<0.05, **p<0.01 compared to vehicle control at each respective time point).</p