Selective suppression of melanoma lacking IFN-γ pathway by JAK inhibition depends on T cells and host TNF signaling.

Therapeutic resistance to immune checkpoint blockers (ICBs) in melanoma patients is a pressing issue, of which tumor loss of IFN-γ signaling genes is a major underlying mechanism. However, strategies of overcoming this resistance mechanism have been largely elusive. Moreover, given the indispensable role of tumor-infiltrating T cells (TILs) in ICBs, little is known about how tumor-intrinsic loss of IFN-γ signaling (IFNγR1KO) impacts TILs. Here, we report that IFNγR1KO melanomas have reduced infiltration and function of TILs. IFNγR1KO melanomas harbor a network of constitutively active protein tyrosine kinases centered on activated JAK1/2. Mechanistically, JAK1/2 activation is mediated by augmented mTOR. Importantly, JAK1/2 inhibition with Ruxolitinib selectively suppresses the growth of IFNγR1KO but not scrambled control melanomas, depending on T cells and host TNF. Together, our results reveal an important role of tumor-intrinsic IFN-γ signaling in shaping TILs and manifest a targeted therapy to bypass ICB resistance of melanomas defective of IFN-γ signaling.

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.

Differential escape mechanisms in cetuximab-resistant head and neck cancer cells.

Acquired cetuximab resistance is a challenge for oncologists treating advanced head and neck carcinoma (HNC). While intrinsic cetuximab resistance mechanism in colorectal cancer is known, resistance in HNC is unclear. We established two different cetuximab resistant HNC cell lines by culturing epidermal growth factor (EGFR) expressing UM-SCC-1 and UM-SCC-6 cell lines in the presence of 5 µg/ml cetuximab. We then explored potential mechanisms of resistance. We found that the 2 cell lines developed resistance by different mechanisms. Specifically, we found that UM-SCC-1 resistant cells (UM-SCC-1R) showed enhanced EGF-induced downstream signals while UM-SCC-6 resistant cells (UM-SCC-6R) demonstrated EGF-independent signaling. Global kinase activity (kinomic) profiling revealed unique signaling differences in the two resistant cell lines. However, both of the resistant lines demonstrated increased phospho-serine 727 and total STAT3 expression compared to the parental lines. STAT3 knockdown promoted increased cytotoxicity both in the presence and absence of cetuximab in the resistant lines suggesting that STAT3 may be a common target in cetuximab resistance.

Myristoylated alanine-rich C-kinase substrate effector domain phosphorylation regulates the growth and radiation sensitization of glioblastoma.

Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol­3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alanine­rich C­kinase substrate (MARCKS) is a 32 kDa intrinsically unstructured protein containing a polybasic (+13) effector domain (ED), which regulates its electrostatic sequestration of phospholipid phosphatidylinositol (4,5)­bisphosphate (PIP2), and its binding to phosphatidylserine, calcium/calmodulin, filamentous actin, while also serving as a nuclear localization sequence. MARCKS ED is phosphorylated by protein kinase C (PKC) and Rho­associated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracycline­inducible system in PTEN­null U87 cells, we demonstrate that MARCKS overexpression suppresses growth and enhances radiation sensitivity in vivo. A new image cytometer, Xcyto10, was utilized to quantify differences in MARCKS ED phosphorylation on localization and its association with filamentous actin. The overexpression of the non­phosphorylatable ED mutant exerted growth­suppressive and radiation­sensitizing effects, while the pseudo­phosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS protein­protein interactions using co­immunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKS­associated proteins, including importin­ß and ku70. On the whole, the findings of this study suggest that the determination of the MARCKS ED phosphorylation status is essential to understanding the impact of MARCKS on cancer progression.

ST6Gal-I sialyltransferase promotes chemoresistance in pancreatic ductal adenocarcinoma by abrogating gemcitabine-mediated DNA damage.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor prognosis. Gemcitabine, as a single agent or in combination therapy, remains the frontline chemotherapy despite its limited efficacy due to de novo or acquired chemoresistance. There is an acute need to decipher mechanisms underlying chemoresistance and identify new targets to improve patient outcomes. Here, we report a novel role for the ST6Gal-I sialyltransferase in gemcitabine resistance. Utilizing MiaPaCa-2 and BxPC-3 PDAC cells, we found that knockdown (KD) of ST6Gal-I expression, as well as removal of surface α2-6 sialic acids by neuraminidase, enhances gemcitabine-mediated cell death assessed via clonogenic assays and cleaved caspase 3 expression. Additionally, KD of ST6Gal-I potentiates gemcitabine-induced DNA damage as measured by comet assays and quantification of γH2AX foci. ST6Gal-I KD also alters mRNA expression of key gemcitabine metabolic genes, RRM1, RRM2, hENT1, and DCK, leading to an increased gemcitabine sensitivity ratio, an indicator of gemcitabine toxicity. Gemcitabine-resistant MiaPaCa-2 cells display higher ST6Gal-I levels than treatment-naïve cells along with a reduced gemcitabine sensitivity ratio, suggesting that chronic chemotherapy selects for clonal variants with more abundant ST6Gal-I. Finally, we examined Suit2 PDAC cells and Suit2 derivatives with enhanced metastatic potential. Intriguingly, three metastatic and chemoresistant subclones, S2-CP9, S2-LM7AA, and S2-013, exhibit up-regulated ST6Gal-I relative to parental Suit2 cells. ST6Gal-I KD in S2-013 cells increases gemcitabine-mediated DNA damage, indicating that suppressing ST6Gal-I activity sensitizes inherently resistant cells to gemcitabine. Together, these findings place ST6Gal-I as a critical player in imparting gemcitabine resistance and as a potential target to restore PDAC chemoresponse.

Enhancement of Cetuximab-Induced Radiosensitization by JAK-1 Inhibition.

BACKGROUND: It is known that cetuximab (an epidermal growth factor receptor [EGFr] inhibitor) is a radiosensitizer. Also, cetuximab is known to only partially inhibit the signal transducer and activator of transcription - 3 (STAT-3); a mediator of protection from apoptosis. Studies were performed to determine if the radiosensitizing effects of cetuximab could be enhanced with the addition of an inhibitor of STAT-3. METHODS/RESULTS: The interaction of JAK-STAT-3 inhibition ([JAK1i]; Calbiochem, LaJolla, CA) and EGFr inhibition (cetuximab) was assessed with and without radiation. Four human head and neck cell lines were studied: UM-SCC-1 and UM-SCC-5, and two modified UM-SCC-5 lines; a STAT-3 knockdown line (STAT-3-2.4) and control (NEG-4.17). Exposure to either 0.5 µg/ml of cetuximab or 1 µM JAK1i for 8 or 24 h resulted in reduced activated STAT-3 (immunoblot), and the combination treatment showed greater reduction in activated STAT-3 compared to the individual treatments. The use of either post-radiation JAK1i (1 µM for 72 h) or post-radiation cetuximab (0.5 µg/ml) enhanced radiation-induced anti-proliferative and apoptotic effects but the greatest enhancement was seen when cells were exposed to both JAK1i and cetuximab post-radiation. Similar results were seen for radiosensitization as assessed by colony formation. Finally, the combination treatment of JAK1i (1 µM) and cetuximab (0.5 µg/ml), following radiation, resulted in an increase of unrepaired radiation-induced DNA double strand breaks at 6 and 24 h after radiation compared to the use of post-radiation JAK1i or cetuximab alone as delineated by neutral comet assay. CONCLUSIONS: These findings suggest that dual inhibition of EGFr (cetuximab) and JAK-STAT-3 (JAK1i) leads to greater radiosensitization than with either cetuximab or JAK1i alone and suggests that this combination treatment may be clinically relevant even for tumors with a marked range of STAT-3 activity.

DNA double strand break repair defect and sensitivity to poly ADP-ribose polymerase (PARP) inhibition in human papillomavirus 16-positive head and neck squamous cell carcinoma.

Patients with human papillomavirus-positive (HPV+) head and neck squamous cell carcinomas (HNSCCs) have increased response to radio- and chemotherapy and improved overall survival, possibly due to an impaired DNA damage response. Here, we investigated the correlation between HPV status and repair of DNA damage in HNSCC cell lines. We also assessed in vitro and in vivo sensitivity to the PARP inhibitor veliparib (ABT-888) in HNSCC cell lines and an HPV+ patient xenograft. Repair of DNA double strand breaks (DSBs) was significantly delayed in HPV+ compared to HPV- HNSCCs, resulting in persistence of γH2AX foci. Although DNA repair activators 53BP1 and BRCA1 were functional in all HNSCCs, HPV+ cells showed downstream defects in both non-homologous end joining and homologous recombination repair. Specifically, HPV+ cells were deficient in protein recruitment and protein expression of DNA-Pk and BRCA2, key factors for non-homologous end joining and homologous recombination respectively. Importantly, the apparent DNA repair defect in HPV+ HNSCCs was associated with increased sensitivity to the PARP inhibitor veliparib, resulting in decreased cell survival in vitro and a 10-14 day tumor growth delay in vivo. These results support the testing of PARP inhibition in combination with DNA damaging agents as a novel therapeutic strategy for HPV+ HNSCC.

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.

Inhibition of STAT-3 results in greater cetuximab sensitivity in head and neck squamous cell carcinoma.

OBJECTIVE: The inhibition of epidermal growth factor receptor (EGFr) with the monoclonal antibody cetuximab reduces cell proliferation and survival which correlates with increased DNA damage. Since the signal transducer and activator of transcription-3 (STAT-3) is involved in the EGFr-induced signaling pathway, we hypothesized that depletion of STAT-3 may augment cetuximab-induced processes in human head and neck cancer cells. MATERIALS AND METHODS: Human head and neck squamous carcinoma cells (UM-SCC-5) were transfected with short hairpin RNA (shRNA) against STAT-3 (STAT3-2.4 and 2.9 cells). A mutated form of this shRNA was transfected for a control (NEG4.17 cells). Radiosensitivity was assessed by a standard colony formation assay. Proliferation was assessed by daily cell counts following treatment and apoptosis was assessed by an annexin V-FITC assay. The alkaline comet assay was used to assess DNA damage. RESULTS: The STAT-3 knockdown cells (STAT3-2.4 and STAT3-2.9 cells) demonstrated enhanced radiosensitivity compared to control NEG4.17 cells, which correlated with increased apoptosis. Also, the STAT-3 knockdown cells demonstrated decreased proliferation with cetuximab treatments compared to control cells (NEG4.17). The increased cetuximab sensitivity of the STAT-3 knockdown cells correlated with increased apoptosis and DNA damage compared to control cells (NEG4.17). CONCLUSION: These studies revealed that the greater anti-proliferative effects and increased cytotoxicity of cetuximab in the STAT3-2.4 and STAT3-2.9 cells compared to control NEG4.17 cells, may be a result of STAT3-mediated effects on cellular apoptosis and DNA damage.

Inhibition of STAT-3 results in radiosensitization of human squamous cell carcinoma.

BACKGROUND: Signal transducer and activator of transcription-3 (STAT-3) is a downstream component of the Epidermal Growth Factor Receptor (EGFr) signaling process that may facilitate the resistance of tumor cells to conventional cancer treatments. Studies were performed to determine if inhibition of this downstream protein produces radiosensitization. METHODS/RESULTS: A431 cells (human squamous cell carcinoma cells with EGFr overexpression) were found to be sensitized to radiation after treatment with STAT-3 small interfering RNA (siRNA). Therefore, a short hairpin RNA (shRNA) against STAT-3 was designed and cloned into a pBABE vector system modified for shRNA expression. Following transfection, clone 2.1 was selected for further study as it showed a dramatic reduction of STAT-3 protein (and mRNA) when compared to A431 parental cells or a negative control shRNA cell line (transfected with STAT-3 shRNA with 2 base pairs mutated). A431 2.1 showed doubling times of 25-31h as compared to 18-24h for the parental cell line. The A431 shRNA knockdown STAT-3 cells A431 were more sensitive to radiation than A431 parental or negative STAT-3 control cells. CONCLUSION: A431 cells stably transfected with shRNA against STAT-3 resulted in enhanced radiosensitivity. Further work will be necessary to determine whether the inhibition of STAT-3 phosphorylation is a necessary step for the radiosensitization that is induced by the inhibition of EGFr.

Anti-EGFR-mediated radiosensitization as a result of augmented EGFR expression.

PURPOSE: Elevated epidermal growth factor receptor (EGFR) expression has correlated with a poor prognosis after standard treatment of several malignancies. However, it is not clear whether the absolute level of EGFR expression affects the radiosensitizing properties of anti-EGFR treatments. A better understanding of this question would be helpful for the design of protocols that deliver these treatments. To explore this question, cells (LS174T) that did not display inherent anti-EGFR treatment-induced radiosensitization were selected for studies that could potentially enhance EGFR expression. MATERIALS AND METHODS: Human colon carcinoma cells (LS174T), which did not show radiosensitization by anti-EGFR treatments, were employed for these studies. (Also, these cells were not responsive to the antiproliferative effects of anti-EGFR treatment.) Using standard transfection techniques (eukaryotic expression vector) as well as an adenoviral construct to enhance EGFR expression, LS174T cells were transduced in a manner that resulted in enhanced expression of EGFR. Subsequently, standard proliferation studies were performed to test the radiosensitizing properties of anti-EGFR treatment (an anti-EGFR monoclonal antibody: IMC-C225). RESULTS: Studies were undertaken to stably transfect LS174T cells with EGFR. The stable transfectants, LS174T.EGFR cells, were responsive to the antiproliferative effects of anti-EGFR treatment, in contrast to the parent LS174T cells. Similar results were demonstrated when the cells were infected with AdEGFR. Additionally, the LS174T.EGFR cells were responsive to the radiosensitizing properties of anti-EGFR treatment (IMC-C225), whereas the parent cells were not. CONCLUSIONS: Although the level of EGFR expression is of prognostic significance in many tumor models, the response of cells to anti-EGFR treatment alone, or combinations of this treatment with radiation or chemotherapy, depends upon many factors that are not necessarily related to the inherent EGFR expression of the tumor cells. However, the studies reported herein, demonstrate that when LS174T cells were transduced to show increased EGFR expression, they became responsive to the radiosensitizing properties of anti-EGFR treatments.

Adenoviral vector-mediated augmentation of epidermal growth factor receptor (EGFr) enhances the radiosensitization properties of anti-EGFr treatment in prostate cancer cells.

PURPOSE: To determine whether an adenoviral vector approach to the augmentation of epidermal growth factor receptor (EGFr) expression results in increased antiproliferative and radiosensitization properties of anti-EGFr antibody therapy in prostate cancer cells. METHODS AND MATERIALS: DU145 and LNCaP human prostate cancer cells were used to test the above question in vitro. An adenoviral vector was utilized to transduce cells with an EGFr transgene (AdEGFr). Immunoblots were performed to measure EGFr expression and EGFr tyrosine phosphorylation. Radiolabeled ligand studies were employed to test binding of epidermal growth factor to EGFr. Scatchard analyses allowed for quantification of the number of EGFrs. Standard immunohistochemistry was performed to assess EGFr expression. Cellular proliferation was assessed after various combinations of treatment. RESULTS: Studies of prostate carcinoma cells infected with AdEGFr demonstrated an increase in EGFr expression. This increase in expression correlated with increased function of EGFr. Specifically, increased EGFr expression also resulted in increased ligand binding, ligand-induced internalization of EGFr, and ligand-induced EGFr tyrosine kinase activity that could be blocked with pre-exposure to IMC-C225 (an anti-EGFr monoclonal antibody). Transduction of the LNCaP cells with AdEGFr did not increase the antiproliferative effects of IMC-C225, but did significantly increase IMC-C225-induced radiosensitization as determined by cell proliferation. CONCLUSIONS: Augmentation of EGFr expression, through an adenoviral vector approach in prostate carcinoma cells, resulted in cells that demonstrated greater IMC-C225-induced radiosensitization compared to cells that were not treated with AdEGFr.