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Human leukemia cells secrete VEGF, which can act in a paracrine manner within the bone marrow microenvironment to promote leukemia cell survival and proliferation. The FLT-3 receptor tyrosine kinase plays an essential role in regulating normal hematopoiesis, but its constitutive activation via mutation in acute leukemias is generally associated with poor outcome. The aim of this study was to investigate interactions between the FLT-3 and VEGF signaling pathways in acute leukemia using cell lines and ex vivo cultures of pediatric acute lymphoblastic leukemia cells following expansion of direct patient explants in immunodeficient mice. Different xenograft lines exhibited variable cell surface FLT-3 expression, as well as basal and FLT-3 ligand-induced VEGF secretion, whereas the MV4;11 cell line, which expresses constitutively active FLT-3, secreted high levels of VEGF. The FLT-3 inhibitor, SU11657, significantly reduced VEGF secretion in three of six xenograft lines and MV4;11 cells, in conjunction with inhibition of FLT-3 tyrosine phosphorylation. Moreover, exposure of xenograft cells to the FLT-3–blocking antibody, D43, also reduced VEGF secretion to basal levels and decreased FLT-3 tyrosine phosphorylation. In terms of downstream signaling, SU11657 and D43 both caused dephosphorylation of extracellular signal-regulated kinase 1/2, with no changes in AKT or STAT5 phosphorylation. Finally, partial knockdown of FLT-3 expression by short interfering RNA also resulted in inhibition of VEGF secretion. These results indicate that FLT-3 signaling plays a central role in the regulation of VEGF secretion and that inhibition of the FLT-3/VEGF pathway may disrupt paracrine signaling between leukemia cells and the bone marrow microenvironment. Mol Cancer Ther; 11(1); 183–93. ©2011 AACR.

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Purpose: We previously demonstrated an association between decreased SMAD4 expression and cetuximab resistance in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to further elucidate the clinical relevance of SMAD4 loss in HNSCC.Experimental Design: SMAD4 expression was assessed by IHC in 130 newly diagnosed and 43 patients with recurrent HNSCC. Correlative statistical analysis with clinicopathologic data was also performed. OncoFinder, a bioinformatics tool, was used to analyze molecular signaling in TCGA tumors with low or high SMAD4 mRNA levels. The role of SMAD4 was investigated by shRNA knockdown and gene reconstitution of HPV-negative HNSCC cell lines in vitro and in vivo.Results: Our analysis revealed that SMAD4 loss was associated with an aggressive, HPV-negative, cetuximab-resistant phenotype. We found a signature of prosurvival and antiapoptotic pathways that were commonly dysregulated in SMAD4-low cases derived from TCGA-HNSCC dataset and an independent oral cavity squamous cell carcinoma (OSCC) cohort obtained from GEO. We show that SMAD4 depletion in an HNSCC cell line induces cetuximab resistance and results in worse survival in an orthotopic mouse model in vivo. We implicate JNK and MAPK activation as mediators of cetuximab resistance and provide the foundation for the concomitant EGFR and JNK/MAPK inhibition as a potential strategy for overcoming cetuximab resistance in HNSCCs with SMAD4 loss.Conclusions: Our study demonstrates that loss of SMAD4 expression is a signature characterizing the cetuximab-resistant phenotype and suggests that SMAD4 expression may be a determinant of sensitivity/resistance to EGFR/MAPK or EGFR/JNK inhibition in HPV-negative HNSCC tumors. Clin Cancer Res; 23(17); 5162–75. ©2017 AACR.

Representative SMAD4 IHC, FaDu negative control, SCC25/SCC61 positive control.

Matrigel colony formation assay of FaDu-mock and FaDu-SMAD4 cells treated with PBS or Cetuximab in combination with JNKi (SP600125) or MEKi (U0126) at indicated concentrations for 7 days. (*p

(A) Western blot confirming the knockdown of SMAD4 levels by stable expression of short-hairpin RNA in SCC25 cell line. (SC: scrambled control shRNA, SMAD4KD: shRNA against SMAD4). (B) Matrigel colony formation assay was performed for SCC25 cell line (controls and SMAD4 knock-downs) treated with PBS, 100nM or 1000nM cetuximab. (C) Western blot showing MAPK and JNK activity in SMAD4-depleted or control SCC25 cells.

Western blot analysis of SCC61L-SC and SMAD4KD cells for phospho-SMAD2 and total SMAD2/3 expression levels. Actin was used as a loading control.

Supplemental Table 1. Summary of patient characteristics with newly diagnosed HNSCC. Supplemental Table 2. Summary of patient characteristics with recurrent and/or metastatic HNSCC. Supplemental Table 3. Injection of FaDu-SMAD4 cells results in lower take rate in mice compared to the control cell line and shows no signs of metastatic lesion.

Both the presence of an abnormal complement of chromosomes (aneuploidy) and an increased frequency of chromosome missegregation (chromosomal instability) are hallmarks of cancer. Analyses of cancer genome data have identified certain aneuploidy patterns in tumors; however, the bases behind their selection are largely unexplored. By establishing time-resolved long-term adaptation protocols, we found that human cells adapt to persistent spindle assembly checkpoint (SAC) inhibition by acquiring specific chromosome arm gains and losses. Independently adapted populations converge on complex karyotypes, which over time are refined to contain ever smaller chromosomal changes. Of note, the frequencies of chromosome arm gains in adapted cells correlate with those detected in cancers, suggesting that our cellular adaptation approach recapitulates selective traits that dictate the selection of aneuploidies frequently observed across many cancer types. We further engineered specific aneuploidies to determine the genetic basis behind the observed karyotype patterns. These experiments demonstrated that the adapted and engineered aneuploid cell lines limit CIN by extending mitotic duration. Heterozygous deletions of key SAC and APC/C genes recapitulated the rescue phenotypes of the monosomic chromosomes. We conclude that aneuploidy-induced gene dosage imbalances of individual mitotic regulators are sufficient for altering mitotic timing to reduce CIN.

Whereas Edwardian male-authored Wildeana tends to represent the Wildean character as a solipsistic bachelor, whose tarnished reputation could affect only his bachelor friends, Mary Elizabeth Braddon’s The Rose of Life (1905) and Julia Frankau’s The Sphinx’s Lawyer (1906) place the Wildean character within the family unit. This allows them to examine the consequences the character’s legal prosecution and stigmatisation could have on his wife. Braddon’s and Frankau’s novels shift the focus from homoeroticism and homosexuality to the catalysing role male influence plays in the development of innate queerness. At the same time, they critically engage with the contemporary belief that female influence might “cure” queerness. Braddon and Frankau avoid demonising and/or oversimplifying the Wildean character’s feelings and note that queerness and the family are by no means oppositional or antithetical units. To achieve this effect, they invariably sacrifice women’s happiness, which allows us to read their novels as more anti-feminist than anti-queer.

Blinatumomab is a BiTE® (bispecific T-cell engager) immuno-oncology therapy, which has demonstrated significant activity in patients with relapsed or refractory B-cell precursor acute lymphoblastic leukemia (R/R B-ALL); however, a subset of patients relapse. Monitoring expression of cluster of differentiation (CD)19 in relapsed patients is critical to inform sequencing of subsequent therapies. The expression of CD19 in 59 pediatric patients with R/R B-ALL was analyzed on the day of diagnosis of R/R B-ALL and on days 15 and 29 of cycle 1 of blinatumomab. Most patients treated with one cycle of blinatumomab retained expression of CD19, and would therefore be eligible for subsequent anti-CD19 CAR T-cell therapy.

Background Blinatumomab is a BiTE ® (bispecific T cell engager) molecule that engages patients' T cells to the CD19 antigen on lymphoid tumor cells. Blinatumomab administered as a 28-day continuous intravenous infusion (cIV) is approved in multiple regions for the treatment of R/R B-ALL in adults and children. Subcutaneous delivery may improve the convenience and satisfaction of patients with R/R B-ALL who are candidates for blinatumomab therapy. Here we report the results from the first cohort of adults with R/R B-ALL receiving SC blinatumomab. Methods In this ongoing multicenter, single arm, open-label, phase 1b dose-finding study (NCT04521231), patients received multiple cycles of SC blinatumomab. Each cycle included a treatment period and a treatment-free interval. In cohort 1, cycle 1, patients received a lower first dose of SC blinatumomab for several days followed by a higher dose multiple times weekly; in subsequent cycles, patients received the higher dose several times weekly during the treatment period. Bone marrow (BM) evaluation was performed on day 27 of each cycle. Results Six patients from cohort 1 were included in this June 22, 2021 data cutoff. Median age was 64 (range 38-83) years. The number of prior therapies ranged from 2-4. Two patients had disease refractory to primary therapy or salvage therapy, 2 patients relapsed after chemotherapy, and 2 patients relapsed after prior allogeneic hematopoietic stem cell transplant. Median BM blast count at study start was 85% (range 28%-95%). Only 1 patient had Preliminary pharmacokinetic results support the SC dosing intervals used in this study and potentially longer intervals. Exposures for SC doses were similar to the efficacious exposures of the approved cIV regimen: mean average concentrations at steady state of 215 and 853 pg/mL for the lower and higher SC dosing regimens of cohort 1, respectively, vs mean steady state concentrations of 228 and 616 pg/mL for 9 and 28 µg/day cIV dosing, respectively. The pharmacodynamic profile following SC blinatumomab of peripheral immune cell redistribution (circulating CD3+ and CD8+ CD69+ T cells), transient cytokine elevation (IL-6, IL-10, IFN-γ) and CD19+ B cell counts declining below the detection limit was consistent with the historical pharmacodynamic profile following cIV blinatumomab. No grade ≥3 cytokine release syndrome events were reported (Table). One patient developed herpes encephalitis and experienced a grade 5 neurological event unrelated to blinatumomab; no other neurological events were reported. Two patients discontinued treatment because of adverse events (injection site reaction in patient with no response, hyperleukocytosis due to disease progression). Three patients had complete hematological response (CR) with no measurable residual disease (MRD) ( Conclusions In this phase 1b dose-finding study, SC blinatumomab has demonstrated encouraging anti-leukemia activity in heavily pretreated patients with R/R B-ALL. Pharmacokinetic and pharmacodynamic results support the use of SC dosing in this population. The safety profile was manageable and consistent with that reported for cIV blinatumomab. Figure 1 Figure 1. Disclosures Gordon: Amgen: Current Employment, Current equity holder in publicly-traded company. Schwartz: Morphosys: Research Funding; Pfizer: Honoraria, Speakers Bureau; Gilead: Other: Travel grants, Speakers Bureau; Jazz Pharmaceuticals: Other: Travel grants, Speakers Bureau; Novartis: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau; BTG International Inc: Membership on an entity's Board of Directors or advisory committees; MSD Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Basilea: Other: Travel grants. Rossi: Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Alexion: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Huguet: Novartis: Other: Advisor; Jazz Pharmaceuticals: Other: Advisor; Celgene: Other: Advisor; BMS: Other: Advisor; Amgen: Other: Advisor; Pfizer: Other: Advisor. Hernández-Rivas: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Wong: Amgen: Current equity holder in publicly-traded company; Amgen: Current Employment. Markovic: Amgen: Current Employment, Current equity holder in publicly-traded company. Katlinskaya: Amgen: Current Employment, Current equity holder in publicly-traded company. Panwar: Amgen: Current Employment, Current equity holder in publicly-traded company. Zugmaier: Micromet/Amgen: Patents & Royalties: Patents 20190300609 and 20130323247 licensed; receives royalties of family members of international applications published as WO2010/052014; WO2010/052013; WO2011/051307; WO2012/055961; WO 2012/062596; WO2014/122251; and WO2015/181683; Amgen: Current Employment; Amgen: Current equity holder in publicly-traded company.

This paper provides an overview of the process of bibliotherapy and suggests some techniques that can be used in implementing it. Before this, autor underlines the differences between clinical and developmental bibliotherapy, and presents a short historical overview of development of this discipline of psychotherapy. Author also quotes a various deffinitions of this term in both meanings and concludes that all of these deffinitions contains a common denominator which is using books in order to helping people. Various chapters of the paper discuss the topics such as: bibliotherapy and children needs, the bibliotherapeutic process, the skills and attributes of a good bibliotherapist, and the limitations of bibliotherapy.

Purpose: We previously demonstrated an association between decreased SMAD4 expression and cetuximab resistance in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to further elucidate the clinical relevance of SMAD4 loss in HNSCC. Experimental Design: SMAD4 expression was assessed by IHC in 130 newly diagnosed and 43 patients with recurrent HNSCC. Correlative statistical analysis with clinicopathologic data was also performed. OncoFinder, a bioinformatics tool, was used to analyze molecular signaling in TCGA tumors with low or high SMAD4 mRNA levels. The role of SMAD4 was investigated by shRNA knockdown and gene reconstitution of HPV-negative HNSCC cell lines in vitro and in vivo. Results: Our analysis revealed that SMAD4 loss was associated with an aggressive, HPV-negative, cetuximab-resistant phenotype. We found a signature of prosurvival and antiapoptotic pathways that were commonly dysregulated in SMAD4-low cases derived from TCGA-HNSCC dataset and an independent oral cavity squamous cell carcinoma (OSCC) cohort obtained from GEO. We show that SMAD4 depletion in an HNSCC cell line induces cetuximab resistance and results in worse survival in an orthotopic mouse model in vivo. We implicate JNK and MAPK activation as mediators of cetuximab resistance and provide the foundation for the concomitant EGFR and JNK/MAPK inhibition as a potential strategy for overcoming cetuximab resistance in HNSCCs with SMAD4 loss. Conclusions: Our study demonstrates that loss of SMAD4 expression is a signature characterizing the cetuximab-resistant phenotype and suggests that SMAD4 expression may be a determinant of sensitivity/resistance to EGFR/MAPK or EGFR/JNK inhibition in HPV-negative HNSCC tumors. Clin Cancer Res; 23(17); 5162–75. ©2017 AACR.

Objectives/Hypothesis: Epidermal growth factor receptor (EGFR) and p16 (a surrogate marker of human papillomavirus [HPV] infection) expression are strong prognostic factors in patients with head and neck squamous cell carcinoma (HNSCC). Study Design: We examined expression levels of total and nuclear EGFR as well as p16 status based on evidence that nuclear EGFR may have a role in DNA damage repair. Methods: An HPV-negative (SQ20B) and an HPV-positive (UMSCC47) HNSCC cell line were examined for EGFR and γH2AX expression. A tissue microarray containing 123 cores obtained from 101 HNSCC tumors was analyzed for EGFR expression by automated quantitative analysis and p16 expression by immunohistochemical staining, and these results were correlated with available clinical data. Results: SQ20B had higher EGFR expression than UMSCC47. Nuclear localization of EGFR on activation with transforming growth factor-α was observed in SQ20B, but not in UMSCC47. SQ20B also had increased γH2AX foci compared to UMSCC47, suggesting that SQ20B has more DNA damage compared to UMSCC47. Total and nuclear EGFR was reliably obtained from 80 of 101 patients. p16 levels were determined in 87 of 101 patients. p16 levels were strongly associated with the oropharyngeal subsite and poorly differentiated histology. Expression of total and nuclear EGFR was higher in p16-negative tumors compared to p16-positive tumors (Wilcoxon rank test, P = .038 and P = .014, respectively). Conclusions: Further studies are required to determine a mechanistic link between these two prognostic factors and the significance of EGFR localization to nucleus in DNA damage repair pathway activation. Laryngoscope, 2012

New agents and treatment strategies that can be safely and effectively integrated into current treatment paradigms for head and neck squamous cell carcinoma (HNSCC) are urgently needed. To date, the anti-EGF receptor (EGFR) monoclonal antibody, cetuximab, is the first and only molecularly targeted therapy to demonstrate a survival benefit for patients with recurrent or metastatic disease. Other anti-EGFR-targeted therapies, including monoclonal antibodies (e.g., panitumumab and zalutumumab) and reversible and irreversible ErbB family tyrosine kinase inhibitors (e.g., lapatinib, afatinib and dacomitinib) are being actively investigated in Phase II and Phase III clinical trials. In addition, validated biomarkers are needed to predict clinical benefit and resistance to anti-EGFR therapy in HNSCC. This review will compare and contrast the mechanisms of action of anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors and also discuss their role in the management of HNSCC and the potential impact of human papillomavirus status in the development of these targeted agents.

Overcoming resistance to chemotherapy is the main therapeutic challenge in the treatment of acute lymphocytic leukemia (ALL). Interactions between leukemia cells and the microenvironment promote leukemia cell survival and confer resistance to chemotherapy. Hypoxia is an integral component of bone marrow (BM) microenvironment. Hypoxia-inducible factor-1α (HIF-1), a key regulator of the cellular response to hypoxia, regulates cell growth and metabolic adaptation to hypoxia. HIF-1α expression, analyzed by Reverse Phase Protein Arrays in 92 specimens from newly diagnosed patients with pre-B-ALL, had a negative prognostic impact on survival (p = 0.0025). Inhibition of HIF-1α expression by locked mRNA antagonist (LNA) promoted chemosensitivity under hypoxic conditions, while pharmacological or genetic stabilization of HIF-1α under normoxia inhibited cell growth and reduced apoptosis induction by chemotherapeutic agents. Co-culture of pre-B ALL or REH cells with BM-derived mesenchymal stem cells (MSC) under hypoxia resulted in further induction of HIF-1α protein and acquisition of the glycolytic phenotype, in part via stroma-induced AKT/mTOR signaling. mTOR blockade with everolimus reduced HIF-1α expression, diminished glucose uptake and glycolytic rate and partially restored the chemosensitivity of ALL cells under hypoxia/stroma co-cultures. Hence, mTOR inhibition or blockade of HIF-1α-mediated signaling may play an important role in chemosensitization of ALL cells under hypoxic conditions of the BM microenvironment.

Epidermal growth factor receptor (EGFR) and p16 (a surrogate marker of human papillomavirus [HPV] infection) expression are strong prognostic factors in patients with head and neck squamous cell carcinoma (HNSCC).We examined expression levels of total and nuclear EGFR as well as p16 status based on evidence that nuclear EGFR may have a role in DNA damage repair.An HPV-negative (SQ20B) and an HPV-positive (UMSCC47) HNSCC cell line were examined for EGFR and γH2AX expression. A tissue microarray containing 123 cores obtained from 101 HNSCC tumors was analyzed for EGFR expression by automated quantitative analysis and p16 expression by immunohistochemical staining, and these results were correlated with available clinical data.SQ20B had higher EGFR expression than UMSCC47. Nuclear localization of EGFR on activation with transforming growth factor-α was observed in SQ20B, but not in UMSCC47. SQ20B also had increased γH2AX foci compared to UMSCC47, suggesting that SQ20B has more DNA damage compared to UMSCC47. Total and nuclear EGFR was reliably obtained from 80 of 101 patients. p16 levels were determined in 87 of 101 patients. p16 levels were strongly associated with the oropharyngeal subsite and poorly differentiated histology. Expression of total and nuclear EGFR was higher in p16-negative tumors compared to p16-positive tumors (Wilcoxon rank test, P = .038 and P = .014, respectively).Further studies are required to determine a mechanistic link between these two prognostic factors and the significance of EGFR localization to nucleus in DNA damage repair pathway activation.

Background. Although online social networking is increasingly used for medical purposes, studies investigating drug use among pregnant females by means of online networks are lacking. Objective. Th e aim of our study was to investigate the extent of inappropriate drug use among pregnant women by creating a Facebook profi le and using it as a tool for interacting with pregnant women. Methods. A Facebook profi le titled “Preserve babies from drugs” was created and maintained by a group of fourth-year pharmacy students for 3 months. Introductory educational material about the principles of drug use in pregnancy, information about health facilities off ering counselling about pregnancy, information about a clinical pharmacology department that off ered counselling and an open-ended questionnaire were posted in the “Notes” section of the Facebook profi le. Results. Of 239 registered pregnant “friends” of the profi le who received the questionnaire from the investigators, 93 responded (39%). Among the respondents, 50 pregnant women (53.8%) reported taking medication(s) during their current pregnancy, and 42 of the respondents reported using one or more drugs improperly. Th e most frequently used drugs were multivitamin and multi-mineral preparations, oral antibiotics, parenteral progesterone and benzodiazepines. Conclusions. Th e Facebook profi le devoted to drug use in pregnancy could be a useful adjunct to eff orts by offi cial health care institutions to identify inappropriate drug use and educate pregnant women appropriately.

Human leukemia cells secrete VEGF, which can act in a paracrine manner within the bone marrow microenvironment to promote leukemia cell survival and proliferation. The FLT-3 receptor tyrosine kinase plays an essential role in regulating normal hematopoiesis, but its constitutive activation via mutation in acute leukemias is generally associated with poor outcome. The aim of this study was to investigate interactions between the FLT-3 and VEGF signaling pathways in acute leukemia using cell lines and ex vivo cultures of pediatric acute lymphoblastic leukemia cells following expansion of direct patient explants in immunodeficient mice. Different xenograft lines exhibited variable cell surface FLT-3 expression, as well as basal and FLT-3 ligand-induced VEGF secretion, whereas the MV4;11 cell line, which expresses constitutively active FLT-3, secreted high levels of VEGF. The FLT-3 inhibitor, SU11657, significantly reduced VEGF secretion in three of six xenograft lines and MV4;11 cells, in conjunction with inhibition of FLT-3 tyrosine phosphorylation. Moreover, exposure of xenograft cells to the FLT-3–blocking antibody, D43, also reduced VEGF secretion to basal levels and decreased FLT-3 tyrosine phosphorylation. In terms of downstream signaling, SU11657 and D43 both caused dephosphorylation of extracellular signal-regulated kinase 1/2, with no changes in AKT or STAT5 phosphorylation. Finally, partial knockdown of FLT-3 expression by short interfering RNA also resulted in inhibition of VEGF secretion. These results indicate that FLT-3 signaling plays a central role in the regulation of VEGF secretion and that inhibition of the FLT-3/VEGF pathway may disrupt paracrine signaling between leukemia cells and the bone marrow microenvironment. Mol Cancer Ther; 11(1); 183–93. ©2011 AACR.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment1,2. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC3,4, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.

The FMS-like tyrosine kinase-3 (FLT-3), which belongs to the class III receptor tyrosine kinase family, is primarily expressed by hematopoietic cells and plays an important role in hematopoiesis. FLT-3 is also expressed in the majority of acute leukemias, in which the presence of FLT-3 activating mutations is associated with poor prognosis. Consequently, there has been a recent surge in the development of FLT-3 inhibitors for the molecular targeting of leukemia, and many of these are now in clinical trials. An improved understanding of how FLT-3 interacts with its ligand, as well as how FLT-3 activating mutations are able to trigger downstream intracellular signaling pathways, will provide greater insight to how small molecule inhibitors may best be utilized and combined with established chemotherapeutic drugs for the management of patients with high-risk acute leukemia.

Introduction: Cetuximab is a monoclonal antibody that increases survival in head and neck squamous cell carcinoma (HNSCC) patients when used in combination with radiation or chemotherapy. However, this EGFR targeted-agent only has modest activity when administered as a monotherapy and a majority of patients develop therapeutic resistance. Previous work has established the TGF-beta signaling pathway has a critical role in HNSCC development and progression. Modulation of this pathway, and more specifically loss of Smad4 expression, is a common means of developing drug resistance in many cancers. To determine the role of Smad4 loss in HNSCC development, metastasis, and cetuximab resistance, Smad4 was knocked down or reexpressed in HNSCC cell lines and evaluated in vitro and in vivo. Methods: Smad4 was knocked-down in SCC61 following stable introduction of Smad4-targeting shRNA. Using stable lentiviral transduction, Smad4 was reexpressed in FaDu, a HNSCC cell line which lacks de novo expression of Smad4. Following modulation, changes in cetuximab sensitivity were determined using Matrigel colony formation assays. The effects of Smad4 modulation on various signal transduction pathways were determined by Western blot analysis with total and phospho-specific antibodies. Changes in metastatic potential were evaluated using an orthotopic in vivo model. The subsequent development of lymph node metastases were monitored utilizing luciferin-based imaging. Key proteins associated with metastasis were also assayed using ELISA and protein antibody arrays. Results: Smad4 loss increased the growth of SCC61 and FaDu and Smad4 status correlated with cetuximab resistance in both cells. Western blot analysis demonstrated Smad4 loss activates JNK and MAPK signaling. Consequently, co-treatment of Smad4 knockdown cells with cetuximab and a JNK inhibitor reversed the previously observed therapeutic resistance. Smad4 knockdown also increased lymph node metastasis in the orthotopic in vivo model. Protein array analysis determined Smad4 knockdown increased the expression of amphiregulin, FGF2, and VEGF, pathways capable of promoting metastasis. Expression of these Smad4-sensitive targets was inhibited with the use of a MAPK inhibitor. Conclusion: We determined Smad4 loss enhances cetuximab resistance and in vivo metastasis development in HNSCC. Additionally, JNK and MAPK pathway activation play an important role in mediating this process. Our results suggest Smad4 downregulation plays a critical role for prognosis of HNSCC patients, and JNK and MAPK inhibition may contribute to improve therapeutic outcome of those patients. Citation Format: Hiroyuki Ozawa, Haixia Cheng, Elana J. Fertig, Jason D. Howard, Ana Markovic, Robert Hughes, Jimena Perez, Harry Quon, Christine H. Chung. Smad4 loss induces cetuximab resistance and increased metastatic potential in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2474. doi:10.1158/1538-7445.AM2014-2474

Head and neck squamous cell carcinoma (HNSCC) is largely divided into two groups based on their etiology, human papillomavirus (HPV)-positive and –negative. Global DNA methylation changes are known to drive oncogene and tumor suppressor expression in primary HNSCC of both types. However, significant heterogeneity in DNA methylation within the groups results in different transcriptional profiles and clinical outcomes. We applied a meta-pathway analysis to link gene expression changes to DNA methylation in distinguishing HNSCC subtypes. This approach isolated specific epigenetic changes controlling expression in HPV− HNSCC that distinguish it from HPV+ HNSCC. Analysis of genes identified Hedgehog pathway activation specific to HPV− HNSCC. We confirmed that GLI1, the primary Hedgehog target, showed higher expression in tumors compared to normal samples with HPV− tumors having the highest GLI1 expression, suggesting that increased expression of GLI1 is a potential driver in HPV− HNSCC. Our algorithm for integration of DNA methylation and gene expression can infer biologically significant molecular pathways that may be exploited as therapeutics targets. Our results suggest that therapeutics targeting the Hedgehog pathway may be of benefit in HPV− HNSCC. Similar integrative analysis of high-throughput coupled DNA methylation and expression datasets may yield novel insights into deregulated pathways in other cancers.

Global methylation changes occur heterogeneously in primary head and neck squamous cell carcinoma (HNSCC). To infer methylation-driven changes in expression in subtypes of HNSCC, we integrated global gene expression and DNA methylation measurements in 44 HNSCC and 25 normal samples using a matrix factorization algorithm. This algorithm incorporated novel models to transform methylation and expression measurements to a common scale. Although unsupervised, this integrated matrix factorization inferred patterns common to gene expression and methylation data that differentiate clinical subtypes within the 44 HNSCC samples, including notably HPV status and tumor site. Informatic analysis of the genes associated with these patterns identified specific pathway activity related to HPV status. This algorithm uniquely linked DNA methylation changes and reactivation of genes in the Hedgehog pathway predominantly in HPV-negative samples. We further examined the pathway by determining expression levels of GLI1, one of the downstream target genes of the Hedgehog pathway, in two independent cohorts. We confirmed that GLI1 expression was higher in tumors compared to normal samples and HPV-negative tumors had higher GLI1 expression compared to HPV-positive tumors, suggesting that increased expression of GLI1 plays a role in HPV-negative HNSCC. Citation Format: Elana J. Fertig, Ana Markovic, Ludmila V. Danilova, Daria A. Gaykalova, Leslie Cope, Christine H. Chung, Joseph A. Califano, Michael F. Ochs. An integrated matrix factorization algorithm for DNA methylation and gene expression identi es HNSCC clinical subtypes and GLI1 signaling speci c to HPV-negative HNSCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2901. doi:10.1158/1538-7445.AM2013-2901

Introduction: Expression of EGFRvIII has been reported in ∼40% of head and neck squamous cell carcinomas (HNSCCs). EGFRvIII lacks the ligand binding domain (exons 2-7), thus this receptor has demonstrated resistance to cetuximab (CTX), a monoclonal antibody against EGFR. Although ligand-independent activation likely drives downstream activation, EGFRvIII-specific pathways are currently unknown. To delineate the downstream effects of EGFRvIII and its contribution to EGFR inhibitor resistance, we transduced an immortalized human keratinocyte cell line (HaCaT), and Chinese hamster ovarian (CHO) cells, which lack EGFR expression to examine the isolated and contextual biological properties of EGFRvIII in vitro and in vivo. Methods: HaCaT and CHO cells were transduced with EGFRvIII, EGFR wild type (EGFR-WT), and an empty vector control. Proliferation of the transduced cells was determined using MTT. Subsequent sensitivity to CTX or gefitinib was determined by colony formation assay. Receptor activation and downstream signaling following ligand stimulation was assessed by Western blot. Subcutaneous hindflank xenografts of transduced HaCaTs were established in immunodeficient mice and resulting tumor growth was monitored. EGFRvIII expression from 30 HNSCC patient samples was also determined by RT-PCR. Results: HaCaT-EGFRvIII demonstrated slower proliferation than HaCaT-EGFR-WT. Ligand-stimulated EGFRvIII was also slower to activate, with EGFR reaching maximal phosphorylation at 30 min compared to 8 hr with EGFRvIII. We also observed that EGFRvIII preferentially signaled through AKT and STAT3, while EGFR utilized both AKT and MAPK. HaCaT-EGFR-WT xenografts exhibited rapid growth, with exponential growth initiating at 4 weeks while HaCaT-EGFRvIII demonstrated slow, linear phase growth at 12 weeks. In isolation, EGFRvIII exhibited a differential phosphorylation pattern compared to EGFR. Tyr 1068 and 1173 were both phosphorylated in EGFR and EGFRvIII, but Tyr 992 and 1086 were not activated in EGFRvIII. EGFR activation in CHO cells was reversible when treated with CTX and gefitinib, while EGFRvIII was only inhibited by gefitinib. However, HaCaT-EGFRvIII cells were not resistant to CTX in vitro. Utilizing RT-PCR, we determined EGFRvIII expression was lower (13%) than previously reported in HNSCC tumor samples. Conclusion: We determined EGFRvIII expression results in a slower proliferation rate in vitro and in vivo compared to EGFR overexpression. Additionally, EGFRvIII preferentially signals downstream through AKT and STAT3. In isolation, EGFRvIII is resistant to CTX inhibition, but does not confer CTX resistance in a fully competent keratinocyte model system. Ultimately, the clinical significance of EGFRvIII in HNSCC is unclear, as EGFR is always expressed in concert with EGFRvIII and its apparent incidence is low. Citation Format: Hiroyuki Ozawa, Ana Markovic, Jason Howard, Harry Quon, Jimena Perez, Robert Hughes, Christine H. Chung. Epidermal growth factor receptor variant III (EGFRvIII) expression initiates slower proliferation and signals through AKT and STAT3 in immortalized keratinocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4271. doi:10.1158/1538-7445.AM2013-4271