Citations

Abstract Hyperactivation of the oncogenic K-RAS pathway leads to increased cell proliferation, tumor growth, and oxidative stress associated with increased ROS production, heightened mitochondrial bioenergetics, and altered cancer metabolisms. Downstream of the K-RAS pathway, Seven-In-Absentia human Homolog (SIAH) is required for proper ERBB/RAS signal transduction. Human peroxiredoxin (PRDX) is an antioxidant protein, whose precise role in modulating oxidative stress during oncogenesis remains to be elucidated. Increased levels of oxidative stress alter the bioenergetics and functional integrity of mitochondrial biology under cellular stress. This study aims to elucidate the biological interaction previously detected between PRDX and SIAH2 via biochemical assays. We plan to investigate the functional role of SIAH2-PRDX interaction in modulating the stress response and adaptive cell survival in K-RAS-driven tumors. Also, we would like to investigate the biological significance of SIAH2-PRDX interaction in response to increased cellular stress in K-RAS-driven human cancer cells treated with standard chemotherapies. Mitochondrial tracking (MITO-tracker®) was used to evaluate mitochondrial integrity of SIAH-proficient and SIAH-deficient human lung (A549) and cervical (HeLa) cancer cells over six days. Immunofluorescence staining and bright field imaging were performed using DOX-inducible human breast (MDA-MB231) and cervical (HeLa) cancer cells to compare changes in organelle integrity and mitochondrial morphology, altered expression of the oxidative stress biomarker and cell death biomarkers in response to induction of the SIAH- proficient and SIAH-deficient states in highly aggressive human cancer cells. By using human pancreatic cancer tissue microarrays (TMAs), we found that the antioxidant protein PRDX is significantly upregulated and co-expressed with SIAH in human pancreatic tumor biospecimens. It seems that PRDX is an indicative biomarker of cellular and oncogenic K-RAS-induced oxidative stress in human pancreatic cancer. By blocking SIAH2 function downstream of the K-RAS signaling pathway, oxidative stress levels were markedly increased, evidenced by mitochondrial aggregations and dysfunction in both human lung and cervical cancer cells. In response to anti-SIAH2 therapy, human TNBC, NSCLC and cervical cancer cells underwent dramatic morphological changes due to the significant loss of cell junction, focal adhesion and cell attachment. These data have shown that anti-SIAH2-based anti-K-RAS and anticancer therapy is highly effective in inducing massive cell death in several highly invasive and metastatic cancer lines. Further investigation into the molecular mechanisms of anti-SIAH2-induced cellular stress and cell death may contribute to the future drug development and validation of a new and effective anti-SIAH2-based targeted therapy against metastatic human cancer in the future. Citation Format: Zena M. Urban, Lauren van Reesema, Minglei Bian, Thomas C. Smyrk, Gloria Peterson, Amy H. Tang. Inhibition of SIAH E3 ligase function leads to increased cell death in metastatic human cancer cells. [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 4442. doi:10.1158/1538-7445.AM2014-4442

Abstract The numerous and diverse genetic lesions in multiple myeloma (MM) make it difficult to discern those genetic abnormalities that may be suitable therapeutic targets. We have observed that the MYC locus is frequently rearranged in MM (about half of both patients and cell lines), suggesting that MYC dysregulation is a common feature of this disease. To evaluate MYC suppression as a therapeutic strategy in MM, we treated human myeloma cell lines with CPI203, a potent and selective inhibitor of bromodomain and extra-terminal (BET) proteins. The bromodomain inhibitor CPI203 has potency and selectivity similar to that of JQ1, but has improved pharmacokinetics for in vivo studies. From our collection of MM cell lines we have identified sensitive and resistant cell lines and characterized their response to CPI203 in detail. A number of conclusions have emerged from this work. First, response correlates with MYC protein levels after 3 days of treatment. MYC levels are significantly suppressed in sensitive lines and much less affected in resistant lines, supporting the hypothesis that MYC suppression alone determines cell fate in MM cell lines. In sensitive cell lines treatment with CPI203 caused a rapid (within 90 min.) decrease in MYC transcription that is consistent with the response being an intrinsic property of the cell lines. Inhibition of MYC transcription was followed by eventual cell cycle arrest and limited apoptosis in these sensitive cell lines. Both sensitive and resistant cell lines exhibited induction of Histone2H2BE gene in response to CPI203 treatment, indicating that drug efflux pumps alone do not mediate resistance. Surprisingly, several of the resistant cell lines exhibited an initial phase of sensitivity to BET inhibition. Upon drug addition, MYC transcription and MYC protein levels are repressed 2- to 5-fold, followed by a gradual recovery of MYC message and protein levels to the initial levels. In this second phase the cells are truly insensitive to BET inhibition: the drug remains active during this incubation and addition of fresh medium with drug does not affect MYC transcription. Furthermore, many aspects of the BET inhibition gene expression profile remain constant throughout this transition between sensitivity and resistance. These results suggest that the recovery phase is not a simple reversal of BET inhibition but rather the emergence of an alternative-signaling pathway that activates MYC expression in the presence of BET inhibition. Presently we are analyzing gene expression data to identify genes and signaling pathways responsible for this “induced resistance”. The identification of such pathways will help us better understand the molecular basis of the response to BET inhibition, which in turn might enable us to identify predictive biomarkers of the therapeutic response. Citation Format: Daniel L. Riggs, Marta Chesi, P Leif Bergsagel. Targeting MYC in multiple myeloma by BET protein inhibition. [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 5489. doi:10.1158/1538-7445.AM2014-5489

Abstract ASCL1 is an important regulatory transcription factor in pulmonary neuroendocrine cell development, but its value as a biomarker of neuroendocrine differentiation in lung adenocarcinoma (AD) and as a potential prognostic biomarker remains unclear. We examined ASCL1expression in lung cancer samples of varied histologic subtype, clinical outcome, and smoking status and compared to expression of traditional neuroendocrine markers. ASCL1 mRNA expression was found almost exclusively in smokers with AD, in contrast to non-smokers. ASCL1 protein expression by immunohistochemistry (IHC) correlated best with synaptophysin compared to chromogranin and CD56/NCAM. Analysis of a compendium of 367 microarray-based gene expression profiles in stage I lung adenocarcinomas identified significantly higher expression levels of the RET oncogene in ASCL1 positive tumors (ASCL1+) compared to ASCL1- tumors (q-value < 10 - 9). High levels of RET expression in ASCL1+ but not in ASCL1- tumors was associated with significantly shorter overall survival in stage 1 (p = 0.007) and in all AD (p = 0.037). RET protein expression by IHC had an association with overall survival in the context of ASCL1 expression. In silico gene set analysis and in vitro experiments by ASCL1 shRNA in AD cells with high endogenous expression of ASCL1 and RET implicated ASCL1 as a potential upstream regulator of the RET oncogene. Also, silencing ASCL1 in AD cells markedly reduced cell growth and motility. These results suggest that ASCL1 and RET expression define a clinically relevant subgroup of approximately 10% of AD characterized by neuroendocrine differentiation. Citation Format: Farhad Kosari, Cristiane M. Ida, Marie Christine Aubry, Lin Yang, Irina V. Kovtun, Janet L. Schaefer Klein, Sandra C. Tomaszek, Stephen J. Murphy, Ping Yang, Dennis Wigle, George Vasmatzis. ASCL1 and RET expression define a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation. [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 5251. doi:10.1158/1538-7445.AM2014-5251

Abstract Introduction: Peripheral blood leukocyte (PBL) telomere length is associated with colon cancer (CC) risk, but the relationship of PBL telomere length to survival in CC patients receiving chemotherapy is unknown. Methods: We measured relative telomere length (RTL) by qPCR of PBL DNA from 2,748 cases (53% male, 85% white, median age 58 yrs) in the North American Phase III Intergroup Trial N0147 that compared disease-free survival (DFS) among completely resected Stage III CC treated with mFOLFOX6 +/- cetuximab. Tumor markers were assessed, and 12% were BRAF mutant; 35% Kras mutant, and 12% deficient mismatch repair (dMMR). Linear and non-linear prognostic associations of PBL RTL to the primary endpoint of DFS were assessed by Cox models. Optimal categories of RTL were developed using cumulative martingale residual plots, designated from shortest to longest RTL (Table 1). Results: Never smokers had longer PBL RTL (p = 0.0006) than past or current smokers. Those with diabetes mellitus (DM) had longer PBL RTL than those without DM (p = 0.0165). PBL RTL was not associated with any tumor markers (MMR status; p = 0.7174; BRAF mutant vs wild-type (wt); p =0.4039; Kras mutant vs wt; p= 0.1149). PBL RTL was significantly associated with DFS in a non-linear form (p = 0.0389). Cases in the longer RTL category had more favorable DFS than those with shorter or longest RTL, (Table), which remained after adjusting for age, sex , T/N stage, grade, tumor markers, and treatment (p = 0.0230). We observed no significant interaction (p = 0.1490) between RTL categories and treatment. Conclusion: We observed a non-linear association between RTL and DFS in stage III CC, suggesting that an optimal range telomere length can influence tumor behavior in CC patients. Future studies of telomere maintenance mechanisms may help to explain the association of RTL with improved DFS. RTL is associated with smoking and DM in CC cases but further studies are needed to determine if either tobacco use or DM affect survival from CC by modulating PBL telomere length. Citation Format: Nivedita Basu, Ruth A. Johnson, Qian Shi, Garth D. Nelson, Julie M. Cunningham, Richard M. Goldberg, Frank A. Sinicrope, Daniel J. Sargent, Steven R. Alberts, Gloria M. Petersen, Stephen N. Thibodeau, Halcyon G. Skinner, Kristin Litzelman, Songwon Seo, Russell J. Vanderboom, Ronald E. Gangnon, Corinne D. Engelman, David N. Rider, Lisa A. Boardman. Telomeres and survival in stage III colon cancers: Findings from NCCTG N0147 (Alliance) study. [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 5102. doi:10.1158/1538-7445.AM2014-5102

Abstract Background. Follicular lymphoma (FL) is an indolent B-cell malignancy with a variable clinical course that may transform to more aggressive forms of lymphoma. Previous modestly powered GWAS have reported multiple FL susceptibility loci in the HLA class I and II regions at 6p21.32-33, but novel non-HLA susceptibility loci and the genetic architecture of FL associations in the HLA region remain to be elucidated. Methods. We conducted the largest GWAS of FL to date consisting of 2,142 cases and 6,221 controls of European ancestry from 22 studies scanned on the Illumina OmniExpress. After imputation of common SNPs using IMPUTE2 and 1,000 Genomes Project v3 data, we conducted a meta-analysis of these data with two previous FL GWAS (n=586 cases, 1,537 controls) and replicated top hits in an additional 629 cases and 4,283 controls. Expression quantitative trait loci (eQTL) analyses were performed to evaluate the effects of associated variants on gene expression. Classical HLA allele imputations and stepwise regression analyses are underway to further characterize the HLA associations. Results. In the joint meta-analysis, the strongest association with FL risk was observed in the HLA region, where a large number of SNPs showed genome-wide significance (P<5x10-8 to P= 1.84x10-84), particularly in HLA class II at 6p21.32 where several HLA eQTLs were identified (P < 0.05). Outside HLA, we identified four novel loci associated with FL at 11q23.3 (CXCR5, rs4938573; P=3.14x10-15), 11q24.3 (ETS1, rs4937362; P=3.33x10-9), 3q13.33 (CD86, rs2681416; P=9.79x10-11) and 3q28 (LPP, rs6444305, P=2.55x10-8). These novel susceptibility loci were located in four biologically relevant genes involved in immune regulation: CXCR5 and its ligand, CXCL13, are involved in guiding B-cells into the B-cell zones of secondary lymphoid organs as well as T-cell migration. ETS1 is expressed in lymphoid cells and regulates immune cell function including differentiation, survival, and proliferation. CD86 and CD80 are classic members of the B7 costimulatory pathway that is important in maintaining immune function, suppression of autoimmunity and antitumor surveillance. LPP encodes a LIM domain-containing protein of the zyxin family and participates in cell adhesion, cell migration, proliferation and transcription dynamics. Conclusions. This large GWAS provides further support for the important role of common genetic variation in non-HLA genes and further evidence of the key role that HLA immune-regulatory genes play in the pathogenesis of FL. Citation Format: Christine F. Skibola, Sonja I. Berndt, James R. Cerhan, Zhaoming Wang, Joseph Vijai, Lucia Conde, Paul de Bakker, Sophia S. Wang, Claire M. Vajdic, Brenda M. Birmann, Susan L. Slager, James McKay, Paige M. Bracci, Alexandra Nieters, Qing Lan, Angela R. Brooks-Wilson, Martha S. Linet, Demetrius Albanes, John J. Spinelli, Roel C.H. Vermeulen, Mark P. Purdue, Meredith Yaeger, Lauren R. Teras, Silvia de Sanjose, Alain Monnereau, Simon Crouch, Jia Nee Foo, Henrik Hjalgrim, Gianluca Severi, Brian K. Link, Kimberly A. Bertrand, Yawei Zhang, Karin E. Smedby, Stephen J. Chanock, Nathaniel Rothman, NHL GWAS Consortium. Meta-analysis of genome-wide association studies identifies novel susceptibility loci for follicular lymphoma. [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 5072. doi:10.1158/1538-7445.AM2014-5072

Abstract Marginal zone lymphomas (MZL) are a group of indolent non-Hodgkin's B-cell lymphomas (NHL) comprising extranodal MZL of mucosa-associated lymphoid tissue (MALT) type, splenic MZL and nodal MZL. MZL comprises 7-12% of adult NHLs. The genetic etiology of MZL is poorly understood. To better understand the genetic risk factors for MZL, we conducted the first genome-wide association study (GWAS) of this lymphoid malignancy. The GWAS included 824 MZL cases and 6,220 controls of European ancestry from 22 studies of NHL. Genotyping was carried out at the Cancer Genomics Research Laboratory (CGR) of NCI using Illumina Human OmniExpress arrays. Standard genotyping quality controls were performed. Imputation of common SNPs was performed using IMPUTE2 software and 1000 Genomes Project release version 3 data. Association testing was subsequently performed using SNPTEST, assuming a log-additive genetic model adjusting for age, gender and three significant eigenvectors. We identified one locus at 6p21.32 within the HLA class II region that attained genome-wide significance (P<5x10-8). When a small number of additional cases (n=58) and controls (n=66) genotyped using Taqman assays were included, rs9461741 (BTNL2) was associated with an increased risk of MZL (per-allele odds ratio = 2.38, 95% confidence interval: 1.74-3.24; P=4.32x10-8). Other SNPs at the 6p21.32 locus were also highly associated with MZL, suggesting that this is unlikely to be a chance finding. Promising signals yet to be confirmed were also observed on chromosomes 3 and 14. The BTNL2 protein has extracellular immunoglobulin domains and is expressed in multiple tissues including various immune organs and the gut and it inhibits T-cell activation. Genetic variability at BTNL2 has been associated with inflammatory and infectious phenotypes, highlighting the importance of this locus for immune diseases. In conclusion, findings from this GWAS identify the HLA class II region as an important susceptibility locus for MZL. Citation Format: Joseph Vijai, Zhaoming Wang, Sonja I. Berndt, Susan L. Slager, James R. Cerhan, Christine Skibola, Sophia S. Wang, Angela R. Brooks-Wilson, Silvia de Sanjose, Paige M. Bracci, Mads Melbye, Bengt Glimelius, Rebecca D. Jackson, Wendy Cozen, Nikolaus Becker, Lauren R. Teras, John J. Spinelli, Jenny Turner, Qing Lan, Mark P. Purdue, Kimberly A. Bertrand, Kenneth Offit, Paolo Vineis, Brian K. Link, Graham G. Giles, Anne Zelenuich-Jacquotte, Alain Monnereau, Maria Grazia Ennas, Demetrius Albanes, Laurie Burdett, Nathaniel Rothman, Stephen J. Chanock, Alexandra Nieters, On behalf of the NHL GWAS Consortium. A genome-wide association study suggests evidence of variants at 6p21.32 associated with marginal zone lymphoma. [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 5071. doi:10.1158/1538-7445.AM2014-5071

Abstract Purpose: The purpose of this study was to evaluate oncologists' preferences for methods to increase delivery of tobacco cessation support to cancer patients. Methods: An email list of 6844 physicians at NCI Designated Cancer Centers was generated using publically available online resources. An online survey was conducted in the fall of 2013 to assess tobacco assessment and cessation practices by physicians, barriers to providing cessation support, and methods to improve access to cessation services for cancer patients. Results of this survey are compared to similar survey findings by the American Society of Clinical Oncology (ASCO) and the International Association for the Study of Lung Cancer (IASLC). Results: Of 6844 electronically delivered survey invitations, we received 887 responses (13%). Consistent with previously published ASCO and IASLC surveys, 90% of physicians agreed that tobacco use by patients affects cancer treatment outcomes (vs. 87-92% in ASCO/IASLC), 90% of respondents reported that they ask patients about their tobacco use (vs. 90% in ASCO/IASLC), and 83% routinely advise tobacco users to quit (vs. 80-82% in ASCO/IASLC). However, only 37% of respondents regularly discussed medications, and 35% actively treated patients (vs. 39-44% in ASCO/IASLC). Most (71%) responding oncologists preferred cessation support be provided at the initial patient consult, and few (11%) were concerned about the use of tobacco cessation medications. Lack of time and lack of available resources were the dominant barriers to the administration of tobacco cessation support to cancer patients by oncologists. Only 10% of oncologists believed they were adequately trained to counsel patients on smoking cessation, but 55% reported that training for someone else in their clinic would increase tobacco cessation support. When asked what provider they prefer to administer cessation support to cancer patients, 16% reported a primary care physician, 18% reported a mid-level (nurse practitioner or physician assistant), 50% reported any other clinical staff, and only 1% preferred to personally provide cessation support. Whereas most (58%) oncologists preferred to have dedicated cessation services within their institution, 38% reported they would use dedicated services either within, or outside their institution. Conclusions: Most oncologists at NCI Designated Cancer Centers recognize the importance of tobacco cessation, but prefer to have tobacco cessation support for cancer patients provided by another trained professional. Citation Format: Alix Pommerenke, Anthony Alberg, Thomas H. Brandon, Ivana Croghan, Michael K. Cummings, Carolyn Dresler, Ellen R. Gritz, Roy Herbst, Scott Leischow, James R. Marshall, Benjamin Toll, Graham Warren. Physician preferences in tobacco cessation support for cancer patients: A survey of physicians at National Cancer Institute Designated Cancer Centers. [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 5049. doi:10.1158/1538-7445.AM2014-5049

Abstract Development of resistance to chemotherapy and immunotherapy is a major obstacle in extending the survival of cancer patients. Although several molecular mechanisms have been identified that can contribute to chemoresistance, the role of immune checkpoint molecules in tumor chemoresistance has received little consideration. Elevated expression of B7-H1 (also named PD-L1) is predictive of an aggressive disease course, including increases in disease progression and cancer-related mortality in a variety of cancers (ovary, breast, liver, pancreas, esophagus, colon and lung). Although the aggressiveness of B7-H1 positive tumors has been attributed to B7-H1's immune-suppressive function, B7-H1 itself may be directly altering tumor cell intracellular signaling pathways promoting acquisition of drug resistance. It has been reported that overexpression of B7-H1 confers chemoresistance in human cancers. However, the mechanisms by which B7-H1 contributes to tumor chemoresistance have yet to be defined. We show that B7-H1 transfected tumor cells are resistant to chemotherapy. This resistance is due to increased activation of MAPK/ERK pathway in tumor cells with B7-H1 overexpression. Inhibition of ERK activation restored drug sensitivity of B7-H1 positive tumor cells. We further analyzed the downstream elements of B7-H1 signaling pathway and identified a new B7-H1 associated protein that is involved in DNA damage repair and MAPK/ERK activation mechanisms. We conclude that B7-H1 mediates tumor chemoresistance by enhancing the activation of MAPK/ERK pathway via its intracellular signaling pathway. Our studies may identify a novel pharmaceutical target for treatment of B7-H1 positive refractory tumor cells. Citation Format: Amanda Orzechowski, Haidong Dong. B7-H1 confers tumor chemoresistance by regulating MAPK/ERK activation. [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 5026. doi:10.1158/1538-7445.AM2014-5026

Abstract Ovarian cancer (OC) is the fifth leading cause of cancer death in women in the US. Müllerian inhibiting substance (MIS) is a glycoprotein hormone that causes Müllerian duct regression through a complex of TGF- β family homologous receptors: a type II ligand-binding receptor (T2R) and a tissue-specific type I receptor (T1R) that initiates downstream signaling. MIS has been proposed as potential therapeutic agent in OC. We have previously shown that specific combination of T2R/T1R receptors are expressed on OC cells, however data is lacking on the MIS-dependent pathways activated in OC cells in a T1R-specific manner. These limitations hinder further development of MIS as a therapy. We sought to determine the downstream signaling pathways activated by MIS in a T1R specific manner. We designed a chimeric OC cell model system in which GM-CSF can function as the ligand to specifically activate candidate T2R/T1R combinations (MISRII with either ALK2, ALK3, or ALK6). RNA-Seq technology was used to identify differentially expressed early and late response genes. Over 15,032 genes were analyzed, restricted to genes in which there were > 32 counts for duplicate samples. Pathway analysis was performed using Ingenuity's Integrative Pathway Analysis Tool (IPA) for genes which were expressed over 2 fold, with an False Discovery Rate (FDR)<0.1 and log (Counts per Million, CPM)>-0.001. We observed 56, 37, and 51 early response (1hr) genes which were up- or down-regulated after stimulation in OC cells engineered to express either ALK2, ALK3, and ALK6, respectively. We identified a subset of 18 of genes (e.g., ID1, SMAD7, FOSB) which were upregulated after treatment in all MISRII/T1R combinations, suggesting redundancy amongst the three T1R partner receptors. These observations will be confirmed by RT-qPCR and immunoblotting. A majority of these redundant genes are known to play roles in TGF-β signaling and cell proliferation pathways. Importantly, as we have previously shown that not all T1R candidates are expressed in OC, we report subsets of genes (e.g., RASSF6, C/EBPBβ, CSMD3) which were uniquely altered after stimulation only in the presence of a specific T1R. This indicates specific pathway activation is dependent upon T1R expression pattern in OC and likely determines response. In addition to previously known TGF- β signaling pathway, we found gene signatures (e.g. FOSB, RASSF6, LRP1B, ID1) suggesting pathways which affect cell proliferation, apoptosis, chemotherapy resistance, and DNA repair in response to MIS. This is the first study to begin to define the specific roles candidate T1R's play in signaling in response to MIS in an OC model. An understanding of these pathways is a necessary prerequisite to examining MIS as a therapy. These preliminary data suggest a role for MIS in apoptosis, cytotoxic drug resistance and DNA repair. This can be used to test combination therapy with currently available drugs and MIS in OC treatment. Citation Format: Qing Zhang, Eati Basal, Jaime I. Davila, Xueqian Yin, Edward B. Leof, William A. Cliby. Comparative RNA-Seq analysis of MIS signaling: Potential relevance as therapeutic strategy in ovarian cancer treatment. [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 3462. doi:10.1158/1538-7445.AM2014-3462

Abstract B cell chronic lymphocytic leukemia (CLL) is an incurable disease and represents a significant health problem in the western world. We and others have reported that primary CLL B cells spontaneously produce increased levels of proangiogenic basic fibroblast growth factor (bFGF) in vitro and that CLL plasma contained elevated levels of bFGF. However, the precise role of bFGF in CLL pathobiology is not clearly understood. In this study we investigated functional implication of the FGF/FGFR signaling axis in CLL B cell biology. We have detected expression of FGFR1 and FGFR3 with comparatively higher levels of the latter receptor, but not FGFR2/FGFR4, in primary CLL B cells. Interestingly, constitutively phosphorylated FGFR and its downstream signaling adaptor protein fibroblast receptor substrate 2-α were also detected in CLL B cells. However, in vitro stimulation of FGFR with recombinant bFGF was unable to increase endogenous phosphorylation levels of FGFR in CLL B cells from most CLL patients tested, suggesting existence of an autocrine FGF/FGFR signaling loop in the leukemic B cells. We know that Axl a known predominant RTK in CLL B cells seems to act as a docking site for multiple cellular kinases/lipase. Therefore, in an effort to determine whether Axl is functionally associated with FGFR in CLL B cells, we first examined if these two RTKs exist in the same molecular complex. Indeed we found that Axl forms a complex with FGFR3 in CLL B cells and is therefore likely to regulate FGFR signaling. Our hypothesis in this regard was further supported when we inhibited Axl phosphorylation in CLL B cells using a high-affinity Axl inhibitor (TP-0903; Tolero). Axl inhibition resulted in significant reduction of FGFR phosphorylation in CLL B cells. Finally, inhibition of FGFR signaling using a high-affinity FGFR inhibitor induced significant levels of apoptosis in CLL B cells (n=20) with a mean LD50 dose of ∼2.5 μM. Of interest, LD50 dose for CLL B cell apoptosis was much lower for the Axl inhibitor (mean LD50=140nM; n=20) compared to the FGFR inhibitor, which we believe occurs partly because TP-0903 seems to exert its inhibitory effect on P-FGFR likely via P-Axl inhibition in CLL B cells. In conclusion, we have detected expression of constitutively active FGFR in primary CLL B cells. Interestingly, our preliminary findings suggest that Axl forms an active RTK complex with FGFR and thus likely regulates FGFR signaling in these leukemic B cells. This is evidenced by the finding that Axl inhibition significantly reduced FGFR phosphorylation accompanied by massive induction of CLL B cell apoptosis, suggesting that targeting dual RTK signaling pathways (Axl and FGFR) in CLL B cells warrants further investigation as a way to develop a more effective and efficient therapeutic intervention for CLL patients. Citation Format: Sutapa Sinha, Justin Boyson, Charla Secreto, Steven L. Warner, Neil E. Kay, Asish K. Ghosh. Fibroblast growth factor receptor is expressed as a constitutively active receptor tyrosine kinase in chronic lymphocytic leukemia B cells and exists in an active complex with Axl: Dual targeting in CLL. [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 3448. doi:10.1158/1538-7445.AM2014-3448