Seema Bhuyan

Abstract 3903: Targeting oral cancer stem cells in the hypoxic niche by BCG infected mesenchymal stem cells

Background: Tumor hypoxia is a major contributing factor in cancer therapeutic failure. The microenvironment of hypoxia/oxidative stress may reprogram cancer cells to highly tumorigenic, metastatic, angiogenic and stem cell-like state. We have characterized in vitro and in vivo models of hypoxia-induced cancer cell reprogramming to cancer stem cell (CSCs) like cells including an immunocompetent model of oral cancer. These CSCs exhibit ABCG2 cell surface marker. Numerous approaches including targeted therapy using oncolytic virus and bacteria have been attempted to target cancer cells in their hypoxic niche without major therapeutic success. Major therapeutic challenges include the inaccessibility of hypoxic niche by therapy-agents and the poor replication of viruses or bacteria intracellular to hypoxic cancer cells. Furthermore, the immune privileged microenvironment of tumor hypoxia could pose a challenge to viral/bacterial-induced immunity against the tumor. In this context, here we have tested a novel stem cell-based approach to deliver BCG infected stem cells to the hypoxic core of tumors. Methods: Human SCC-25 cell line-derived xenograft in NOD/SCID mice exhibit hypoxic zones, where ABCG2+ resides (1). SCC-25 tumor-bearing mice were injected i.v. with CD271+ bone marrow mesenchymal stem cells (BM-MSCs). We recently found that CD271+ BM-MSCs could be infected with M. tuberculosis. The BCG infected CD271+ BM-MSCs were injected to SCC-25 xenograft-bearing mice. The mice were sacrificed on day-20 of stem cell injection and then evaluated for the hypoxic CSCs. Next, we have developed a 4-NQO induced oral cancer cell line, which was injected orthotropic to the tongue of congenic mice. The mice developed tumors, which was hypoxic. In these tumor-bearing mice, BCG-infected murine CD271 BM-MSCs were injected i.v., and animals were monitored for four weeks for tumor growth. On day-20 of injection, mice were sacrificed, and the tumors were collected. In a control group, BCG alone was injected. Results: In both the SCC-25 and an immunocompetent mouse model, we found that BCG or CD271+ BM-MSCs injection alone did not result in marked anti-tumor activity and or elimination of ABCG2+ cells from their hypoxic niche. In contrast, the injection of BCG infected CD271+ BM-MSCs led to marked reduction of tumor growth. Importantly, we observed marked replication of BCG intracellular to CSCs. These results indicate that CD271+ BM-MSCs facilitated the transfer of BCG to cancer cells residing in the hypoxic zone. Importantly, the transfer of BCG was seen more in the immunocompetent model, suggesting potential role of immune system. Conclusions: Our findings indicate that hypoxic CSCs may be targeted by a few BCG infected CD271 BM-MSCs. We propose this could be a novel therapeutic approach to target drug resistance cancer stem cells residing in the hypoxic niche of tumors.1. Bhuyan R et al, Cancer Research, 2016; Volume 76, Issue 14 Supplement, pp. 935. Note: This abstract was not presented at the meeting. Citation Format: Bidisha Pal, Seema Bhuyan, Jaishree Garhyan, Hong Li, Rashmi Bhuyan, Herman Yeger, Bikul Das. Targeting oral cancer stem cells in the hypoxic niche by BCG infected mesenchymal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3903. doi:10.1158/1538-7445.AM2017-3903

Abstract 2681: Oral micro biome enhances stemness in oral cancer cells by activating Toll like receptor signaling

Background: The resistance and progression of cancers after chemotherapy to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Recent studies suggest, microbiomes can induce a cascade of host events to either support or inhibit tumor growth. Specially, in oral cancer, chemotherapy treatment may alter the oral microbial flora, which may favor or inhibit tumor growth. Hence, it is importantl to develop novel experimental approaches to study the role of oral microbial flora in oral cancer stemness (self-renewal and undifferntiated state of cancer stem cells). Importantly, patients in developing area, including Assam, where KaviKrishna laboratory is located, may have distinct oral microbial flora that could favor oral cancer growth. Hence, it is important to include patients from developing countries for such studies. Our previous research showed that chemotherapy ehances stemenss in many cancer cell types, including oral squamous cell carcinoma cell line SSC-25. The stemness switch is characterized by enhanced expression of stemness associated genes including Nanog, Lin28A/B, Oct-4, MYC, HIF-2alpha and inflammation associated genes including Toll like receptor (TLR) 2/4. Here we investigated the role of oral microbiomes in the TLR mediated stemness switch of oral cancer cells. Methods: SCC-25 oral cancer cell line was treated with bacterial product lipopoly saccharide (LPS), and the stemness switch evaluated by isolation of ABCG2+ cells and expression of stemness associated genes by these cells. Capacity of interaction of tumor stromal cells with mesenchymal stem cells was also evaluated. Additionally, we obtained sputum from oral cancer subjects undergoing chemotherapy. The patients were from the Kamrup district of Assam, where KaviKrishna laboratory is located. The sputum was processed and then added to the culture medium of SCC-25 cells. These post-sputum treated SCC-25 cells were subjected to phenotypic stemness switch analysis. Results: We found, LPS and sputum treatment led to the enhanced stemness of ABCG2+ cells, including the high expression of TLR2/4, MYC, Nanog, Sox-2, and HIF-2alpha. Importantly, sputum derived from oral cancer subjects under remission showed inhibitory activity on ABCG2+ cell self-renewal. In contrast, sputum obtained from oral cancer subjects with relapse showed enhanced stemness of ABCG2+ cells, and also increased tumorigenic potential. The post-sputum treated ABCG2+ cells exhibited high expression of TLR2/4 and associated increase of HIF-2alpha and MYC transcriptional activity. The sputum treated with broad spectrum antibiotic ciprofloxacin did not enhance the stemness and TLR2/4 signaling of SCC-25 cells. Conclusion: These results indicate that oral microbiomes may differentially influence the stemness of oral cancer cells. We also conclude that live bacteria present in the sputum may be required to enhance stemness in a TLR2/4 dependent manner. Note: This abstract was not presented at the meeting. Citation Format: Joyeeta Talukdar, Rashmi Bhuyan, Bidisha Pal, Sorra Sandhya, Hong Li, Seema Bhuyan, Sukanya Garhyan, Debabrat Baishya, Anupam Sarma, Jyotirmoy Phukan, Amal Kataki, Bikul Das. Oral micro biome enhances stemness in oral cancer cells by activating Toll like receptor signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2681. doi:10.1158/1538-7445.AM2017-2681

Abstract 4020: Identification and characterization of Novel MYC and p53 target molecules from medicinal plants of North East India

Background: Identification and characterization of novel molecular inhibitors that target key regulators of cancer stem cell (CSCs) self-renewal is promising in cancer therapeutics. MYC and p53, the two major cellular transcription factors, acts as key molecular regulators in stem cells and cancer stem cells self-renewal. While MYC enhances, p53 inhibits the self-renewal of stem cells. Thus, developing novel molecules that could potentially target either MYC and or p53 might potential therapeutic application. However, the search for small molecules that could target the self-renewal aspect of these two proteins has been elusive. Previously; we demonstrated that isoprenoid molecule squalene could enhance bone marrow hematopoietic and mesenchymal stem cells (MSCs) by modulating MYC. Importantly, we found that ursolic acid, an isoprenoid molecule found in Tulsi plant, inhibited HIF-1alpha, a transcription factor regulated by MYC and p53. Hence, having these prior experiences on isoprenoids, we speculate that novel isoprenoid may exist that could modulate MYC’s role in cancer self-renewal. We are especially interested in identifying novel isoprenoid molecules (metabolites of mevalonate pathway) found in medicinal plants of North East India, where KaviKrishna laboratory is located. Methods: For the study, we proposed to develop an in vitro self-renewal based assay platform(henceforth known as STEM-TECH platform) to identify novel molecules that act on self-renewal aspect of MYC and p53. In this assay, we subjected the oral cancer cell line SCC-25 to 3-D tumoringeic growth using methylcellulose based method. Using this assay, we have screened 20 herbal extracts of North East India, and selected 3 herbal extracts for further evaluation. For the in vivo study, we added these herbal extracts to drinking water of C57BL/6 mice treated with 4NQO (an oral carcinogen) and FVB/N mice with MYC-induced thymic lymphoma. The mice were observed for 10 weeks and subjected to evaluation of tumor growth, and also evaluation of cancer stem cells using ABCG2, a cell surface marker expressed by cancer stem cells. Results: We found that herbal extracts from Tulsi and Soalu exhibited strong anti-tumor activity in the in vivo mouse models. Importantly, these extracts exhibited the ability to inhibit the self-renewal of MYC driven ABCG2+ cancer cells (1). Also, these extracts were then subjected to in vitro Stem-Tech assay and we found that one herbal extract could modulate the transcriptional binding of MYC. Importantly, we found that squalene, an isoprenoid found in several herbal extracts in NE India could modulate MYC activity by an unknown mechanism. Conclusion: Our results indicate that the vast, untapped herbal plants available in India’s remote North East contain valuable herbal medicinal plant having potential MYC inhibitor. 1. Das B et al. MYC through HIF-2alpha regulates the self-renewal program in cancer stem cells (under review). Note: This abstract was not presented at the meeting. Citation Format: Sora Sandhya, Joyeeta Talukdar, Bidisha Pal, Seema Bhuyan, Debabrat Baishya, Bikul Das. Identification and characterization of Novel MYC and p53 target molecules from medicinal plants of North East India [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4020. doi:10.1158/1538-7445.AM2017-4020

Abstract 935: Oral cancer cells may hijack stem cell altruism to survive during extreme hypoxia, and exposure to chemotherapeutic drugs

The mechanism of tumor hypoxia induced reprogramming of cancer cells are not understood well. Hypoxia might activate evolutionary preserved cellular defense mechanism that could contribute to tumor progression and metastasis. We recently described that during hypoxia/oxidative stress, human embryonic stem cells (hES) exhibit an altruistic defense mechanism, where a few cells reprogram to a highly undifferentiated state, and secrete glutathione to protect rest of the community of cells from oxidative stress induced DNA damage (Das B et al, Stem Cells, 2012). The altruistic stem cells phenotype exhibited high HIF-2alpha and low p53 activity. After a few weeks, the reprogrammed cells, although highly fit to survives, underwent spontaneous apoptosis/differentiation by re-activating the p53/MDM2 oscillation. Thus, the reprogrammed cells sacrificed its own fitness to enhance the fitness of the rest of the community, an altruistic behavior (Das B et al. Stem Cells, 2012). Here, we investigated the potential hijacking of the altruistic defense mechanism by oral cancer cells during exposure to hypoxia. We exposed the four oral cancer cell lines SCC-25, SCC-15, SCC-9 and SCC-5 to extreme hypoxia followed by re-oxygenation for 72 hours. We found that while majority of cancer cells underwent apoptosis, a few cancer cell lines survived, exhibited side-population (SP) phenotype, high level of HIF-2alpha, Nanog, Sox-2 and MYC. The SP cells exhibited migratory activity, as well as high tumorigenic and metastatic activity in NOD/SCID mice. ChIP assay indicated that HIF-2alhpa interact with MYC and NOTCH1. The conditioned media of SP cells exhibited high level of glutathione, and ability to protect non-SP cells from cisplatin-induced toxicity. These results indicate that HIF-2alpha and MYC may cooperate to reprogram a few oral cancer cells to altruistic stemness phenotype. The altruistic phenotype that exhibited cytoprotective activity against cisplatin mediated toxicity. Thus, similar to bacteria, where altruistic biofilm exhibit novel drug resistance mechanism, stem cell altruism may serve as a novel drug resistance mechanism in oral cancer. Citation Format: Rashmi Bhuyan, Hong Li, Sukanya Gayan, Bidisha Pal, Reza Bayat-Mokhtari, Jyotirmoy Phukan, Debabrata Baishya, Anupam Sarma, Joyeeta Talukdar, Manaf Muhammad Alkurdi, Wael Tasabehji, Seema Bhuyan, Gayatri Gogoi, Ista Pulu, Herman Yeger, Bikul Das. Oral cancer cells may hijack stem cell altruism to survive during extreme hypoxia, and exposure to chemotherapeutic drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 935.

Abstract 4324: Undenatured whey protein isolate exhibit chemopreventive activity against HPV-16 induced carcinogenesis of CD271+ oral mucosa stem cells

Oral cancer, and associated HPV-16 infection, and tobacco smoking is a major public health concern in the Kamrup district of India. To facilitate research on oral cancer chemoprevention, we have set up a collaborative research team between Forsyth Institute, an oral medicine specialized center affiliated to Harvard School of Dental Medicine, and KaviKrishna Laboratory, a non-for profit private laboratory set up located in Guwahati, the major city of Kamrup district, India. To study the cellular and molecular mechanism of HPV-16 mediated carcinogenesis, we have developed an in vitro model of HPV-16 mediated carcinogenesis of oral squamous stem cells. In this model (Bhuyan et al. The potential role of oral mucosa stem cell altruistic behavior as the initiating event of malignant transformation. AACR abstract Control # 16-A-6618-AACR), the treatment of oral mucosa cells derived from healthy volunteer with HPV-16 derived E6 protein led to expansion of a p53 deficient CD271+ expressing oral mucosa stem cells (OMSC). Noted that CD271 cell surface marker was recently identified as a putative marker for OMSCs. Using this in vitro carcinogenesis model, we evaluated the potential chemopreventive role of dietary whey protein. The E6 treated p53 deficient CD271+ cells were treated with DMEM/F12 media containing 0.2% of Immunocal, an undenatured whey protein (Tsai WY et al. Nutr Cancer 2000. PMID: 11525598). The p53 status as well as in vitro self-renewal activity of the CD271+ cells were examined by ELISA, transcriptional activity assay, and methylcellulose based clonogenic assay. We also fed Immunocal 20 gm/daily for 6 months to 5 individual with oral leukoplakia lesion with HPV-16 positivity. The CD271+ cells obtained from the leukoplakia lesion of clinical subjects were subjected to p53 status, and clonogenic assay. The data was compared with CD271+ cells obtained from leukoplakia lesion of subjects taking regular diet without whey protein supplements. We found that addition of Immunocal whey protein led to 8-fold decrease in the expansion of CD271+ cells following E6 treatment in the in vitro model of OMSC culture. Importantly, Immunocal treatment prevented the suppression of p53 in OMSCs. In the preliminary clinical study, the dietary intake of Immunocal led to complete loss of leukoplakia lesion in the 4/5 individual. In contrast, the control subjects exhibited the presence of CD271+ cells in the leukoplakia lesion having low p53 status, and high clonogenic activity. We are incorporating a larger number of subjects to study the potential chemopreventive activity of whey protein, using the locally available affordable whey protein extracts. Conclusion: This study indicate that whey protein extract, which is available in the local villages of Kamrup district could serve as a chemopreventive agent against oral cancer. Citation Format: Sora Sandhya, Rika Tsuchida, Sukanya Gayan, Rashmi Bhuyan, Joyeeta Talukdar, Bidisha Pal, Seema Bhuyan, Jugal Ch Das, Amal Ch Kataki, Debabrata Baishya, Bikul Das. Undenatured whey protein isolate exhibit chemopreventive activity against HPV-16 induced carcinogenesis of CD271+ oral mucosa stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4324.

Abstract 4073: The potential role of oral mucosa stem cell altruistic behavior as the initiating event of malignant transformation

Oral cancer presents a major health burden across the globe, specially in developing countries. Kamrup, a district of India, where the KaviKrishna laboratory is located has the highest incidence of oral cancer in the entire world. The mechanism of oral cancer carcinogenesis process is not clearly known. Previous studies indicate the potential role of HPV-16 virus as well as tobacco consumption as major contributing factors of oral cancer. In mouse, the carcinogen agent 4-NQO was found to induce oral cancer having similar phenotype as human oral mucosa cancer. 4-NQO may exert tobacco like oxidative stress toxicity to oral mucosa. Hence, developing both in vitro and in vivo models of HPV-16 and 4-NQO mediated oral carcinogenesis might help to understand the initiating event of oral carcinogenesis. In this study, we propose that oral mucosa stem cells (OMSCs) could be the target of HPV-16, and 4-NQO induced carcinogensis. Thus, in vitro treatment of oral mucosa cells of healthy human volunteers with HPV-16 protein E6, and 4-NQO led to expansion of CD271+ cells, which are enriched in OMSCs. Importantly, the expanded CD271+ cells exhibited high HIF-2alpha, low p53, and high glutathione secretion, a phenotype of stem cell altruism that we recently described in human embryonic stem cells (Das B et al. Stem Cells, 2012). In human ES cells, the altruistic reprogramming served as an initiating event of malignant transformation by altering p53/MDM2 oscillation, in an abnormal state of low p53 and high MDM2. Therefore, we performed a complete evaluation of E6 protein and 4-NQO treated CD271+ oral mucosa cells for altruistic behavior, including low p53 and high MDM2 state. For this purpose, the carcinogen treated oral mucosa cells were subjected to immunomagnetic sorting for CD271+ cells. We found that post-carcinogen treated CD271+ cells exhibited in vitro self-renewal activity, high GSH secretion, and importantly activation of a HIF-2alpha/MYC co-operation. ChiP assay revealed the MYC binding to HIF-2alpha, and Sox-2, an stemness associated transcription factor. Importantly, HIF-2alpha was important for the reversible but prolonged suppression of p53 for more than two weeks. We also found that the high HIF-2alpha and low p53 expressing CD271+ cells could be enriched in a ABCG2+ population. Thus, we were able to enrich a CD271+/ABCG2+ cell population in oral mucosa cells of both human and mouse. Based on these findings we propose to use HPV-16 and 4NQO derived carcinogenesis models to study the potential role of altruistic reprogramming in the malignant transformation of oral mucosa stem cells to oral cancer stem cell like cells. Our study may unravel a novel mechanism of malignant transformation, the failure of altruistic stem cells to sacrifice its fitness (altruism) as a potential initiating event of malignant transformation of stem cells to cancer stem cells. Citation Format: Sukanya Gayan, Hong Li, Rashmi Bhuyan, Sora Sandhya, Joyeeta Talukdar, Bidisha Pal, Jaishree Garhyan, Wael Tasabehji, Manaf Muhammad Alkurdi, Heidar Zohrehei, Seema Bhuyan, Anupam Sarma, Gayatri Gogoi, A.C. Kataki, Rakesh Bhatnagar, Debabrata Baishya, Bikul Das. The potential role of oral mucosa stem cell altruistic behavior as the initiating event of malignant transformation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4073.

Abstract 251: Stem cell altruism may serve as a novel drug resistance mechanism in oral cancer

Background: The mechanism of oral squamous cell carcinoma resistance to platinum-based chemotherapy is not clearly known. Previous studies indicated that glutathione (GSH), a cellular antioxidant may detoxify cisplatinum (CDDP), a commonly used chemotherapy agent in oral cancer. Our previous research in human embryonic stem cells (hESCs) indicated the altruistic behavior of ABCG2+ hESCs that secrete high level of GSH to protect other hESCs exposed to oxidative stress (Das B et al. Stem Cells, 2012). Here we investigated if CDDP exposure lead to altruistic stem cell reprogramming of ABCG2+ oral squamous cell carcinoma cells and subsequent GSH-mediated resistance against CDDP. Methods: Two oral squamous cell cancer cell lines SCC-25 and SCC-15 were treated with 3-10 uM CDDP for three-days, and then subjected to flow cytometry and immunomagnetic sorting based evaluation of ABCG2+ cells. The conditioned media (CM) obtained from ABCG2+ cells were examined for GSH content. The CM treated cancer cell lines were examined for resistance against CDDP toxicity. Next, the post-CDDP treated ABCG2+ cells were examined for enhanced stemness phenotype that corresponds to altruistic stem cell phenotype (Das B et al, Stem Cells 2012). Results: We found that CDDP treatment increases the ABCG2+ self-renewal capacity of SCC-25 and SCC-15 cells as measured by serial transplantation assay. The CM of the post-CDDP treated cells exhibited high level of GSH. When the SCC-15 and SCC-25 cells were treated with CM plus CDDP, the cancer cells exhibited 10-15-fold increase in resistance against CDDP toxicity. Next, the post-CDDP treated SCC-25 and SCC-15 cells exhibited enhanced stemness reprogramming phenotype characterized by very high HIF-2alpha, Sox-2 and Nanog transcriptional activity. Furthemore, we found increased expression of EMT (epithelial mesenchymal transition) marker expression including Snail, Twist and vimentin as evaluated by flow cytometry. siRNA HIF-2alpha treatment led to marked loss in the in vivo self-renewal capacity of ABCG2+ SCC-25 and SCC-15 cells. We then noted that post-CDDP ABCG2+ cells exhibited reversible state, as after two weeks of culture, most of the cells either differentiated or underwent apoptosis. Conclusions: These results indicate that oral cancer cells exhibit altruistic defense mechanism to resist the toxicity of CDDP. The altruistic defense mechanism involved high secretion of GSH. Thus, we suggest that similar to bacterial altruism as a mechanism of drug resistance, stem cell altruism may also serve as a novel mechanism of drug resistance in cancer. Citation Format: Bidisha Pal, Reza Bayat-Mokhtari, Hong Li, Rashmi Bhuyan, Joyeeta Talukdar, Sora Sandhya, Anupam Sarma, Wael Tasabehji, Seema Bhuyan, Sukanya Gayan, Amal Ch Kataki, Debabrata Baishya, Herman Yeger, Dean W. Felsher, Bikul Das. Stem cell altruism may serve as a novel drug resistance mechanism in oral cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 251.

Abstract 1529: MYC and HIF-2alpha cooperates in oral squamous carcinoma cell self-renewal during hypoxia

Oral squamous cell carcinoma (OSCC) is a global health challenge. The overall survival rate of this devastating disease has not significantly changed, and the biology of this type of cancer also remain largely unexplored. To enhance the understanding of this disease through global heath cancer research collaborative effort, we have set up an international collaboration between Forsyth Institute, Cambridge, and KaviKrishna laboratory, which is located in Kamrup district of Assam, having highest incidence of oral cancer in the world. Through this collaborative effort we intend to explore the role of hypoxia in oral cancer progression, metastasis and therapy resistance. Method: We used an oral squamous cell carcinoma cell line SSC25 for this purpose, and performed several experiments using a well-described in vitro assay of CSC self-renewal under hypoxia. Results: We identified a rare ABCG2+ expressing, highly tumorigenic cell population in SSC-25 having cancer stem cell (CSC) like characteristics in the in vivo serial transplantation assay, as well as high metastatic activity to bone marrow of NOD/SCID mice. The ABCG2+ cells, when exposed to hypoxia ( Citation Format: Hong Li, Joyeeta Talukdar, Sora Sandhya, Seema Bhuyan, Sukanya Gayan, Anupam Sarma, Reza Bayat Mokhtari, Dean W. Felsher, Herman Yeger, Bikul Das. MYC and HIF-2alpha cooperates in oral squamous carcinoma cell self-renewal during hypoxia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1529. doi:10.1158/1538-7445.AM2015-1529