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Prostate cancer is the second most common cause of cancer related deaths in men in the United States. Pathogenesis is driven by the androgen receptor (AR), which has led to front-line treatment modalities that are based on androgen deprivation therapy (ADT). About 10-20% of all prostate cancers evolve to resist ADT and are classified as castration-resistant prostate cancer (CRPC) indicating the continued need for new treatment options. Bromodomain-containing protein 4 (BRD4) is an acetylated-chromatin associating protein that is involved in transcriptional elongation, mRNA splicing, epigenetic bookmarking, and super-enhancer activity. The BRD4 protein has been shown to both bind and colocalize with AR at androgen response elements (AREs) on chromatin. Furthermore, elevated BRD4 expression is prognostic of increased prostate specific antigen (PSA) levels following radical prostatectomy and is correlated with higher Gleason scores and poor overall survival. The intertwined activity of BRD4 with multiple essential driver mechanisms of prostate cancer suggests it may be a key target for developing novel therapeutics. Using our ultra-high throughput cell-based screening platform, which directly measures degradation of pathogenic proteins upon exposure to diverse chemical libraries, we identified a series of novel monovalent BRD4 degraders that was optimized to produce our lead compound PLX-3618. A cancer cell panel screen for antiproliferative effects of PLX-3618 indicated enhanced sensitivity in subsets of prostate cancer lines. PLX-3618 elicited selective, rapid, and deep degradation of BRD4 protein in prostate cancer cell models, without degrading the closely related BRD2 and BRD3 proteins. Addition of either proteosome or neddylation inhibitors blocked BRD4 degradation indicating a ubiquitin-proteosome system mediated clearance mechanism. Degradation of BRD4 led to sustained multimodal inhibition of the AR pathway and disruption of key oncogene enhancer networks. When compared to pan-BET inhibitors, treatment of select prostate cancer cell lines with PLX-3618 resulted in increased levels of the tumor suppressors p53 and p21, an aberrant DNA damage response, and substantially amplified apoptosis. Finally, in in vivo preclinical models of prostate cancer, PLX-3618 showed far superior efficacy over a pan-BET inhibitor. Taken together, the selective degradation of BRD4 via the potent monovalent degrader PLX-3618 represents a novel strategy in treating prostate cancer. Citation Format: Kenneth Steadman, Gregory S. Parker, Geoffray Leriche, Sarah Fish, Julia Toth, Mary E. Spalding, Elizabeth Daniele, Aleksandar Jamborcic, Xiaoming Li, E Adam Kallel, Farhana Barmare, Kenneth Chng, Erika Green, Michael Hocker, Elliot Imler, Yi Zhang, Peggy A. Thompson, Simon Bailey. PLX-3618, a potent, selective monovalent BRD4 degrader demonstrates activity in models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 421.

Abnormalities in nuclear shape are a well-known feature of cancer, but their contribution to malignant progression remains poorly understood. Here, we show that depletion of the cytoskeletal regulator, Diaphanous-related formin 3 (DIAPH3), or the nuclear membrane–associated proteins, lamin A/C, in prostate and breast cancer cells, induces nuclear shape instability, with a corresponding gain in malignant properties, including secretion of extracellular vesicles that contain genomic material. This transformation is characterized by a reduction and/or mislocalization of the inner nuclear membrane protein, emerin. Consistent with this, depletion of emerin evokes nuclear shape instability and promotes metastasis. By visualizing emerin localization, evidence for nuclear shape instability was observed in cultured tumor cells, in experimental models of prostate cancer, in human prostate cancer tissues, and in circulating tumor cells from patients with metastatic disease. Quantitation of emerin mislocalization discriminated cancer from benign tissue and correlated with disease progression in a prostate cancer cohort. Taken together, these results identify emerin as a mediator of nuclear shape stability in cancer and show that destabilization of emerin can promote metastasis. Significance: This study identifies a novel mechanism integrating the control of nuclear structure with the metastatic phenotype, and our inclusion of two types of human specimens (cancer tissues and circulating tumor cells) demonstrates direct relevance to human cancer. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6086/F1.large.jpg. Cancer Res; 78(21); 6086–97. ©2018 AACR.

BACKGROUND Acquired resistance to antiepidermal growth factor receptor (anti-EGFR) therapy may be caused by EGFR–v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ErbB2) heterodimerization and pathway reactivation. In preclinical studies, inhibiting ErbB2 blocked this resistance mechanism and resensitized cells to anti-EGFR therapy. Cetuximab targets EGFR, whereas lapatinib inhibits both EGFR and ErbB2. The objective of this phase 1 trial was to assess the safety, dose-limiting toxicities (DLTs), and maximum tolerated doses (MTDs) of cetuximab and lapatinib in patients with solid tumors. METHODS Patients received standard weekly cetuximab with escalating lapatinib doses of 750 mg, 1000 mg, or 1250 mg daily in 3-week cycles. DLTs were monitored through the end of cycle 2. Pretreatment and post-treatment tumor biopsies and germline DNA samples were obtained for correlative studies. RESULTS Twenty-two patients were enrolled, and 18 patients each were evaluable for toxicity and response. Fifty-nine percent of patients had received prior anti-EGFR therapy. Common toxicities included rash and diarrhea. No patient experienced a DLT at the highest dose level, and no grade 4 toxicity was observed. Response included no complete responses, 3 partial responses, 9 patients with stable disease, and 6 patients with disease progression, for an overall response rate of 17% and a clinical benefit rate of 67%. The clinical benefit rate in patients who had previously received anti-EGFR therapy was 70%. The mean treatment duration was 4.7 cycles (range, 1-14 cycles). Decreased expression of EGFR/ErbB2 pathway components after treatment was correlated with response, whereas increased expression in the PI3K, Jak/Stat, and MAPK pathways occurred in nonresponders. CONCLUSIONS The combination of cetuximab and lapatinib was well tolerated, had the expected toxicities, and exhibited notable clinical activity, including in patients who had received previous anti-EGFR therapy. Further clinical study of this combination is warranted. Cancer 2015;121:1645–1653. © 2015 American Cancer Society.

Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous SNPs resulting in the amino acid changes at V12M, Q141K and D620N. To determine whether the SNPs have an effect on drug transport, human embryonic kidney cells (HEK-293) were stably transfected with full length ABCG2 coding wild-type or SNP variants of ABCG2. In 4-day cytotoxicity assays with mitoxantrone, topotecan, SN-38 or diflomotecan, cells transfected with wild-type R482 ABCG2 showed IC50 values up to 1.2-fold to 5-fold higher than cells expressing comparable levels of Q141K ABCG2, suggesting that the Q141K SNP affects drug transport. FTC-inhibitable mitoxantrone efflux normalized to ABCG2 surface expression as assayed by the anti-ABCG2 antibody 5D3 was significantly lower in cells transfected with Q141K ABCG2 than in those transfected with wild-type R482 ABCG2 (P = 0.0048). Values for V12M and D620N ABCG2 were comparable to those for wild-type R482 ABCG2. The vanadate-sensitive ATPase activity of ABCG2 was assayed in Sf9 insect cells infected with wild-type or SNP variants of ABCG2. Basal ATPase activity in cells transfected with Q141K ABCG2 was 1.8-fold lower than in cells transfected with wild-type ABCG2, but was comparable among cells expressing wild-type, V12M or D620N ABCG2. Confocal studies of ABCG2 localization revealed higher intracellular staining in the Q141K transfectants than in cells transfected with wild-type or V12M ABCG2. Decreased transport of Hoechst 33342 was observed in Sf9 cells expressing V12M ABCG2; however, this was not true in HEK-293 cells expressing V12M ABCG2. These results suggest that the Q141K SNP affects the transport efficiency of ABCG2 and may result in altered pharmacokinetics or drug-resistance profiles in clinical oncology.

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered followed by activation of the photosensitizer by exposure to a light source of a given wavelength. This, in turn, generates reactive oxygen species that induce cellular apoptosis and necrosis in tumor tissue. Based on our earlier finding that the photosensitizer pheophorbide a is an ABCG2 substrate, we explored the ability of ABCG2 to transport photosensitizers with a structure similar to that of pheophorbide a. ABCG2-overexpressing NCI-H1650 MX50 bronchoalveolar carcinoma cells were found to have reduced intracellular accumulation of pyropheophorbide a methyl ester and chlorin e6 compared to parental cells as measured by flow cytometry. The ABCG2 inhibitor fumitremorgin C was found to abrogate ABCG2-mediated transport. Intracellular fluorescence of hematoporphyrin IX, meso-tetra(3-hydroxyphenyl)porphyrin, and meso-tetra(3-hydroxyphenyl)chlorin was not substantially affected by ABCG2. ABCG2-overexpressing cells also displayed decreased intracellular fluorescence of protoporphyrin IX generated by exogenous application of 5-aminolevulinic acid. Mutations at amino acid 482 in the ABCG2 protein known to affect substrate specificity were not found to impact transport of the photosensitizers. In cytotoxicity assays, ABCG2-transfected HEK-293 cells were 11-fold, 30-fold, 4-fold, and >7-fold resistant to PDT with pheophorbide a, pyropheophorbide a methyl ester, chlorin e6, and 5-aminolevulinic acid, respectively. ABCG2-transfected cells were not resistant to PDT with meso-tetra(3-hydroxyphenyl) chlorin. Neither multidrug resistance-associated protein 1 expression nor P-glycoprotein expression appreciably decreased the intracellular fluorescence of any of the photosensitizers examined as determined by flow cytometry. The results presented here implicate ABCG2 as a possible cause for cellular resistance to photodynamic therapy.

Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate based on Abcg2−/− knockout mouse studies (J. W. Jonker et al., Proc. Natl. Acad. Sci. USA, 99: 15649–15654, 2002). We developed a functional assay for ABCG2 using PhA and the ABCG2 inhibitor fumitremorgin C. In selected cell lines expressing high levels of P-glycoprotein, multidrug resistance-associated protein 1, or ABCG2, PhA transport was observed only in cells expressing ABCG2. Fumitremorgin C-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. We found that 100 μm of the cyclin-dependent kinase inhibitor UCN-01 or 1 μm of the P-glycoprotein inhibitor tariquidar inhibited ABCG2-mediated PhA transport. In 4-day cytotoxicity assays, ABCG2-mediated resistance to SN-38 and topotecan was abrogated in ABCG2-transfected HEK-293 cells treated with 1 μm tariquidar, and ABCG2-transfected cells were 6–7-fold resistant to UCN-01. PhA is an ABCG2-specific substrate with potential value in measuring ABCG2 function and expression in clinical samples.

Non-AIDS-defining cancers (NADCs) now exceed rates of AIDS-defining cancers in HIV-positive patients. Treatment of NADCs may be complicated by drug-drug interactions between antiretrovirals and chemotherapy. Docetaxel is a widely used anticancer agent that is primarily metabolized by CYP3A4 enzymes and used to treat NADCs. A preclinical in vivo assessment was performed to gain a better understanding of CYP3-mediated drug-drug interactions between antiretrovirals and docetaxel, as well as to assess any alterations in gene expression with these combinations.Docetaxel (20 mg/kg i.v.) was administered to male FVB mice in the presence and absence of dexamethasone (10 mg/kg p.o. ×4d), efavirenz (25 mg/kg p.o. ×4d), ketoconazole (50 mg/kg p.o.), or ritonavir (12.5 mg/kg p.o.). At various time points, plasma and liver tissue were harvested. Docetaxel concentrations were determined by LC/MS/MS. Pharmacokinetic parameters were calculated. Liver tissue RNA was used to evaluate alterations in Cyp3a11 and Abcb1a gene expression.Docetaxel exposure was altered by CYP3A4 inhibitors but not by inducers. The CYP3A4 inducers efavirenz and dexamethasone did not have a significant effect on docetaxel exposure (AUC). However, the CYP3A4 inhibitors ritonavir and ketoconazole resulted in a 6.9- and 3.1-fold increase in AUC, respectively. Alterations in gene expression did not account for the altered docetaxel exposure.Docetaxel exposure was significantly altered by CYP3A4 inhibitors. Until a definitive clinical trial is performed, docetaxel should be used with caution in patients on a ritonavir-containing antiretroviral regimen or an alternative antineoplastic therapy or antiretroviral regimen should be considered.

Supplemental Digital Content is available in the text., Objectives Response to the oncology drug gemcitabine may be variable in part due to genetic differences in the enzymes and transporters responsible for its metabolism and disposition. The aim of our in-silico study was to identify gene variants significantly associated with gemcitabine response that may help to personalize treatment in the clinic. Methods We analyzed two independent data sets: (a) genotype data from NCI-60 cell lines using the Affymetrix DMET 1.0 platform combined with gemcitabine cytotoxicity data in those cell lines, and (b) genome-wide association studies (GWAS) data from 351 pancreatic cancer patients treated on an NCI-sponsored phase III clinical trial. We also performed a subset analysis on the GWAS data set for 135 patients who were given gemcitabine+placebo. Statistical and systems biology analyses were performed on each individual data set to identify biomarkers significantly associated with gemcitabine response. Results Genetic variants in the ABC transporters (ABCC1, ABCC4) and the CYP4 family members CYP4F8 and CYP4F12, CHST3, and PPARD were found to be significant in both the NCI-60 and GWAS data sets. We report significant association between drug response and variants within members of the chondroitin sulfotransferase family (CHST) whose role in gemcitabine response is yet to be delineated. Conclusion Biomarkers identified in this integrative analysis may contribute insights into gemcitabine response variability. As genotype data become more readily available, similar studies can be conducted to gain insights into drug response mechanisms and to facilitate clinical trial design and regulatory reviews.

ABCG2 is a transporter with potential importance in cancer drug resistance, drug oral absorption, and stem cell biology. In an effort to identify novel inhibitors of ABCG2, we examined the ability of commercially available bisindolylmaleimides (BIM) and indolocarbazole protein kinase inhibitors (PKI) to inhibit ABCG2, given the previous demonstration that the indolocarbazole PKI UCN-01 interacted with the transporter. At a concentration of 10 μmol/L, all of the compounds tested increased intracellular fluorescence of the ABCG2-specific substrate pheophorbide a in ABCG2-transfected HEK-293 cells by 1.3- to 6-fold as measured by flow cytometry; the ABCG2-specific inhibitor fumitremorgin C increased intracellular fluorescence by 6.6-fold. In 4-day cytotoxicity assays, wild-type ABCG2-transfected cells were not more than 2-fold resistant to any of the compounds, suggesting that the PKIs are not significantly transported by ABCG2. BIMs I, II, III, IV, and V, K252c, and arcyriaflavin A were also able to inhibit [125I]iodoarylazidoprazosin labeling of ABCG2 by 65% to 80% at 20 μmol/L, compared with a 50% to 70% reduction by 20 μmol/L fumitremorgin C. K252c and arcyriaflavin A were the most potent compounds, with IC50 values for inhibition of [125I]iodoarylazidoprazosin labeling of 0.37 and 0.23 μmol/L, respectively. K252c and arcyriaflavin A did not have any effect on the ATPase activity of ABCG2. Four minimally toxic compounds—BIM IV, BIM V, arcyriaflavin A, and K252c—reduced the relative resistance of ABCG2-transfected cells to SN-38 in cytotoxicity assays. We find that indolocarbazole and BIM PKIs directly interact with the ABCG2 protein and may thus increase oral bioavailability of ABCG2 substrates. [Mol Cancer Ther 2007;6(6):1877–85]

2616 Background: Acquired resistance to anti-EGFR MoAb therapy may be via EGFR-ErbB2 heterodimerization and pathway reactivation. Dual anti-EGFR treatment was recently found to be active in colon cancer. We performed a phase I trial of CET and LAP to determine the DLTs, MTD, and clinical activity of the combination. Methods: Pts received CET at 400mg/m2 then 250 mg/m2 weekly, combined with daily LAP in a 3+3 dose escalation trial. LAP dose levels (DL) were (1) 750mg, (2) 1000mg, and (3) 1250mg. Cycles lasted 3 weeks, toxicity was assessed through C2, and pts were restaged every 2 cycles. Baseline and post-C1 tumor biopsies were analyzed for phosphoprotein activation in 36 EGFR/ErbB2 pathway proteins. Germ-line pharmacogenetic (PGx) variations were correlated with efficacy and toxicity. Results: Between 10/2010 and 10/2012, 22 pts were enrolled - colon (8), lung (8), head and neck (4), and anal cancers (2) - and 59% had prior anti-EGFR therapy. 18 pts were evaluable for toxicity, and 18 for response. Mean treatment was 3.8 cycles (range 1 - 12); 3 patients are still on trial. One DLT occurred at DL1 (gr 3 rash) and DL2 (gr 3 diarrhea). No pt on DL3 experienced a DLT. No pt experienced a gr 4 toxicity; gr 3 toxicities anytime on therapy included rash (17%), diarrhea (6%), fatigue (6%), lymphopenia (6%), and hypomagnesemia (6%). Rash was experienced by 94% of pts (gr 1=50%, gr 2=28%, and gr 3=17%). Partial responses (PR) occurred in 4 pts (22%), stable disease (SD) in 8 (44%), and disease progression in 6 (33%), for a clinical benefit rate of 67%. Seven of 13 (54%) pts on prior EGFR therapy had SD or PR. Down-regulation of phosphorylated EGFR/ErbB2 pathway components correlated with response; distinct pathway components were up-regulated in non-responders, including PI3K, Jak/Stat, MAPK, and IGF. PGx variants in FcRII correlated with response (p=0.045); no variants correlated with toxicity. Conclusions: The RP2D is CET 250 mg/m2 weekly and LAP 1250mg daily. Treatment was well tolerated with few grade 3 toxicities and a significant 67% clinical benefit rate. Non-responders showed up-regulation of EGFR pathway components – components which are druggable, warranting further study. A trial in colorectal cancer is ongoing. Clinical trial information: NCT01184482.

TPS2624 Background: Therapeutic blockade of Epidermal Growth Factor Receptor (EGFR) signaling with the monoclonal antibody cetuximab is clinically effective in the treatment of patients with metastatic squamous cell carcinoma of the head and neck or KRAS wildtype colorectal cancer. However, these patients eventually become resistant to this therapy. An exploration of the EGFR signaling network using an EGFR network-focused small interfering RNA library identified potential regulators of resistance to EGFR-targeted therapies. The ABL1 gene was identified as a central node to target in this complex genomic pathway. In a preclinical EGFR-expressing cancer cell line model, targeting c-abl, the gene product of ABL1, using nilotinib was found to be highly synergistic in decreasing cell survival when combined with anti-EGFR targeted therapy. Methods: We have initiated an open-label Phase I study for patients who progressed after standard therapies for metastatic KRAS wildtype colorectal cancer or metastatic head and neck squamous cell carcinoma. Enrolled patients must have adequate performance status and organ function. Treatment consists of cetuximab 400 mg/m2 on day 1, then 250 mg/m2 once weekly, and nilotinib twice daily, starting on day 1, according to a traditional 3+3 dose escalation, from 200mg to 300mg BID. Patients are restaged every 2 cycles (every 8 weeks). The primary endpoint is the maximum tolerated dose (MTD) of nilotinib when used in conjunction with cetuximab. Secondary endpoints are clinical benefit rate (defined as rates of stable disease, partial response, and complete response) and response rate. Additionally, biopsies of metastases obtained prior to and after initiation of therapy will be used to establish primary tumor cell cultures using conditional cellular reprogramming to permit the dynamic study of signaling and drug sensitivity through an evaluation of evidence of a drug effect on EGFR signaling and on Antibody-Dependent Cell-Mediated Cytotoxicity. An additional 10 colorectal cancer patients will be treated as an expansion cohort at the MTD. This expansion cohort data may be used to plan a Phase II trial in the future.

2590 Background: Preclinical research has shown that one mechanism of acquired resistance to CTX is via EGFR-ErbB2 heterodimerization, which can reactivate oncogenic pathways. LPT is a dual EGFR and ErbB2 intracellular tyrosine kinase inhibitor. A phase I translational clinical study was performed to determine the maximum tolerated dose (MTD), dose limiting toxicities (DLTs), and clinical activity of CTX and LPT in patients with EGFR-driven solid tumor malignancies that can be treated with CTX. Methods: Patients (Pts) were enrolled in a 3+3 dose escalation trial. Prior CTX therapy was allowed. CTX was given at 400mg/m2 on Cycle 1, Day 1 (C1D1), then 250 mg/m2 weekly. LPT dose levels (DL) were (1) 750mg, (2) 1000mg, and (3) 1250mg orally daily. Rash management included daily sunblock, steroid cream, and doxycycline. Cycle length was 21 days, and patients were assessed for toxicity through the end of C2, and for efficacy after every 2 cycles. Fresh tumor biopsies were obtained at baseline and at the end of C1 to compare EGFR and ErbB2 expression levels and EGFR related pathway activation. DNA from blood samples was analyzed for pharmacogenetic (PGx) variations and correlations with toxicity and pharmacokinetics (PK). Results: Between October, 2010 to January 2012, 13 pts were enrolled, with colon (4), lung (3), head and neck (3), and anal cancers (3); 10 were evaluable for toxicity. Treatment-related toxicities of any grade included: rash (67%), diarrhea (42%), fatigue (33%), nausea/vomiting (17%), and dehydration (8%). DLTs included G3 rash in 1 of 6 pts on DL1, and G3 diarrhea despite optimal therapy in 1 of 4 pts on DL2. Enrollment to DL2 continues. Of 7 pts evaluable for response, 1 had an uPR, 3 had SD, including 1 with SD of ≥4 cycles, and 3 had DP. Both patients with uPR and prolonged SD were treated on DL1. Tumor EGFR-ErbB2 and EGFR pathway phosphorylation analyses and PGx results will be presented. Conclusions: The combination of CTX and LPT is well tolerated with expected toxicities. Efficacy was seen even on DL 1. Phase II clinical studies in CTX-sensitive diseases such as colon and head and neck cancer are planned.

5586 Background: Pharmacogenetic (PGx) research attempts to explain variations in the efficacy and toxicity of chemotherapy based on germline and tumor genetic variations. Standard treatment for lo...

e16023 Background: Patients with LAH&NSCC caused by the human papillomavirus (HPV) have higher survival rates when treated with induction chemotherapy or concurrent cisplatin chemoradiotherapy. No ...

5557 Background: Salivary gland tumors are an uncommon and morphologically diverse group of neoplasms that are highly resistant to cytotoxic chemotherapy. Response rates to multi-agent combinations...

Background: The cancer stem cell (CSC) hypothesis holds that CSCs generate tumor daughter cells via asymmetric cell division, evade immune surveillance, and are resistant to anticancer treatment, and thus remain viable to regenerate a population of malignant clones after therapy. While CSCs’ inherent resistance to chemotherapy is a central tenet of the cancer stem cell hypothesis, little research has focused on this critical question. Methods: Identification and separation of putative head and neck cancer squamous cell carcinoma (H&N SCC) CSCs was performed via flow cytometry of the SCC9 cell line using both Hoechst 33342 dye (‘side population’) and antibodies against CD44, CD2, CD3, CD10, CD16, CD18, CD31, CD64, and CD140b (‘CD44+/lineage-‘). Expression of genes characterizing a stem cell phenotype as well as drug transporters known to mediate chemotherapy drug resistance was measured via RT-PCR and compared between subpopulations. The SCC9 cell line was incubated with various chemotherapy agents in attempt to enrich the CSC subpopulation, including cisplatin, paclitaxel, ixabepilone, depsipeptide, methotrexate, SN38, and topotecan, followed by flow cytometric sorting. The resultant CSC subpopulation was compared between drug treated and untreated controls to determine whether the previous drug exposure enriched the CSC subpopulation. Results: Use of Hoechst dye did not identify a ‘side population,’ likely due to low expression of the ABCG2 transporter in H&N CSCs. Using CD44+/lineage- markers, a CSC subpopulation representing 8.5% on average of total cells was identified. Variant expression of stem cell genes in this subpopulation included ACTC1, BMP3, CCND2, COL9A1, DLL3, FGF3, GDF2, GDF3, PDX1, KRT15, SOX1, TERT, and WNT1. Significant expression of drug transporters in the CSC subpopulation included ABCA3, SLC16A2, SLC16A3, SLC22A1, SLC28A2, SLC2A1, SLC25A13, and SLCO1B3. ABCB1 expression was increased in the CSC subpopulation but still showed relatively low RNA expression. There was no difference in expression of ABCC1 and ABCG2. Incubation with cytotoxic agents at various concentrations (IC25 to IC75) for 24 to 96 hours showed no clear pattern of enriching or depletion of the CSC subpopulation. Conclusion: CD44+/lineage- can identify a putative CSC subpopulation in H&NSCC which preferentially expresses stem cell pathway genes as well as drug transporters. This increased transporter expression does not, however, induce a drug resistance phenotype in this in vitro cell line model. Other in vitro and/or in vivo methodologies should be explored to better analyze and characterize treatment resistance of CSCs in order to further test the validity of the cancer stem cell hypothesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3320.