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Significance In breast cancer, the chronically stiffening mechanical microenvironment promotes tumor growth/invasion. However, the molecular details and integration of cancer mechanotransduction signaling pathways are not well understood. We find that nontumorigenic breast epithelial cells are mechanically sensitive and respond to acute mechanical stimuli with the generation of ROS-stimulated calcium signaling in a microtubule-dependent manner. These results establish that epithelial cells conserve a mechanotransduction pathway from muscle and bone (X-ROS). Using common breast cancer genetic mutations, we further report that oncogene activation reduces X-ROS pathway mechanoresponsiveness at the level of ROS sensitivity. This definition of how oncogene activation disrupts highly conserved mechanotransduction signaling mechanisms could improve the understanding of altered tumor cell responses to the mechanical microenvironment and reveal new therapeutic opportunities., Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

Multiple anal human papillomavirus (HPVs) may increase the risk of anal cancer among men who have sex with men (MSM) living with human immunodeficiency virus (HIV). The Jaccard Similarity Index (JSI) was explored as a measure of multiple HPV persistence.The TRUST/RV368 cohort enrolled MSM living with and without HIV in Abuja and Lagos, Nigeria. Participants with anal swabs at baseline, 3- and 12-month visits were tested for high- and low-risk HPVs using a next-generation sequencing assay. Persistence of the same HPV genotypes over time was calculated using the JSI and categorized into high, medium, and low similarity tertiles. Factors associated with higher versus lower similarity were estimated with multivariable ordinal logistic regression and reported as adjusted odds ratios (aORs) and 95% confidence intervals (CIs).Of the 225 participants, median age was 25 years (interquartile range, 22-29 years), 62% were living with HIV, median HPVs was 3 (interquartile range, 2-5), and HPV6 (28%), HPV16 (26%), HPV11 (23%), and HPV45 (20%) were most prevalent. Fifty-three percent of participants had highly similar HPVs at 3 months, and the similarity was associated with HIV (aOR, 3.11; 95% CI, 1.6-5.9) and recent receptive sex (aOR, 1.9; 95% CI, 1.0-3.5). By 12 months, 20% had highly similar HPVs, and it was associated with 12 years or longer since anal coital debut (aOR, 6.8; 95% CI, 3.1-5.2), self-reported genital warts (aOR, 3.1; 95% CI, 1.5-6.6), and 200 or less CD4 cells/mm3 (aOR, 13.3; 95% CI, 2.7-65.2) for those living with HIV.Studies evaluating the JSI as a predictor of high-grade intraepithelial lesions would further confirm its applicability as a quantitative measure of multiple HPV persistence.

The technical challenges of imaging non-adherent tumor cells pose a critical barrier to understanding tumor cell responses to the non-adherent microenvironments of metastasis, like the bloodstream or lymphatics. In this study, we optimized a microfluidic device (TetherChip) engineered to prevent cell adhesion with an optically-clear, thermal-crosslinked polyelectrolyte multilayer nanosurface and a terminal lipid layer that simultaneously tethers the cell membrane for improved spatial immobilization. Thermal imidization of the TetherChip nanosurface on commercially-available microfluidic slides allows up to 98% of tumor cell capture by the lipid tethers. Importantly, time-lapse microscopy demonstrates that unique microtentacles on non-adherent tumor cells are rapidly destroyed during chemical fixation, but tethering microtentacles to the TetherChip surface efficiently preserves microtentacle structure post-fixation and post-blood isolation. TetherChips remain stable for more than 6 months, enabling shipment to distant sites. The broad retention capability of TetherChips allows comparison of multiple tumor cell types, revealing for the first time that carcinomas beyond breast cancer form microtentacles in suspension. Direct integration of TetherChips into the Vortex VTX-1 CTC isolation instrument shows that live CTCs from blood samples are efficiently captured on TetherChips for rapid fixation and same-day immunofluorescence analysis. Highly efficient and unbiased label-free capture of CTCs on a surface that allows rapid chemical fixation also establishes a streamlined clinical workflow to stabilize patient tumor cell samples and minimize analytical variables. While current studies focus primarily on CTC enumeration, this microfluidic device provides a novel platform for functional phenotype testing in CTCs with the ultimate goal of identifying anti-metastatic, patient-specific therapies.

Background: Clinical breast cancer imaging inevitably focuses on tumor growth rather than the metastatic dissemination that is a greater challenge for patient survival. Emerging preclinical evidence indicates that chemotherapy can elevate levels of circulating tumor cells (CTCs) and increase metastatic recurrence. Targeting metastatic phenotypes of CTCs could reveal therapeutic strategies to reduce metastasis that would be overlooked by measurements of tumor growth.Methods: We investigated how the FDA-approved microtubule-depolymerizing Vinca alkaloid, Vinorelbine, affects three metastatic phenotypes of reattachment, clustering and microtentacles (McTNs). Microfluidic cell tethering technology (TetherChip) was used to measure Vinorelbine effects on McTNs and clustering while xCelligence impedance was used for reattachment assays. Bioluminescence imaging monitored tumor growth and metastasis in mice. ANGLE Parsortix and Vortex Biosciences VTX-1 were used to capture live tumor cells from blood samples for confocal microscopy to rapidly measure tumor cell responses to Vinorelbine.Results: We demonstrate that a focused (1h) Vinorelbine treatment is sufficient to inhibit reattachment, clustering and McTNs in non-adherent breast tumor cells. Quantitative analysis of treated non-adherent cells reveals that McTNs are significantly lower in number (p= 0.012) and shorter in length (p= 0.000034). Treating mice with Vinorelbine (5mg/kg) for only 24h did not significantly affect primary tumor survival. However, median metastatic tumor survival after injection of circulating tumor cells extended from 8 weeks to 30 weeks after a focused 24h treatment with Vinorelbine. Microtentacle inhibition by Vinorelbine was also detectable within 1h, using live tumor cells isolated from blood samples and analyzed with confocal microscopy for McTNs on TetherChip microfluidic surfaces. As few as 11 tumor cells were sufficient to yield 90% power to detect this 1h Vinorelbine drug response, demonstrating feasibility with the small number of tumor cells available from patient biopsies.Conclusions: This study establishes a proof-of-concept that targeted microtubule disruption can selectively inhibit metastasis and reveals that existing FDA-approved therapies could have anti-metastatic actions that are currently overlooked when focusing exclusively on tumor growth. Moreover, the anti-metastatic actions of Vinorelbine could be detected in less than one hour using microfluidic TetherChip technology, establishing a new approach for precision medicine aimed at reducing metastasis.

Long noncoding RNA differentiation antagonizing nonprotein coding RNA (lncRNA-DANCR) is associated with poor prognosis in multiple cancers, and promotes cancer stemness and invasion. However, the exact mechanisms by which DANCR promotes non-small cell lung cancer (NSCLC) remain elusive. In this study, we determined that DANCR knockdown (KD) impeded cell migration and reduced stem-like characteristics in two NSCLC cell lines, A549 and H1755. Wnt signaling was shown to promote NSCLC proliferation, stemness, and invasion, therefore, we hypothesized that DANCR may regulate these activities through induction of the Wnt/&beta, catenin pathway. DANCR KD reduced &beta, catenin signaling and protein expression, and decreased the expression of &beta, catenin gene targets c-Myc and Axin2. One of the well-defined functions of lncRNAs is their ability to bind and inhibit microRNAs. Through in silico analysis, we identified tumor suppressor miR-216a as a potential binding partner to DANCR, and confirmed this binding through coimmunoprecipitation and luciferase-reporter assays. Furthermore, we show that DANCR-induced &beta, catenin protein expression may be blocked with miR-216a overexpression. Our findings illustrate a role of DANCR in NSCLC migration and stemness, and suggest a novel DANCR/miR-216a signaling axis in the Wnt/&beta, catenin pathway.

Aggressive cellular phenotypes such as uncontrolled proliferation and increased migration capacity engender cellular transformation, malignancy and metastasis. While genetic mutations are undisputed drivers of cancer initiation and progression, it is increasingly accepted that external factors are also playing a major role. Two recently studied modulators of breast cancer are changes in the cellular mechanical microenvironment and alterations in calcium homeostasis. While many studies investigate these factors separately in breast cancer cells, very few do so in combination. This current work sets a foundation to explore mechano-calcium relationships driving malignant progression in breast cancer. Utilizing real-time imaging of an in vitro scratch assay, we were able to resolve mechanically-sensitive calcium signaling in human breast cancer cells. We observed rapid initiation of intracellular calcium elevations within seconds in cells at the immediate wound edge, followed by a time-dependent increase in calcium in cells at distances up to 500μm from the scratch wound. Calcium signaling to neighboring cells away from the wound edge returned to baseline within seconds. Calcium elevations at the wound edge however, persisted for up to 50 minutes. Rigorous quantification showed that extracellular calcium was necessary for persistent calcium elevation at the wound edge, but intercellular signal propagation was dependent on internal calcium stores. In addition, intercellular signaling required extracellular ATP and activation of P2Y2 receptors. Through comparison of scratch-induced signaling from multiple cell lines, we report drastic reductions in response from aggressively tumorigenic and metastatic cells. The real-time scratch assay established here provides quantitative data on the molecular mechanisms that support rapid scratch-induced calcium signaling in breast cancer cells. These mechanisms now provide a clear framework for investigating which short-term calcium signals promote long-term changes in cancer cell biology.

Background Our next generation sequencing (NGS)-based human papillomavirus (HPV) genotyping assay showed a high degree of concordance with the Roche Linear Array (LA) with as little as 1.25 ng formalin-fixed paraffin-embedded-derived genomic DNA in head and neck and cervical cancer samples. This sensitive genotyping assay uses barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+ applicable to population studies, but it’s diagnostic performance has not been tested in cases with multiple concurrent HPV infections. Methods We conducted a cross-sectional study to compare the positive and negative predictive value (PPV and NPV), sensitivity and specificity of the NGS assay to detect HPV genotype infections as compared to the LA. DNA was previously extracted from ten anal swab samples from men who have sex with men in Nigeria enrolled on the TRUST/RV368 cohort study. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs. low-risk HPV type-specific infections. Results In total there were 94 type-specific infections detected in 10 samples with a median of 9.5, range (9 to 10) per sample. Using the LA as the gold standard, 84.4% (95% CI: 75.2–91.2) of the same anal type-specific infections detected on the NGS assay had been detected by LA. The PPV and sensitivity differed significantly for high risk (PPV: 90%, 95% CI: 79.5–96.2; sensitivity: 93.1%, 95% CI: 83.3–98.1) as compared to low risk HPV (PPV: 73%, 95% CI: 54.1–87.7; sensitivity: 61.1, 95% CI: 43.5–76.9) (all p 0.05). The NGS assay detected 10 HPV genotypes that were not among the 37 genotypes found on LA (30, 32, 43, 44, 74, 86, 87, 90, 91, 114). Conclusions The NGS assay accurately detects multiple HPV infections in individual clinical specimens with limited sample volume and has extended coverage compared to LA. Electronic supplementary material The online version of this article (doi:10.1186/s12985-017-0771-z) contains supplementary material, which is available to authorized users.

PURPOSE Noninvasive therapies, such as probiotics that promote local immunity and reduce persistence of high-risk human papillomavirus (HR-HPV), would circumvent challenges in implementing anal cancer screening where same-sex behavior is stigmatized and criminalized. We describe the persistence of HR-HPV and its relationship with the anal microbiota among Nigerian men who have sex with men (MSM). METHODS Anal swabs from HIV-uninfected and -infected MSM who were enrolled in the TRUST/RV368 cohort were HPV genotyped at enrollment and at 3 and 12 months. Participants who presented with the same type(s) of HR-HPV across all visits were categorized as having persistent infections. The anal microbiota composition at baseline of HR-HPV–positive individuals was evaluated. Unsupervised K-means clustering of the 15 most abundant bacterial taxa identified 2 microbial clusters (MC-1 and MC-2). Unadjusted associations of 12-month HR-HPV persistence was evaluated by MC membership and HIV status using χ2 tests and logistic regression. RESULTS One hundred nine HIV-infected and 41 HIV-uninfected participants—contributing 284 baseline HR-HPV infections—were observed for a median of 12 months (interquartile range, 11.5-12.6 months). MC-1 was dominated by Prevotella and MC-2 consisted of a diverse set of anaerobic bacteria ( Finegoldia, Corynebacterium, Peptoniphilus, and Anerococcus). At 12 months, 45% (67 of 150) of MSM had 86 persistent HR-HPV and 55% (83 of 150) cleared all baseline HR-HPV. HPV16 (21%), HPV45 (13%), and HPV51 (12%) were the most persistent. Persistence of any HR-HPV was nonsignificantly higher in MC-1 (odds ratio [OR], 1.6; 95% CI, 0.8 to 3.1). Persistent HPV16 was more abundant in MC-2 versus MC-1 (OR, 3.9; 95% CI, 1.2 to 12.0), whereas HPV45 and HPV51 were similarly distributed across both MCs. Compared with HIV-uninfected, persistence (OR, 1.6; 95% CI, 0.8 to 3.3) and MC-2 membership (OR, 1.3; 95% CI, 0.8 to 2.0) were nonsignificantly higher among HIV-infected participants. CONCLUSION Anal HPV16 has the highest annual persistence and is associated with a low Prevotella anal microbiota, a potentially modifiable cofactor.

Background Anal precancers and cancers can be detected during screening with high-resolution anoscopy (HRA). The sensitivity of HRA depends on the burden and duration of human papillomavirus (HPV) among those screened as well as anoscopist proficiency, which is highly correlated with prior screening experience. Our objective was to compare the identification and type of HPV and the likelihood of HRA-detected precancer for men who have sex with men (MSM) undergoing their first HRA-screening in Nigeria. Methods MSM were recruited from an HIV test-and-treat cohort, TRUST/RV368, into a new anal cancer screening program. Anal swabs obtained during screening underwent Ion Torrent next-generation sequencing using barcoded HPV PCR broad-spectrum primers 5+/6+ to detect up to 161 HPVs. All high-risk (HR) HPVs and the most abundant low-risk (LR)-HPVs were evaluated as type-specific infections with some categorized as belonging to a multiple infection. HRA screening results included benign, low-grade squamous intraepithelial lesions (LSIL), or HSIL as detected by cytology or histology. Multivariable logistic regression was used to assess the association of HPV and other cofactors with any SIL. Results Among 342 MSM, 60% were HIV-infected, 89% were under 35 years of age, and 51% had 8 or more years since anal coital debut. Of those with SIL, 89% had LSIL and only 11% had HSIL. Prevalence of any HPV and high-risk (HR)-HPV was 92% and 74%, respectively. The most prevalent genotypes in rank order were HPV6 (31%), HPV16 (23%), HPV42 (20%), HPV11 (18%), HPV45 (18%), and HPV51 (17%). For multiple HR-HPVs, 31% had a single HR-HPV, 32% had 2-3, and 10% had 4 or more. Low-risk HPVs, type 6 and/or 11, were common (42%) and were significantly associated with SIL (adjusted odds ratio [aOR]:1.8, 95% confidence interval [CI]: 1.1–3.1) together with perianal warts (aOR:6.7, 95% CI: 3.3–13.5). In contrast, HR-HPV and multiple HR-HPVs were not significantly associated with SIL (all p > 0.05). Conclusions Detection of HSIL was low. Although HR-HPV was abundant, HSIL development also depends on the duration of HR-HPV infections and the anoscopist's level of experience. As our cohort ages and the anoscopist becomes more skilled, detection of HSIL will likely improve.

Ovarian carcinomas are categorized into five major histotypes, each characterized by distinct differences in grade at diagnosis, presentation with metastatic disease, and clinical responses to treatments. Microtentacles (McTNs), microtubule-based extensions of the plasma membrane, were initially described on cells of nongynecologic primaries with high metastatic potential but have not been extensively explored in ovarian carcinomas. In this study, we investigated whether ovarian cancer cells exhibit McTNs as well as the effect of microtubule-targeted drugs on ovarian cancer McTNs. We analyzed 3 immortalized ovarian surface epithelium cell lines (IOSE), 8 clear-cell (OCCC), 3 low-grade serous (LGS), and 6 high-grade serous (HGS) ovarian cancer cells for McTN phenotype, length, and number, using a novel tethering platform in which cells are suspended but stationary, allowing for analysis via confocal microscopy. Additionally, selected cell lines were also treated with microtubule-targeting agents, including paclitaxel, ixabepilone, vinblastine, and colchicine and the effects on McTN dynamics and functions were analyzed. Tubulin subtype expression and post-translational modifications (PTM) were characterized by Western blot. Unpaired t-tests were used to describe differences between McTN length and number. We observed 4 patterns of McTN expression: absent (A), symmetric-short (SS), symmetric-long (SL), and tufted (T). In some cases, multiple morphologies were observed within the same cell line. LGS and OCCC expressed fewer McTNs per cell than HSC. McTNs were shorter among OCCC compared to HGS; whereas LGS expressed a range of McTN lengths similar to those observed in HGS. We also observed differences in the expression of the PTM between the different ovarian cancer subtypes, including alterations in alpha tubulin-stabilizing modifications such as glu-tubulin and acetylated tubulin, as well as proteins involved in actin cortex stability. Increased acetylated tubulin was associated with increased McTN number and length in HGS cells. Microtubule-destabilizing drugs such as colchicine and vinblastine led to a decrease in McTN formation. Ovarian cancer metastasis typically occurs through shedding of the main tumor into the peritoneal space, and the spread of disease is the leading cause of mortality. Studying McTN function and response to chemotherapeutics allows us to improve our understanding of ovarian cancer metastasis and the effect of microtubule-targeting compounds on the spread of ovarian cancer. Citation Format: Cong (Ava) Fan, Sulan Wu, Jocelyn Reader, Eleanor Claire-Higgins Ory, Cornell Lee, McMillan Ching, Trevor Mathias, Julia Ju, Rachel Lee, Michele Vitolo, Stuart Martin, Christopher Jewell, Amy Fulton, Gautam Rao, Mark Carey, Dana M. Roque. Characterization of microtentacle phenotype and function in ovarian carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B68.

The metastatic cascade consists of multiple complex steps, but the belief that it is a linear process is diminishing. In order to metastasize, cells must enter the blood vessels or body cavities (depending on the cancer type) via active or passive mechanisms. Once in the bloodstream and/or lymphatics, these cancer cells are now termed circulating tumor cells (CTCs). CTC numbers as well as CTC clusters have been used as a prognostic marker with higher numbers of CTCs and/or CTC clusters correlating with an unfavorable prognosis. However, we have very limited knowledge about CTC biology, including which of these cells are ultimately responsible for overt metastatic growth, but due to the fact that higher numbers of CTCs correlate with a worse prognosis; it would seem appropriate to either limit CTCs and/or their dissemination. Here, we will discuss the different cancer treatments which may inadvertently promote the mobilization of CTCs and potential CTC therapies to decrease metastasis.

Purpose: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition. Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo. Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor–induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML. Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234–47. ©2017 AACR.

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5(+)/6(+) (BSGP)5(+)/6(+). To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.

The oral second-generation bis-aryl urea fms-like tyrosine kinase 3 (FLT3) inhibitor quizartinib (AC220) has favorable kinase selectivity and pharmacokinetics. It inhibits mutant and wild-type FLT3 in vivo at 0.1 and 0.5 µM, respectively, and has shown favorable activity and tolerability in phase I and II trials in acute myeloid leukemia, with QT prolongation as the dose-limiting toxicity. Co-administration with chemotherapy is planned. We characterized interactions of quizartinib with the ATP-binding cassette (ABC) proteins ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein). Its effects on uptake of fluorescent substrates and apoptosis were measured by flow cytometry, binding to ABCB1 and ABCG2 drug-binding sites by effects on [125I]iodoarylazidoprazosin ([125I]-IAAP) photolabeling and ATPase activity, and cell viability by the WST-1 colorimetric assay. Quizartinib inhibited transport of fluorescent ABCG2 and ABCB1 substrates in ABCG2- and ABCB1-overexpressing cells in a concentration-dependent manner, from 0.1 to 5 µM and from 0.5 to 10 µM, respectively, and inhibited [125I]-IAAP photolabeling of ABCG2 and ABCB1 with IC50 values of 0.07 and 3.3 µM, respectively. Quizartinib at higher concentrations decreased ABCG2, but not ABCB1, ATPase activity. Co-incubation with quizartinib at 0.1 to 1 µM sensitized ABCG2-overexpressing K562/ABCG2 and 8226/MR20 cells to ABCG2 substrate chemotherapy drugs in a concentration-dependent manner in cell viability and apoptosis assays. Additionally, quizartinib increased cellular uptake of the ABCG2 substrate fluoroquinolone antibiotic ciprofloxacin, which also prolongs the QT interval, in a concentration-dependent manner, predicting altered ciprofloxacin pharmacokinetics and pharmacodynamics when co-administered with quizartinib. Thus quizartinib inhibits ABCG2 at pharmacologically relevant concentrations, with implications for both chemosensitization and adverse drug interactions. These interactions should be considered in the design of treatment regimens combining quizartinib and chemotherapy drugs and in choice of concomitant medications to be administered with quizartinib.