Matthew Sung

Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer

Prostate cancer growth depends on androgen receptor signaling. Androgen ablation therapy induces expression of constitutively active androgen receptor splice variants that drive disease progression. Taxanes are a standard of care therapy in castration-resistant prostate cancer (CRPC); however, mechanisms underlying the clinical activity of taxanes are poorly understood. Recent work suggests that the microtubule network of prostate cells is critical for androgen receptor nuclear translocation and activity. In this study, we used a set of androgen receptor deletion mutants to identify the microtubule-binding domain of the androgen receptor, which encompasses the DNA binding domain plus hinge region. We report that two clinically relevant androgen receptor splice variants, ARv567 and ARv7, differentially associate with microtubules and dynein motor protein, thereby resulting in differential taxane sensitivity in vitro and in vivo. ARv7, which lacks the hinge region, did not co-sediment with microtubules or coprecipitate with dynein motor protein, unlike ARv567. Mechanistic investigations revealed that the nuclear accumulation and transcriptional activity of ARv7 was unaffected by taxane treatment. In contrast, the microtubule-interacting splice variant ARv567 was sensitive to taxane-induced microtubule stabilization. In ARv567-expressing LuCap86.2 tumor xenografts, docetaxel treatment was highly efficacious, whereas ARv7-expressing LuCap23.1 tumor xenografts displayed docetaxel resistance. Our results suggest that androgen receptor variants that accumulate in CRPC cells utilize distinct pathways of nuclear import that affect the antitumor efficacy of taxanes, suggesting a mechanistic rationale to customize treatments for patients with CRPC, which might improve outcomes.

ERG induces taxane resistance in castration-resistant prostate cancer

Taxanes are the only chemotherapies used to treat patients with metastatic castration-resistant prostate cancer (CRPC). Despite the initial efficacy of taxanes in treating CRPC, all patients ultimately fail due to the development of drug resistance. In this study, we show that ERG overexpression in in vitro and in vivo models of CRPC is associated with decreased sensitivity to taxanes. ERG affects several parameters of microtubule dynamics and inhibits effective drug-target engagement of docetaxel or cabazitaxel with tubulin. Finally, analysis of a cohort of 34 men with metastatic CRPC treated with docetaxel chemotherapy reveals that ERG-overexpressing prostate cancers have twice the chance of docetaxel resistance than ERG-negative cancers. Our data suggest that ERG plays a role beyond regulating gene expression and functions outside the nucleus to cooperate with tubulin towards taxane insensitivity. Determining ERG rearrangement status may aid in patient selection for docetaxel or cabazitaxel therapy and/or influence co-targeting approaches.

NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

Significance Nicotinamide adenine dinucleotide (NAD+) is an endogenous small molecule that has effects on diverse processes, including obesity, lifespan, and cancer. A major goal is to identify the NAD+-regulated cellular pathways that may mediate these effects. In this study, we demonstrate that NAD+ regulates the microtubule cytoskeleton. We find that these effects are mediated by the mitochondrial sirtuin-3. Our findings have implications for many clinically used chemotherapeutics that target microtubules, as we demonstrate that high NAD+ levels can reduce sensitivity to these drugs. These results are also significant because they demonstrate for the first time that NAD+, a molecule regulated by age, diet, and disease state, can influence basic microtubule functions. Nicotinamide adenine dinucleotide (NAD+) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD+-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD+. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD+ levels. We find that these effects of NAD+ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD+ on microtubule polymers. Taken together, these data demonstrate that NAD+ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents.

Abstract 3095: The development of CPI as a novel, next-generation DNA-targeting payload for ADCs

DNA targeting drugs represent one of cornerstones of anti-cancer therapy for both hematologic and solid tumor indications. Low potency anti-DNA compounds (e.g. platins, anthracyclines) are included in many standard-of-care (SOC) regimens, however their modest activity and overall toxicity profiles limit their therapeutic potential. To increase the therapeutic window for DNA-damaging agents, high potency anti-DNA compounds with enhanced anti-tumor activity have been delivered to tumors as payloads of targeting modalities such as antibody-drug conjugates (ADCs). Herein, we describe the development of a novel DNA-damaging compound comprised of a dimeric structure of cyclopropylpyrrolo[e]indolones (CPIs) that was designed to alkylate DNA and generate toxic interstrand crosslinks (ICLs). In response to the CPI-induced formation of ICLs, CPI treatment of cells primarily activates the Fancomia anemia DNA damage response pathway, whereas other successful DNA-damaging ADC payloads such as calicheamicin activate double-strand break response pathways. CPI shows ~860-fold greater potency than calicheamicin in a panel of cell lines derived from a broad spectrum of tumor indications. Importantly, this new CPI payload retains potent activity in calicheamicin- and SOC-resistant tumor models (including overcoming overexpression of drug efflux pumps). When evaluated as payloads on anti-CD33 targeting ADCs, the CPI conjugate showed dramatically improved efficacy over the corresponding calicheamicin conjugates in MDR+ tumor models. As a site-specific conjugate, the CPI ADC shows enhanced in vivo stability and possesses a wider therapeutic window than the corresponding conventional calicheamicin conjugate and other leading DNA-damaging conjugates on the CD33 platform. Citation Format: Jennifer Kahler, Maureen Dougher, Jane Xu, Matthew Doroski, Andreas Maderna, Russell Dushin, Stephane Thibault, Mauricio Leal, Madan Katragadda, Christopher J. O9Donnell, Matthew Sung, Puja Sapra. The development of CPI as a novel, next-generation DNA-targeting payload for ADCs [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 3095. doi:10.1158/1538-7445.AM2017-3095

Gemtuzumab Ozogamicin (GO) Inclusion to Induction Chemotherapy Eliminates Leukemic Initiating Cells and Significantly Improves Survival in Mouse Models of Acute Myeloid Leukemia

Gemtuzumab ozogamicin (GO) is an anti-CD33 antibody-drug conjugate for the treatment of acute myeloid leukemia (AML). Although GO shows a narrow therapeutic window in early clinical studies, recent reports detailing a modified dosing regimen of GO can be safely combined with induction chemotherapy, and the combination provides significant survival benefits in AML patients. Here we tested whether the survival benefits seen with the combination arise from the enhanced reduction of chemoresidual disease and leukemic initiating cells (LICs). Herein, we use cell line and patient-derived xenograft (PDX) AML models to evaluate the combination of GO with daunorubicin and cytarabine (DA) induction chemotherapy on AML blast growth and animal survival. DA chemotherapy and GO as separate treatments reduced AML burden but left significant chemoresidual disease in multiple AML models. The combination of GO and DA chemotherapy eliminated nearly all AML burden and extended overall survival. In two small subsets of AML models, chemoresidual disease following DA chemotherapy displayed hallmark markers of leukemic LICs (CLL1 and CD34). In vivo, the two chemoresistant subpopulations (CLL1+/CD117− and CD34+/CD38+) showed higher ability to self-renewal than their counterpart subpopulations, respectively. CD33 was coexpressed in these functional LIC subpopulations. We demonstrate that the GO and DA induction chemotherapy combination more effectively eliminates LICs in AML PDX models than either single agent alone. These data suggest that the survival benefit seen by the combination of GO and induction chemotherapy, nonclinically and clinically, may be attributed to the enhanced reduction of LICs.

Abstract 923: Analysis of microtubule perturbations and androgen receptor localization in circulating tumor cells from castration resistant prostate cancer patients as predictive biomarkers of clinical response to docetaxel chemotherapy

Although the recent availability of novel treatment for castration resistant prostate cancer (CRPC) has shown improvements in overall survival, CRPC is incurable and lethal disease. It has been recognized that tumor progression despite castrate levels of androgens is still associated with active signaling from the androgen receptor (AR) which affects gene transcription following its nuclear accumulation. Taxanes bind to and stabilize cellular microtubules (MTs) perturbing the fine and intricate organization of the microtubule network thus, impairing MT-based cell processes like cell division and intracellular trafficking. Our recent work showed that AR translocation from the cytoplasm to the nucleus is MT-dependent and that taxane treatment sequesters AR into the cytoplasm inhibiting the receptors transcriptional activity. While this novel mechanism of taxane activity may explain the clinical efficacy of taxanes in CRPC, clinically we still fail to understand the molecular basis of patient response or resistance to taxanes. We hypothesized that engagement of the MT-AR pathway by the taxanes can be used as read-out of effective drug-target engagement in cancer cells and can be used as biomarker predictive of taxane activity in CRPC patients. To assess the predictive role of these biomarkers, we are currently conducting a prospective clinical study in which we isolate circulating tumor cells (CTCs) from the peripheral blood of CRPC patient undergoing docetaxel treatment. We have enrolled 50 of the 80 total patients, from which we isolated CTCs before (at baseline) and during docetaxel treatment (at day 8 of first cycle of docetaxel and upon relapse) using two different approaches: the EpCAM-based immunomagnetic enrichment (CellSearch) and the ficolling technique to isolate unenriched PBMCs inclusive of CTCs. To avoid issues with leucocyte contamination we subject the EpCAM captured cells are to staining for DAPI , PSMA, CD-45, AR and Tubulin and image them by confocal microscopy. CTCs are defined as nucleated, PSMA+, CD-45- cells. The unenriched PBMC population is also subjected to the same multiplex confocal microscopy protocol. Microtubule network morphology and AR subcellular localization in then assessed in CTCs and each biomarker alone and in combination is correlated with clinical response to docetaxel treatment defined by PCWG2 recommendation. So far we have collected and analyzed baseline and cycle 1-Day 8 samples from all 50 patients and relapse samples from 31 patients. Determination of effective drug-target engagement on C1D8 may allow early detection of molecular response to treatment, or lack of molecular response, which will ultimately allow for chemotherapy customization for the individual patient. Citation Format: Shinsuke Tasaki, Matthew Sung, Alexandre Matov, Giuseppe Galletti, Elan Diamond, Neil Bander, Kathy Zhou, Scott Tagawa, David Nanus, Paraskevi Giannakakou. Analysis of microtubule perturbations and androgen receptor localization in circulating tumor cells from castration resistant prostate cancer patients as predictive biomarkers of clinical response to docetaxel chemotherapy. [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 923. doi:10.1158/1538-7445.AM2014-923

Abstract 3492: Using CTCs to interrogate mechanisms of taxane resistance in the prospective TAXYNERGY clinical trial in prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate cancer progression into castration-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR) signaling despite surgical and chemical androgen ablation. The taxanes represent the only class of chemotherapy that improves overall survival in CRPC patients. Despite their success, CRPC patients do progress on taxane treatment rendering taxane-resistant tumors. The molecular mechanisms underlying clinical taxane resistance in CRPC have not been well elucidated due to the lack of available tumor tissue to study. Circulating tumor cells (CTCs) represent a liquid biopsy of the original tumor and isolation of them can lead to their molecular characterization potentially revealing predictive biomarkers for taxane sensitivity or resistance. Here, we use a geometrically enhanced differential immunocapture (GEDI) microfluidic device that couples an anti-prostate specific membrane antigen (PSMA) antibody with optimized 3D geometry to capture and isolate live CTCs from whole blood of CRPC patients. The GEDI-microfluidic device was shown to have a 2-400 fold higher sensitivity for CTC capture than the FDA-approved CellSearch® system. We have previously shown that CRPC patient CTCs can be used to derive functional information that correlates to clinical response to taxane chemotherapy, namely AR subcellular localization status. We have developed a suite of other functional assays that can be performed on live GEDI-captured CTCs that enable their molecular characterization and allow us to test specific mechanistic hypotheses based on our extensive preclinical data. Included, and herein described, are the determination of AR subcellular localization, extent of effective drug-target engagement assessed by microtubule bundling, identification of RNA species relevant to the mechanism of taxane resistance and computer vision algorithms that will allow for enriched and automated analysis of high-volume image sets of GEDI-captured CTCs. In addition, we will be testing the hypothesis that distinct AR splice variants may affect patient sensitivity to taxane-based chemotherapy. This suite of assays are being rigorously applied in a phase II clinical trial in which chemotherapy-naive CRPC patients will be initially treated with either docetaxel or cabazitaxel and clinically evaluated for an early switch to the other taxane following disease progression. This prospective, randomized, multi-site clinical trial will enroll 100 CRPC patients within one year. Patients will be followed until relapse and each patient will have 15 independent GEDI assays performed across five time points from baseline to chemotherapy crossover to relapse. The depth of coverage this suite of assays provides will offer unique insights for potential mechanisms of clinical taxane resistance and predictive biomarkers for taxane sensitivity in CRPC patient CTCs. Citation Format: Matthew S. Sung, Ada Gjyrezi, Guang Yu Lee, Alexandre Matov, Giuseppe Galletti, Matthew Loftus, Yusef Syed, Timothy Lannin, Atanas Hristov, Christopher Mason, Scott Tagawa, Brian Kirby, David Nanus, Paraskevi Giannakakou. Using CTCs to interrogate mechanisms of taxane resistance in the prospective TAXYNERGY clinical trial in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3492. doi:10.1158/1538-7445.AM2013-3492

Abstract 696: BRCA1-mediated ubiquitination of tubulin may serve as a signal for taxol-induced apoptosis

The taxanes are among the most effective chemotherapeutic agents used for the treatment of solid tumors, yet eventually most tumors become resistant to taxane chemotherapy. Mechanisms responsible for clinical resistance to taxanes have not been fully elucidated. Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor gene, whose expression has been clearly correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer (NSCLC). However, the molecular mechanism underlying the relationship between BRCA1 expression and taxane activity remains unclear. BRCA1 is a nuclear shuffling protein with many more described nuclear actions than cytoplasmic. BRCA1 has been shown to regulate the cell9s ability to undergo apoptosis and that increased BRCA1 cytoplasmic localization correlates with increased apoptotic functions. In association with BARD1, the BRCA1/BARD1 complex functions as an E3 ubiquitin ligase. Recent literature suggests γ-tubulin is a target of this complex resulting in mono-ubiquitination of γ-tubulin which regulates its centrosomal localization and function. Here, we test the hypothesis that either α- or β-tubulin also serve as a target for the BRCA1/BARD1 E3 ubiquitin ligase activity which regulates the cell9s sensitivity to Taxol. To approach this, we generated a stable-BRCA1 knockdown (B1-KD) of the NSCLC cell line A549 in which an 80% knockdown of BRCA1 results in resistance to Taxol when compared to the parental line, phenocopying the clinical data correlating low BRCA1 expression with taxane resistance. The B1-KD line exhibited aberrant centrosomal amplification and activity likely due to the γ-tubulin mislocalization. Then, we looked at the ability of BRCA1 to ubiquinate α- or β- tubulin subunits that form the microtubule cytoskeleton. Tubulin immunoprecipitation experiments revealed that the B1-KD results in a loss of tubulin ubiquitination when compared to the parental line. Taxol treatment in both cell lines seems to increase tubulin ubiquitination suggesting that the residual BRCA1 in the knockdown line is enough to ubiquitinate tubulin. Currently, we are looking at mechanisms linking tubulin ubiquitination to cell death, mainly capthesin-B-, calpain- or caspase-mediated apoptosis. Initial experiments suggest that capthesin-B inhibition does not inhibit Taxol-induced cell death in both the parental and B1-KD A549 cell line. We propose a model in which Taxol-induced tubulin ubiquitination, via the BRCA1/BARD1 complex, serves as the signal to initiate downstream cell death signaling pathways in the presence of Taxol treatment. This model would assign a strong cytoplasmic role for BRCA1 and show BRCA1 regulation of apoptosis may exist outside of the nucleus. It might also provide insight into how lung cancer cells become resistant to taxanes with the potential to disrupt these resistance mechanisms and enhance therapeutic efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 696. doi:10.1158/1538-7445.AM2011-696

Abstract 1113: The role of BRCA1 in taxane resistance

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The taxanes are among the most effective chemotherapeutic agents used for the treatment of solid tumors, yet eventually most tumors become resistant to taxane chemotherapy. Mechanisms responsible for clinical resistance to taxanes have not been fully elucidated. Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor gene with DNA damage repair activity, whose expression has been clearly correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer (NSCLC). However, the molecular mechanism underlying the relationship between BRCA1 expression and taxane activity remains unclear. In addition to its DNA damage repair activity, BRCA1 was shown to ubiquitinate γ-tubulin and affect centrosomal nucleation of microtubules. Here, we test the hypothesis that reduced BRCA1 expression will enhance microtubule nucleation (due to centrosome hyperactivity) which in turn, will produce hyper-acetylated microtubules (since this post-translational modification is specific for and accumulates on the microtubule polymer) making them non-responsive to taxanes. To this end, we developed a lentiviral shRNA-knockdown system of BRCA1 in a NSCLC model cell line, A549. Real-time PCR and Western Blot analyses confirmed 80% BRCA1 knockdown in the A549-BRCA1-KD cell line. Microtubule regrowth experiments following nocodazole treatment with subsequent washout showed an increase in centrosomally-nucleated microtubules (i.e. centrosomal hyperactivity) in the A549-BRCA1-KD cells compared to controls. These experiments also showed that the large majority of the microtubules that regrew were acetylated. Western Blot analysis showed that A549-BRCA1-KD cells indeed contain increased Ac-Tubulin content compared to controls. Interestingly, analysis of 155 NSCL patient samples from the randomized Neoadjuvant/Adjuvant Taxol Carboplatin Hope (NATCH) trial revealed that patients with high basal Ac-Tubulin in the adjuvant arm had a significant shorter time to progression suggesting that Ac-Tubulin content is a negative predictor for taxane efficacy. To assess taxane sensitivity in the A549-BRCA1-KD cell line, we treated cells for 72 hrs with paclitaxel and used two different cell viability assays, Cell-Titer Glo and Sulforhodamine B. Both showed A549-BRCA1-KD cells have a modest resistance (2-3 fold) to paclitaxel treatment, recapitulating the clinical data. Currently, we are correlating BRCA1 expression levels with Ac-Tubulin content in the NATCH patients as well as developing an siRNA screen to assess signaling pathways that modulate taxane sensitivity in the BRCA1-KD model. Elucidation of how lung cancer cells become resistant to taxanes will provide us with insight into how to potentially reverse these resistance mechanisms and enhance therapeutic efficacy. (Supported by NCI 5P01CA116676 and T32CA062948) 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 1113.