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Background: Triple negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, and despite recent treatment advances, clinical outcomes remain poor, particularly in the metastatic setting. TNBC is a biologically heterogeneous entity, and up to 50% of TNBC express the androgen receptor (AR). Gene expression subtyping has identified a subset of AR-TNBC with a luminal AR-driven (LAR) phenotype. Consequently, there has been great interest in translating androgen receptor signaling inhibitors (ARSIs) approved for use in prostate cancer such as enzalutamide and bicalutamide into the management of LAR-TNBC. However, early phase clinical trials of anti-androgens in metastatic AR-TNBC have had modest results. While multiple mechanisms of anti-androgen resistance have been identified in prostate cancer, including AR amplification and over-expression, emergence of AR splice variants (AR-Vs), and transition to AR independent growth, molecular determinants of anti-androgen resistance in TNBC remain poorly understood, and pre-clinical models to study acquired ARSI resistance in LAR-TNBC have been limited. Methods: A novel pre-clincial model of acquired ARSI resistance was derived from the LAR subtype MDA-MB-453 cell line through serial passaging with increasing concentrations of enzalutamide. Celltiter Blue viability assays were used to determine enzalutamide IC50 of parental and enzalutamide-resistant (EnzR) cell lines. RNA was extracted from the parental and resistant cells, reverse transcribed and expression of AR, splice-variants and canonical AR target genes were evaluated using RT-qPCR. Immunofluorescence was used to investigate the amount and nuclear localization of the AR protein within the parental and enzalutamide resistant cells. Results: EnzR MDA-MB-453 cells were derived through culture in increasing concentrations of enzalutamide for >6 months, and found to have a significant increase in enzalutamide IC50 compared to the parental cell line. EnzR MDA-MB-453 cells had a 4.5 fold increase in full-length AR expression at the transcriptional level compared to the parental cell line. AR protein expression and nuclear localization were also increased in EnzR cells compared to the parental cell line. Canonical AR targets including NKX3.1 were downregulated in response to enzalutamide in parental but not EnzR cells. While pathogenic AR splice variants (AR-Vs) implicated in ARSI resistance in prostate cancer were detected at low levels in MDA-MB-453 cells, no increase in AR-V expression was observed in the EnzR cell line. Conclusions: We have developed a novel pre-clinical model of anti-androgen/ARSI resistance in LAR-TNBC, and identified AR overexpression and persistent AR signaling associated with acquired enzalutamide resistance in LAR-TNBC. While pathogenic AR splice variants have been implicated in ARSI resistance in metastatic prostate cancer and are detected in the MDA-MB-453 cell line model, enzalutamide resistance was not associated with increased AR splice variant expression in this model. Future work will investigate mechanisms leading to AR overexpression as well as combination therapeutic strategies to overcome acquired ARSI resistance. Citation Format: Hannah Krause, Marina N. Sharifi, Serena K. Wolfe, Jamie M. Sperger, Kari B. Wisinski, Ruth O’Regan, Joshua Lang. Increased androgen receptor expression as a mechanism of acquired anti-androgen resistance in androgen receptor-positive triple negative breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-14.

Inhibitors of poly(ADP-ribose) polymerase (PARP) proteins potentiate antitumor activity of platinum chemotherapy. This study sought to determine the safety and tolerability of PARP inhibitor talazoparib with carboplatin and paclitaxel.We conducted a phase I study of talazoparib with carboplatin AUC5-6 and paclitaxel 80 mg/mForty-three patients were treated. The MTD for both schedules was talazoparib 250mcg daily. The main toxicity was myelosuppression including grade 3/4 hematologic treatment-related adverse events (TRAEs). Dose modification occurred in 87% and 100% of patients for schedules A and B, respectively. Discontinuation due to TRAEs was 13% in schedule A and 10% in B. Ten out of 22 evaluable patients in schedule A and 5/16 patients in schedule B had a complete or partial response. Twelve out of 43 patients received ≥6 cycles of talazoparib after induction, with a 13-month median duration of maintenance.We have established the recommended phase II dose of Talazoparib at 250mcg on a 3- or 7-day schedule with carboplatin AUC6 and paclitaxel 80 mg/m

Background: Somatic genomic alterations that activate the phosphatidylinositol-3-kinase (PI3K) pathway signaling are found in 30-50% of breast cancers, and the p110a-specific inhibitor alpelisib is approved for use in combination with fulvestrant in PIK3CA mutated hormone receptor-positive (HR+) metastatic breast cancer. However, preclinical and clinical data has demonstrated significant variability in response to alpelisib and other PI3K inhibitors even in the presence of hotspot PIK3CA activating mutations, likely reflecting functional differences between somatic alterations, bypass signaling mechanisms, and intratumor clonal heterogeneity. Thus, there is an ongoing need for novel predictive biomarkers to help guide patient selection for these therapies. Circulating tumor cells (CTCs) represent an accessible source of tumor derived analytes that allow for the interrogation of protein level readouts of PI3K pathway activation, may better reflect the biologic heterogeneity of metastatic disease than single site solid tumor biopsy, and are amenable to longitudinal analysis. Here, we report the development of an assay to evaluate the expression of activated AKT (AKT pS473) in CTCs as a putative biomarker of sensitivity to PI3K inhibitor therapies in metastatic breast cancer. Methods: Peripheral blood for CTC isolation was collected serially from patients with metastatic breast cancer and known somatic PI3K pathway mutation status. Samples were processed using VERSA (Versatile Exclusion-based Rare Sample Analysis), a microfluidic technology that integrates CTC capture and downstream analysis. Following fixation, CTCs were captured immunomagnetically with antibodies for EpCAM and Trop2, permeabilized and stained on chip for pan-AKT plus AKT pS473. Quantitative fluorescent microscopy was used to enumerate CTCs, defined as cytokeratin positive cells with intact nuclei and negative for a group of normal blood cell markers (CD45/CD11b/CD34/CD66b), and to quantify activated AKT protein expression in CTCs and matched peripheral blood mononuclear cells (PBMCs). Results: 100% of patients with somatic PIK3CA mutations in our pilot cohort had CTCs with detectable phospho-AKT expression, and in 50% of patients, the median phospho-AKT/pan AKT ratio was higher in CTCs compared to matched peripheral blood cells, suggestive of increased PI3K pathway activity. All patients demonstrated heterogeneity in phospho-AKT expression and phospho-AKT/pan-AKT ratio among individual CTCs at a single timepoint and across timepoints in longitudinal analysis. Conclusion: We report here the feasibility of quantitative and longitudinal detection of activated AKT in EpCAM/Trop2 captured CTCs from metastatic breast cancer with somatic PIK3CA mutations. Future work will prospectively evaluate multiple clinical applications of this assay in patients receiving alpelisib plus endocrine therapy for PIK3CA mutated HR+ metastatic breast cancer, as well as expanding the assay to include the detection of additional phospho-protein readouts of PI3K pathway activity in CTCs. In addition to the potential to complement solid biopsy PIK3CA mutation status as a predictive biomarker of sensitivity to PI3K therapies, this assay has the unique potential to provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs while on treatment, which may serve as an early biomarker of clinical response or resistance. Citation Format: Marina N Sharifi, Jamie M Sperger, Cole Gilsdorf, Serena K Wolfe, Amanda Parkes, Kari B Wisinski, Ruth M O'Regan, Joshua M Lang. Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-35.

Background: Phosphatidylinositol-3-kinase (PI3K) activating mutations are found in 30-40% of breast cancers, and the PI3K inhibitor alpelisib is FDA-approved for PIK3CA-mutated hormone-receptor positive metastatic breast cancer (MBC). However, rates of intrinsic alpelisib resistance are as high as 40%, and there is an ongoing need for novel biomarkers to help guide patient selection. Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) may better reflect the heterogeneity of metastatic disease than tissue biopsy, and are amenable to longitudinal analysis. By combining targeted ctDNA sequencing with CTC transcriptional profiling and quantitation of PI3K pathway phospho-proteins, we have developed the first multiplex assay to simultaneously assess genomic alterations and CTC PI3K pathway activity via liquid biopsy. Methods: Peripheral blood was collected from MBC patients with known PIK3CA mutation status serially prior to starting new standard therapies and on treatment. Cell-free DNA was extracted from plasma and sequenced using a custom capture panel (IDT). CTCs were isolated with VERSA microfluidic technology integrating capture with downstream analysis. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA-seq, or staining on chip for phospho-AKT pS473 and total AKT or phospho-rpS6 pS235/S236 and total rpS6, followed by quantification of single cell phospho/total protein mean fluorescence ratios. Results: In a pilot cohort of 14 patients, mean CTC phospho-AKT/total AKT protein expression ratio was higher in patients with PIK3CA mutations compared to patients without mutations (0.37 vs 0.26, p Conclusion: We have demonstrated the feasibility of a comprehensive liquid biopsy for simultaneous monitoring of PI3K pathway mutations in ctDNA and PI3K pathway signaling in CTCs via transcriptional profiling and phospho-protein expression in patients with MBC. Future work will prospectively evaluate this assay in patients receiving alpelisib for PIK3CA-mutated MBC. This has the potential to complement PIK3CA mutation status as a biomarker of sensitivity to PI3K therapies, and may also provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs on treatment. Citation Format: Marina N. Sharifi, Kyle T. Helzer, Jamie M. Sperger, Matthew L. Bootsma, Hannah Krause, Cole S. Gilsdorf, Serena K. Wolfe, Zachary Kauffman, Amye J. Tevaarwerk, Mark E. Burkard, Amanda M. Parkes, Ruth M. O'Regan, Kari B. Wisinski, Shuang G. Zhao, Joshua M. Lang. Simultaneous longitudinal assessment of PIK3CA genomic mutations and PI3K pathway activity in circulating tumor cells in metastatic breast 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 1955.

Background Expert knowledge is often shared among multidisciplinary academic teams at tumor boards (TBs) across the country, but these conversations exist in silos and do not reach the wider oncology community. Objective Using an oncologist-only question and answer (Q&A) website, we sought to document expert insights from TBs at National Cancer Institute–designated Comprehensive Cancer Centers (NCI-CCCs) to provide educational benefits to the oncology community. Methods We designed a process with the NCI-CCCs to document and share discussions from the TBs focused on areas of practice variation on theMednet, an interactive Q&A website of over 13,000 US oncologists. The faculty translated the TB discussions into concise, non–case-based Q&As on theMednet. Answers were peer reviewed and disseminated in email newsletters to registered oncologists. Reach and engagement were measured. Following each Q&A, a survey question asked how the TB Q&As impacted the readers’ practice. Results A total of 23 breast, thoracic, gastrointestinal, and genitourinary programs from 16 NCI-CCC sites participated. Between December 2016 and July 2021, the faculty highlighted 368 questions from their TBs. Q&As were viewed 147,661 times by 7381 oncologists at 3515 institutions from all 50 states. A total of 277 (75%) Q&As were viewed every month. Of the 1063 responses to a survey question on how the Q&A affected clinicians’ practices, 646 (61%) reported that it confirmed their current practice, 163 (20%) indicated that a Q&A would change their future practice, and 214 (15%) reported learning something new. Conclusions Through an online Q&A platform, academics at the NCI-CCCs share knowledge outside the walls of academia with oncologists across the United States. Access to up-to-date expert knowledge can reassure clinicians’ practices, significantly impact patient care in community practices, and be a source of new knowledge and education.

Hormone receptor (HR) positive breast cancer has a high propensity for late recurrences that might be prevented with longer durations of endocrine therapy (ET). However, trials evaluating extended adjuvant ET have produced somewhat conflicting results. Additionally, ET is associated with not only day to day side effects that can impact quality of life, but more detrimental effects that can cause significant morbidity. Although patients with higher stage disease are at greater risk of late recurrences, even patients with stage 1 disease have a significant risk of recurrence after 5 years. Current guidelines recommend extending therapy for patients with node-positive disease, but recommendations for patients with node-negative disease are less clear. This has led to the development of various genomic tests to aid oncologists in further individualizing their approach when it comes to deciding which subpopulation of patients with HR-positive breast cancer may benefit from extending their endocrine therapy beyond 5 years. There are several assays that are prognostic of recurrences years 6 to 10 following diagnosis. Additionally, the breast cancer index has been shown to be predictive of extended ET in patients who have completed 5 years of tamoxifen. None of the available assays are, to date, predictive of recurrence after 10 years. Genomic testing is not appropriate for all patients, especially if the results will not predict the choice of further treatment. Ultimately, genomic testing should help facilitate shared decision making between the patient and oncologist.

Endocrine therapy has been the standard of care for patients with metastatic hormone receptor (HR)-positive, HER2-negative breast cancer since the 1970s, improving survival while avoiding the toxicities associated with cytotoxic chemotherapy. However, all HR-positive tumors ultimately develop resistance to endocrine therapy. Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have more recently become an important component of the management of this breast cancer subtype, significantly delaying time to the disease progression and improving survival when combined with endocrine therapy. However, as with endocrine therapy alone, treatment resistance remains a universal phenomenon. As more women receive CDK4/6 inhibitors as part of their treatment, the management of de novo and acquired resistance to combined CDK4/CDK6 inhibitor plus endocrine therapy regimens has emerged as an important clinical challenge. Several resistance mechanisms have been described, including alterations in the CDK4/6/cyclin D complex or its major effector retinoblastoma protein (pRb), bypass signaling through other cyclin/CDK complexes and activation of upstream signaling pathways, in particular the PI3K/mTOR pathway, but robust biomarkers to predict resistance remain elusive, and the role for continuing CDK4/6 inhibitors after progression remains under investigation. Novel strategies being evaluated in clinical trials include the continuation of CDK4/6 inhibitors through progression, as well as triplet therapy combinations with PI3K inhibitors or immune checkpoint inhibitors.

Introduction: The androgen receptor (AR) is expressed in up to 50% of patients with triple negative breast cancer (TNBC), and a luminal androgen receptor (LAR) subtype has been identified that is dependent on androgen receptor signaling, but AR protein expression by immunohistochemistry (IHC) has been a suboptimal biomarker for response to anti-androgen therapies in AR-TNBC and there remains an unmet need for biomarkers to identify patients likely to benefit from anti-androgens. We have developed a multiplex liquid biopsy encompassing AR protein expression and downstream gene expression analysis in circulating tumor cells (CTCs). We report here the longitudinal evaluation of this liquid biopsy as part of a phase I trial (NCT03090165) of the anti-androgen bicalutamide with selective CDK4/6 inhibitor ribociclib in metastatic AR-TNBC. Methods: Peripheral blood was collected (n=11) prior to treatment, after two cycles of treatment, and at progression and processed using the VERSA (Versatile Exclusion-based Rare Sample Analysis) microfluidic chip that integrates CTC capture and downstream analysis. For protein expression, fixed CTCs were captured immunomagnetically and stained on chip for AR followed by quantitative fluorescent microscopy. For gene expression analysis, live CTCs were captured immunomagnetically followed by RNA isolation on chip, reverse transcription, and RT-qPCR for a panel of 40 luminal AR genes. Results: AR protein and transcript were detected in CTCs from 10/11 patients, and AR target gene expression in 8/11 patients. The pathologic ligand independent AR splice variant AR-V7 was detected in 2/11 patients. Pretreatment CTC number was higher in patients with anti-androgen resistant (range 3-753) than anti-androgen sensitive (range 0-30) disease. Among patients with low AR IHC (50%) on biopsy, AR-V7 was detected in CTCs of 2/3 of patients with resistant disease and 0/2 of patients with sensitive disease. Conclusions: This study demonstrates the feasibility of a longitudinal multiplex liquid biopsy as a pharmacodynamic biomarker of AR pathway activity in AR-TNBC, and suggests that AR pathway activity in CTCs may provide additional information to solid tumor biopsy AR expression in predicting sensitivity to anti-androgens. This work also demonstrates for the first time the detection of AR-V7 in CTCs in anti-androgen resistant AR-TNBC, suggesting that AR-V7 expression may be a mechanism of anti-androgen resistance in AR-TNBC as it is in advanced prostate cancer. Citation Format: Marina N. Sharifi, Serena K. Wolfe, Jamie M. Sperger, Jennifer L. Schehr, Saswati Bhattacharya, Kari B. Wisinski, Joshua M. Lang, Ruth M. O'Regan. Multiplex liquid biopsy for AR pathway activity in metastatic androgen receptor-positive triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 590.

New anti-HER2 therapies have greatly improved clinical outcomes in HER2-positive breast cancer, but tumor resistance to HER2-directed therapies remains a significant problem that has spurred the development of a number of targeted therapies directed at non-HER2 pathways in this disease. Here we review major mechanisms of resistance to anti-HER2 therapies identified in the preclinical and clinical setting, as well as the preclinical and clinical status of the major categories of novel non–HER2-targeted therapies in clinical development. These include CDK4/6 inhibitors, PI3K/mTOR pathway inhibitors, antiangiogenic agents, inhibitors of bypass signaling through non-HER growth factors, including insulin-like growth factor 1 receptor, fibroblast growth factor receptor, and c-MET, and advances in immunotherapy, including checkpoint inhibitors and cancer vaccines.

34 Background: Management of BCR PCa requires accurate assessment of location and extent of recurrent disease. FACBC has been shown to be a sensitive modality for detection and localization of recurrent disease but treatment guidelines are based on the findings of conventional (conv) imaging, including computed tomography, magnetic resonance imaging, or bone scintigraphy, and little is known about how prior treatment impacts FACBC findings and concordance with conv scans. Methods: This single-center retrospective study included 137 patients (pts) who had FACBC for BCR at the University of Wisconsin-Madison from 10/2017-10/2019. Clinical, pathological, imaging, and treatment data were collected by chart review. Pts were classified by type of primary treatment for localized PCa, either radical prostatectomy (RP) or radiation therapy (RT). Findings of conv scans performed within 4 weeks prior or any time after FACBC were collected. Results: 105 pts had RP and 32 pts had RT as their primary PCa treatment. Gleason score and PSA at diagnosis were similar between groups. Median PSA at time of FACBC was higher in the RT compared to RP group (3.3 vs 0.7 ng/dL) and median time from initial diagnosis to FACBC was longer (70 vs 55 months). Frequency of (+) FACBC findings was higher in the RT group (66% vs 47%); only 3% of pts in the RT group had a (-) FACBC compared to 29% in the RP group. The rate of (+) lesions in the prostate/prostate bed was higher in the RT group (41% vs 22%), while the rate of (+) lesions in pelvic nodes and distant sites was similar between groups. Of 69 pts who also had conv imaging, 61% had concordant conv imaging findings. In the RT group, conv and FACBC findings were similar in 47% of pts and not similar in 28%. In the RP group, conv and FACBC findings were similar in 26% of pts and not similar in 17%. Management after FACBC is listed in table. Median time from FACBC to first (+) conv scans was 6 (range: 0-18) and 5 (range: 0-17) months for RT and RP groups, respectively. Conclusions: In this large retrospective cohort, pts treated with initial RT had a longer median time from diagnosis to FACBC and higher median PSA at the time of FACBC compared to the RP group. RT patients had a higher rate of (+) FACBC findings but were more likely to continue on observation. The median time from FACBC to first (+) conv scan was 5-6 months, supporting the role of FACBC in earlier detection of recurrent disease in both groups of patients. Further analysis of concordance between FACBC and conv imaging is in process. [Table: see text]

Autophagy is a highly conserved self-degradative process that has a key role in cellular stress responses and survival. Recent work has begun to explore the function of autophagy in cancer metastasis, which is of particular interest given the dearth of effective therapeutic options for metastatic disease. Autophagy is induced upon progression of various human cancers to metastasis and together with data from genetically engineered mice and experimental metastasis models, a role for autophagy at nearly every phase of the metastatic cascade has been identified. Specifically, autophagy has been shown to be involved in modulating tumor cell motility and invasion, cancer stem cell viability and differentiation, resistance to anoikis, epithelial-to-mesenchymal transition, tumor cell dormancy and escape from immune surveillance, with emerging functions in establishing the pre-metastatic niche and other aspects of metastasis. In this review, we provide a general overview of how autophagy modulates cancer metastasis and discuss the significance of new findings for disease management.

SummaryAutophagy is a conserved catabolic process that plays a housekeeping role in eliminating protein aggregates and organelles and is activated during nutrient deprivation to generate metabolites and energy. Autophagy plays a significant role in tumorigenesis, although opposing context-dependent functions of autophagy in cancer have complicated efforts to target autophagy for therapeutic purposes. We demonstrate that autophagy inhibition reduces tumor cell migration and invasion in vitro and attenuates metastasis in vivo. Numerous abnormally large focal adhesions (FAs) accumulate in autophagy-deficient tumor cells, reflecting a role for autophagy in FA disassembly through targeted degradation of paxillin. We demonstrate that paxillin interacts with processed LC3 through a conserved LIR motif in the amino-terminal end of paxillin and that this interaction is regulated by oncogenic SRC activity. Together, these data establish a function for autophagy in FA turnover, tumor cell motility, and metastasis.

Macro-autophagy is an ancient and highly conserved self-degradative process that plays a homeostatic role in normal cells by eliminating organelles, pathogens, and protein aggregates. Autophagy, as it is routinely referred to, also allows cells to maintain metabolic sufficiency and survive under conditions of nutrient stress by recycling the by-products of autophagic degradation, such as fatty acids, amino acids, and nucleotides. Tumor cells are more reliant than normal cells on autophagy for survival in part due to their rapid growth rate, altered metabolism, and nutrient-deprived growth environment. How this dependence of tumor cells on autophagy affects their progression to malignancy and metastatic disease is an area of increasing research focus. Here, we review recent work identifying critical functions for autophagy in tumor cell migration and invasion, tumor stem cell maintenance and therapy resistance, and cross-talk between tumor cells and their microenvironment.

Background: To date, the use of anti-androgens in the subset of triple negative breast cancers (TNBC) that express androgen receptor (AR) has shown modest response rates, indicating anti-androgen-resistance in the majority of these tumors. Based on data that Cyclin D kinase (CDK) inhibitors reverse resistance to anti-androgens in prostate cancer cell lines, we hypothesize that the use of CDK inhibition may enhance the activity of anti-androgens in AR-positive TNBC. Methods: Key eligibility include: patients with centrally confirmed AR-positive TNBC, defined as AR expression >0%; 0 to 1 line of prior therapy for metastatic disease; and measurable disease. Patients are treated with bicalutamide 150mg orally once daily plus ribociclib at one of 3 dose levels (see table). Table 1Dose levelBicalutamideRibociclib1150mg orally once daily (D1 to 28)400mg daily (D1 to 21)2150mg orally once daily (D1 to 28)400mg daily (D1 to 28)3150mg orally once daily (D1 to 28)600mg daily (D1 to 21) Results: AR expression was positive by trial criteria in 74% of screened patients. Three patients have been accrued at each dose level. Median age is 56 and 6 and 3 patients were treated in first and second-line settings, respectively. Median AR expression was 50% (range 5 to 75%). Toxicity data is available for 6-patients treated on dose levels 1 and 2. No dose-limiting toxicities were noted. As anticipated with ribociclib, the most common toxicity is neutropenia (1 patient grade 4 and 2 patients grade 3). Two patients experienced grade 3 hypertension and 1 experienced grade 3 lymphopenia. Grade 2 or lower toxicities included fatigue, nausea, hyperglycemia and mucositis. One patient experienced grade 1 QT interval prolongation. Conclusion: The combination of bicalutamide and ribociclib is tolerable without unexpected toxicities. Data on the 3-patients treated at dose level 3 and dose expansion will be included. Phase 2 dosing schedule will be decided based on phase 1 results. Citation Format: Sharifi M, Wisinski KB, Burkard ME, Tevaarwerk AJ, Tamkus D, Chan N, Truica C, Danciu O, Hoskins K, O'Regan RM. Phase I trial of bicalutamide and ribociclib in androgen receptor-positive triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT1-02-01.

BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesis—two processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunction—caused by defects in mitophagy—can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

Chlamydomonas reinhardtii is a unicellular green alga that is a key model organism in the study of photosynthesis and oxidative stress. Here we describe the large-scale generation of a population of insertional mutants that have been screened for phenotypes related to photosynthesis and the isolation of 459 flanking sequence tags from 439 mutants. Recent phylogenomic analysis has identified a core set of genes, named GreenCut2, that are conserved in green algae and plants. Many of these genes are likely to be central to the process of photosynthesis, and they are over-represented by sixfold among the screened insertional mutants, with insertion events isolated in or adjacent to 68 of 597 GreenCut2 genes. This enrichment thus provides experimental support for functional assignments based on previous bioinformatic analysis. To illustrate one of the uses of the population, a candidate gene approach based on genome position of the flanking sequence of the insertional mutant CAL027_01_20 was used to identify the molecular basis of the classical C. reinhardtii mutation ac17. These mutations were shown to affect the gene PDH2, which encodes a subunit of the plastid pyruvate dehydrogenase complex. The mutants and associated flanking sequence data described here are publicly available to the research community, and they represent one of the largest phenotyped collections of algal insertional mutants to date.

Highlight CHLI1 is indispensable for MgCh function in C. reinhardtii, while CHLI2 is not involved in MgCh activity. Inactivation of MgCh due to CHLI1 deficiency results in altered nuclear gene expression., The oligomeric Mg chelatase (MgCh), consisting of the subunits CHLH, CHLI, and CHLD, is located at the central site of chlorophyll synthesis, but is also thought to have an additional function in regulatory feedback control of the tetrapyrrole biosynthesis pathway and in chloroplast retrograde signaling. In Arabidopsis thaliana and Chlamydomonas reinhardtii, two genes have been proposed to encode the CHLI subunit of MgCh. While the role of CHLI1 in A. thaliana MgCh has been substantially elucidated, different reports provide inconsistent results with regard to the function of CHLI2 in Mg chelation and retrograde signaling. In the present report, the possible functions of both isoforms were analyzed in C. reinhardtii. Knockout of the CHLI1 gene resulted in complete loss of MgCh activity, absence of chlorophyll, acute light sensitivity, and, as a consequence, down-regulation of tetrapyrrole biosynthesis and photosynthesis-associated nuclear genes. These observations indicate a phenotypical resemblance of chli1 to the chlh and chld C. reinhardtii mutants previously reported. The key role of CHLI1 for MgCh reaction in comparison with the second isoform was confirmed by the rescue of chli1 with genomic CHLI1. Because CHLI2 in C. reinhardtii shows lower expression than CHLI1, strains overexpressing CHLI2 were produced in the chli1 background. However, no complementation of the chli1 phenotype was observed. Silencing of CHLI2 in the wild-type background did not result in any changes in the accumulation of tetrapyrrole intermediates or of chlorophyll. The results suggest that, unlike in A. thaliana, changes in CHLI2 content observed in the present studies do not affect formation and activity of MgCh in C. reinhardtii.

Macro-autophagy is a major catabolic process in the cell used to degrade protein aggregates, dysfunctional organelles and intracellular pathogens that would otherwise become toxic. Autophagy also generates energy and metabolites for the cell through recycling of degraded autophagosomal cargo, which can be particularly important for cell viability under stress. The significance of changes in the rates of autophagic flux for cellular function and disease is being increasingly appreciated, and interest in measuring autophagy in different experimental systems is growing accordingly. Here, we describe key methodologies used in the field to measure autophagic flux, including monitoring LC3 processing by western blot, fluorescent cell staining, and flow cytometry, in addition to changes in the levels or posttranslational modifications of other autophagy markers, such as p62/Sqstm1 and the Atg5–Atg12 conjugate. We also describe what cellular stresses may be used to induce autophagy and how to control for changes in the rates of autophagic flux as opposed to inhibition of flux. Finally, we detail available techniques to monitor autophagy in vivo.

Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. A light-sensitive, phytoene-accumulating mutant, pds1-1, was isolated in Chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase (PDS) gene. PDS catalyzes the second step in carotenoid biosynthesis--the conversion of phytoene to ζ-carotene. Decreased accumulation of downstream colored carotenoids suggested that the pds1-1 mutant is leaky for PDS activity. A screen for enhancers of the pds1-1 mutation yielded the pds1-2 allele, which completely lacks PDS activity. A second independent null mutant (pds1-3) was identified using DNA insertional mutagenesis. Both null mutants accumulate only phytoene and no other carotenoids. All three phytoene-accumulating mutants exhibited slower growth rates and reduced plating efficiency compared to wild-type cells and white phytoene synthase mutants. Insight into amino acid residues important for PDS activity was obtained through the characterization of intragenic suppressors of pds1-2. The suppressor mutants fell into three classes: revertants of the pds1-1 point mutation, mutations that changed PDS amino acid residue Pro64 to Phe, and mutations that converted PDS residue Lys90 to Met. Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 90 and 143 are in close contact in the active PDS enzyme and have important roles in its structural stability and/or activity.

Introduction: Autophagy, a catabolic process, can play a crucial role in cancer cell survival. Modifying this process has shown to be an effective mechanism of inducing cell death. In order to explore this concept, we induced autophagy with one agent (Akt-inhibitor MK-2206), and decreased autophagosomal trafficking with other agents (Paclitaxel and Nocadazole) by disrupting the microtubule networks. We hypothesized that by modifying autophagy through this combination, it would lead to an accumulation of autophagosomes and eventual cell death in a synergistic manner. Methods: SCC 61 and Cal 27 HNSCC cell lines were used to explore this hypothesis. SCC 61 cells were transduced with an m-Cherry GFP LC3B vector in order to detect autophagosomes under both fixed and live cell confocal microscopy. Cell viability and apoptosis assays were performed. A siRNA ATG 7 KO SCC 61 cell line was used to detect if in fact autophagy was important for this therapeutic approach. Results: There was an accumulation of autophagosomes with the combination of an Akt inhibitor and microtubule disrupters. A decrease in fusion of autophagosome to lysosome was seen when cells were treated with an agent, which disrupted the microtubule networks (Paclitaxel) but even more dramatically when treated with the combination (MK-2206 and Paclitaxel). Moreover, accumulation of autophagosomes occurred prior to induction of apoptosis with combination treatment. These effects were decreased with siRNA ATG 7 KO in SCC 61 cells, which were more resistant to combination treatment compared to control. Discussion: Manipulating autophagy with Akt inhibition (induction) and Paclitaxel (decreasing autophagic trafficking) leads to an accumulation of autophagosomes and eventual cell death in a synergistic manner. This therapeutic approach of altering the normal autophagic process warrants further investigation. Citation Format: Omar G. Ahmed, Wen-Liang Kuo, Tai-Fen Wei, Jeannette S. Messer, Marina Sharifi, Madhavi Nagilla, Kay Macleod, Ezra E.W. Cohen. Modifying autophagy through combination treatments as a potential therapeutic strategy in head and neck squamous cell carcinoma (HNSCC). [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 316. doi:10.1158/1538-7445.AM2014-316

Macro-autophagy is a catabolic process important for degradation of damaged organelles, protein aggregates and intracellular recycling of proteins and lipids. Autophagy is thought to suppress tumor initiation by promoting genome stability and limiting necrosis and inflammation, but conversely is utilized by tumor cells to survive nutrient stress, hypoxia, and cytotoxic therapies, promoting growth of established tumors. However, whether autophagy impacts the metastatic process is not well understood. To examine the role of autophagy in breast cancer metastasis, essential autophagy genes atg5 and atg7 were stably knocked down the aggressive and metastatic 4T1 mouse mammary tumor cell line using shRNA. Inhibition of autophagy did not reduce cell proliferation or survival under stress in vitro or primary tumor growth in vivo, but markedly reduced metastasis to the lungs and liver. Surprisingly, this was not due reduced survival in the circulation or at the secondary site, as atg5 and atg7-deficient cells were able to colonize the lungs to the same extent as control cells when injected into the circulation in a tail vein experimental metastasis assay, suggesting that inhibition of autophagy impairs escape from the primary tumor in this model. Indeed, inhibition of autophagy prevented cell spreading, migration and invasion through collagen in transwell assays of 4T1 cells, MDA-MB-231 human breast cancer cells, and B16.F10 melanoma cells. This was associated with failure of RhoA-mediated protrusion, impaired focal adhesion disassembly, and accumulation of the key focal adhesion component paxillin. We show that paxillin is degraded by autophagy, and siRNA-mediated knockdown of paxillin in autophagy-deficient cells rescued their spreading and motility, suggesting that impaired focal adhesion disassembly and migration in autophagy-deficient cells was the result of failure of autophagic degradation of paxillin. Intriguingly, active RhoA is localized at autophagosomes, and inhibition of RhoA activity inhibits not only cellular protrusion but also autophagic flux in metastatic cells, suggesting that RhoA may be involved in upstream regulation of autophagic degradation of paxillin to promote focal adhesion disassembly. In sum, we have identified a novel and unexpected function for autophagy in focal adhesion disassembly and tumor cell motility that is required for metastasis in vivo. This suggests that while autophagy may also promote the survival of disseminating tumor cells during metastasis, autophagy is in fact required for escape from the primary tumor. Citation Format: Marina N. Sharifi, Christopher Collier, Lauren Drake, Hong Chen, Stephanie Mui, Kay F. Macleod. Autophagy is required for focal adhesion turnover, tumor cell motility and metastasis. [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 321. doi:10.1158/1538-7445.AM2014-321

BNIP3 is a hypoxia-inducible protein involved in mitochondrial dynamics and mitophagy that is up-regulated at pre-malignant stages of various human cancers, including breast cancer but down-regulated at later stages of tumorigenesis. To examine the role of BNip3 in tumor progression, we crossed BNip3 null mice to the MMTV-PyMT mouse model of breast cancer. Loss of BNip3 promoted primary tumor growth in vivo and tumor cell proliferation in vitro and was associated with increased aerobic glycolysis and reduced mitochondrial respiration. BNip3 null tumor cells also showed increased invasive properties that were cell intrinsic and transplantable to wild-type host mice. Consistently, there was reduced latency to lung metastasis in the MMTV-PyMT;BNip3-/- mice and increased numbers of metastases compared to control mice. BNip3 null tumors exhibited increased mitochondrial mass, but a greater fraction of mitochondria were dysfunctional with reduced membrane potential and decreased metabolite uptake compared to BNip3 wild-type tumor cells. Consistently, increased proliferation of BNip3 null tumor cells was associated with increased carbon flux from glucose to lipid and nucleic acids. Interestingly, although mitochondria were less efficient at uptake of pyruvate and glutamine, the rate of production of Krebs cycle intermediates was increased. This indicates that defective mitochondria due to inefficient mitophagy can induce the Warburg effect, as originally proposed by Warburg himself. Loss of BNip3 also resulted in more hypoxic tumors that expressed increased levels of Hif-1α and its target genes and showed increased angiogenesis, although blood vessels were abnormal possibly explaining increased tumor hypoxia, thus suggesting a negative feedback loop between Hif-1α and its target gene, BNip3. Inhibition of glycolysis or inhibition of HIF activity reduced proliferation in BNip3 null tumor cells to the levels observed in control cells. Significantly, BNip3 null tumor cells underwent autophagic flux as efficiently as wild-type cells demonstrating for the first time that inhibition of mitophagy has a distinct tumorigenic consequence to general inactivation of autophagy, as reported elsewhere. These results demonstrate that BNip3 has tumor and metastasis suppressor properties required to maintain mitochondrial integrity, promote oxidative metabolism and mitigate against the metastasis promoting activities of hypoxia and glycolytic metabolism. Citation Format: Aparajita Hoskote Chourasia, Kristin Tracy, Michelle Boland, Marina Sharifi, Kay F. Macleod. BNIP3 suppresses mammary tumorigenesis and metastasis through negative regulation of Warburg effect and HIF-1α. [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 4316. doi:10.1158/1538-7445.AM2014-4316

The phosphoinositol-3 kinase (PI3K) pathway is highly dysregulated in squamous cell carcinoma of the head and neck (SCCHN). While inhibitors of the PI3K/AKT pathway are being developed in cancer, their efficacy does not appear to be related to the presence of mutations or amplification in pathway genes. The PI3K pathway is a major regulator of macro-autophagy, an evolutionarily conserved catabolic process that degrades cellular materials to promote cellular homeostasis and survival under stress. Employing a panel of SCCHN cell lines, we observed a significant correlation between the activity of PI3K/AKT inhibitors and their ability to induce autophagy. More specifically, resistance to these inhibitors was associated with accumulation of p62/SQSTM1, a pleotropic protein that is consumed during autophagy, while loss of autophagy was, for the first time, found to be due to silencing of an essential autophagy gene, ATG7. Moreover, modulating ATG7 and p62/SQSTM1 could regulate sensitivity to PI3K/AKT inhibitors, underscoring a mechanistic link between autophagy and drug sensitivity. Analysis of human tissues revealed progressive accumulation of p62/SQSTM1 in a significant proportion of cancer samples compared to normal tissue, suggesting that defective autophagy has relevance to SCCHN. These findings are further validated by analysis of TCGA data confirming homozygous deletion and mRNA down-regulation of ATG7 in 10.0% of SCCHN samples. Taken together, these data indicate that p62/SQSTM1 levels modulate sensitivity to PI3K/AKT inhibitors; cancers vary in their capacity to undergo autophagy through epigenetic modification and, when deficient, accumulate p62/SQSTM1; and expression of autophagy-related proteins may serve as markers for resistance to PI3K/AKT inhibitors in SCCHN.

e13532 Background: Though there have been many advances in the treatment of HNSCC, the 5-year survival rate remains at 50%. This is partly due to molecular alterations such as the activation of the PI3k-AKT pathway in HNSCC, which has been associated with treatment failure. We stained human tissue samples and saw increased expression of p-AKT and pS6 in HNSCC compared to normal and dysplasia. This led us to investigate the role of the AKT inhibitor MK-2206 in combination with an already established chemotherapeutic agent, paclitaxel. Methods: Cell viability and Combination index were used to determine synergy in 4 HNSCC cell lines. PI exclusion and Annexin/PI staining were used to determine cell death and apoptosis, respectively. Autophagy was measured through changes in LC3BII protein levels using western blot analysis and a stably transfected GFP-LC3BII cell line was used to detect changes in protein localization under confocal microscopy. Results: All cell lines were sensitive to paclitaxel while only some were sensitive to MK-2206. However, there was effective synergy to the combination treatment in all cell lines. Apoptotic synergy was observed in some cell lines indicating that the combination stimulates cell death in the appropriate context. In one autophagy competent cell line, MK-2206 induced robust autophagy while paclitaxel blocked a latter step in the pathway leading to an accumulation of autophagosomes prior to induction of apoptosis. Conclusions: Paclitaxel and MK-2206 appears to be an effective combination in HNSCC possibly through their effect on autophagy trafficking, leading to an accumulation of autophagosomes and eventual apoptosis. Clinical trials examining this combination are warranted.