Search

Fig S2. SLC35F2 Expression Determines Sensitivity to YM155

Supplementary Table S2. Drugs and their concentrations used in the isogenic drug screen.

Correlation of tumor androgen levels with SLC35F2 transcript expression in PDX models

Supplementary Figure 1. Distribution of gene alterations in Breast TCGA and verification of expression of MCF10A cells. Supplementary Figure 2. Analysis of the MCF10A drug screen. Supplementary Figure 3. Significance of overlap between interactions found in this study and in the CGP. Supplementary Figure 4. Response of isogenic engineered cells to dasatinib. Supplementary Figure 5. Dasatinib sensitivity of CML versus AML cancer cell lines. Supplementary Figure 6. Verification of LYN knockdown via siRNA and response of LYN T319I to dasatinib. Supplementary Figure 7. Co-­�expression of MYC and LYN in breast cancer cell lines.

Androgen receptor (AR) signaling is fundamental to prostate cancer and is the dominant therapeutic target in metastatic disease. However, stringent androgen deprivation therapy regimens decrease quality of life and have been largely unsuccessful in curtailing mortality. Recent clinical and preclinical studies have taken advantage of the dichotomous ability of AR signaling to elicit growth-suppressive and differentiating effects by administering hyperphysiologic levels of testosterone. In this study, high-throughput drug screening identified a potent synergy between high-androgen therapy and YM155, a transcriptional inhibitor of survivin (BIRC5). This interaction was mediated by the direct transcriptional upregulation of the YM155 transporter SLC35F2 by the AR. Androgen-mediated YM155-induced cell death was completely blocked by the overexpression of multidrug resistance transporter ABCB1. SLC35F2 expression was significantly correlated with intratumor androgen levels in four distinct patient-derived xenograft models, and with AR activity score in a large gene expression dataset of castration-resistant metastases. A subset of tumors had significantly elevated SLC35F2 expression and, therefore, may identify patients who are highly responsive to YM155 treatment.Implications: The combination of androgen therapy with YM155 represents a novel drug synergy, and SLC35F2 may serve as a clinical biomarker of response to YM155. Mol Cancer Res; 15(5); 521–31. ©2017 AACR.

Purpose: Small-cell neuroendocrine prostate cancer (SCNPC) exhibits an aggressive clinical course and incidence rates seem to be increasing following resistance to potent androgen receptor (AR) antagonists. Currently, treatment options are limited and few model systems are available to identify new approaches for treatment. We sought to evaluate commonalities between SCNPC and other aggressive neuroendocrine carcinomas to identify therapeutic targets. Experimental Design: We generated whole transcriptome RNA-sequencing data from AR-active prostate cancers (ARPCs) and SCNPCs from tumors collected at rapid autopsy and two other neuroendocrine carcinomas, Merkel cell carcinoma (MCC), and small-cell lung cancer. We performed cross-tumor comparisons to identify conserved patterns of expression of druggable targets. We tested inhibitors to highly upregulated drug targets in a panel of prostate cancer cell lines and in vivo patient-derived xenograft (PDX) models. Results: We identified BCL2 as highly upregulated in SCNPC compared with ARPC. Inhibitors targeting BCL2 induced apoptotic cell death in SCNPC cell lines at nanomolar concentrations while ARPC cell lines were resistant. Treatment with the BCL2 inhibitor navitoclax leads to a reduction of growth of SCNPC PDX tumors in vivo, whereas ARPC PDX models were more resistant. We identified Wee1 as a second druggable target upregulated in SCNPC. Treatment with the combination of navitoclax and the Wee1 inhibitor AZD-1775 repressed the growth of SCNPC PDX resistant to single-agent BCL2 inhibitors. Conclusions: The combination of BCL2 and Wee1 inhibition presents a novel therapeutic strategy for the treatment of SCNPC.

Tumors that overexpress the MYC oncogene, including most receptor triple-negative breast cancers, frequently demonstrate aneuploidy, numerical chromosome alterations associated with highly aggressive cancers. Aneuploidy is also associated with rapid tumor evolution and poor patient outcome. We identify that MYC overexpression induces reversible defects in microtubule nucleation and mitotic spindle assembly, in TNBCs and other epithelial cells, promoting chromosome segregation defects, micronuclei and chromosomal instability (CIN). High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in cells with MYC overexpression; whereas TPX2 depletion blocks mitotic progression, induces cell death and prevents tumor growth. Attenuating MYC expression reverses mitotic defects, even in established breast tumor cell lines, implicating an ongoing role for high MYC in the persistence of CIN in cancers. Our studies implicate the MYC oncogene as a regulator of spindle assembly and identify a new MYC-TPX2 synthetic-lethal interaction in TNBC that could represent a future therapeutic strategy in MYC-overexpressing cancers. Moreover, our studies suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution. Citation Format: Goga A, Rohrberg J, Corella A, Taileb M, Kilinc S, Jokisch M-L, Camarda R, Zhou A, Balakrishnan S, Chang AN, Klein-Connolly H. MYC dysregulates mitotic spindle function in triple-negative breast cancer creating a dependency on TPX2 [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 P3-09-01.

Prostate cancer (PC) is driven by androgen receptor (AR) activity, a master regulator of prostate development and homeostasis. Frontline therapies for metastatic PC deprive the AR of the activating ligands testosterone (T) and dihydrotestosterone (DHT) by limiting their biosynthesis or blocking AR binding. Notably, AR signaling is dichotomous, inducing growth at lower activity levels, while suppressing growth at higher levels. Recent clinical studies have exploited this effect by administration of supraphysiological concentrations of T, resulting in clinical responses and improvements in quality of life. However, the use of T as a therapeutic agent in oncology is limited by poor drug-like properties as well as rapid and variable metabolism. Here, we investigated the antitumor effects of selective AR modulators (SARMs), which are small-molecule nonsteroidal AR agonists developed to treat muscle wasting and cachexia. Several orally administered SARMs activated the AR program in PC models. AR cistromes regulated by steroidal androgens and SARMs were superimposable. Coregulatory proteins including HOXB13 and GRHL2 comprised AR complexes assembled by both androgens and SARMs. At bioavailable concentrations, SARMs repressed MYC oncoprotein expression and inhibited the growth of castration-sensitive and castration-resistant PC in vitro and in vivo. These results support further clinical investigation of SARMs for treating advanced PC.

Androgen receptor (AR) signaling is fundamental to prostate cancer and is the dominant therapeutic target in metastatic disease. However, stringent androgen deprivation therapy regimens decrease quality of life and have been largely unsuccessful in curtailing mortality. Recent clinical and preclinical studies have taken advantage of the dichotomous ability of AR signaling to elicit growth-suppressive and differentiating effects by administering hyperphysiologic levels of testosterone. In this study, high-throughput drug screening identified a potent synergy between high-androgen therapy and YM155, a transcriptional inhibitor of survivin (BIRC5). This interaction was mediated by the direct transcriptional upregulation of the YM155 transporter SLC35F2 by the AR. Androgen-mediated YM155-induced cell death was completely blocked by the overexpression of multidrug resistance transporter ABCB1. SLC35F2 expression was significantly correlated with intratumor androgen levels in four distinct patient-derived xenograft models, and with AR activity score in a large gene expression dataset of castration-resistant metastases. A subset of tumors had significantly elevated SLC35F2 expression and, therefore, may identify patients who are highly responsive to YM155 treatment. Implications: The combination of androgen therapy with YM155 represents a novel drug synergy, and SLC35F2 may serve as a clinical biomarker of response to YM155. Mol Cancer Res; 15(5); 521–31. ©2017 AACR.

Clinical trials of high-dose androgen (HDA) therapy for prostate cancer (PC) have shown promising efficacy but are limited by lack of criteria to identify likely responders. To elucidate factors that govern the growth-repressive effects of HDAs, we applied an unbiased integrative approach using genetic screens and transcriptional profiling of PC cells with or without demonstrated phenotypic sensitivity to androgen-mediated growth repression. Through this comprehensive analysis, we identified genetic events and related signaling networks that determine the response to both HDA and androgen withdrawal. We applied these findings to develop a gene signature that may serve as an early indicator of treatment response and identify men with tumors that are amenable to HDA therapy.

Triple-negative breast cancer (TNBC), in which cells lack expression of the estrogen receptor (ER), the progesterone receptor (PR) and the ERBB2 (also known as HER2) receptor, is the breast cancer subtype with the poorest outcome. No targeted therapy is available against this subtype of cancer owing to a lack of validated molecular targets. We previously reported that signaling involving MYC-an essential, pleiotropic transcription factor that regulates the expression of hundreds of genes-is disproportionally higher in triple-negative (TN) tumors than in receptor-positive (RP) tumors. Direct inhibition of the oncogenic transcriptional activity of MYC has been challenging to achieve. Here, by conducting a shRNA screen targeting the kinome, we identified PIM1, a non-essential serine-threonine kinase, in a synthetic lethal interaction with MYC. PIM1 expression was higher in TN tumors than in RP tumors and was associated with poor prognosis in patients with hormone- and HER2-negative tumors. Small-molecule PIM kinase inhibitors halted the growth of human TN tumors with elevated MYC expression in patient-derived tumor xenograft (PDX) and MYC-driven transgenic mouse models of breast cancer by inhibiting the oncogenic transcriptional activity of MYC and restoring the function of the endogenous cell cycle inhibitor, p27. Our findings warrant clinical evaluation of PIM kinase inhibitors in patients with TN tumors that have elevated MYC expression.

Summary: Tumors that overexpress the MYC oncogene are frequently aneuploid, a state associated with highly aggressive cancers and tumor evolution. However, how MYC causes aneuploidy is not well understood. Here, we show that MYC overexpression induces mitotic spindle assembly defects and chromosomal instability (CIN) through effects on microtubule nucleation and organization. Attenuating MYC expression reverses mitotic defects, even in established tumor cell lines, indicating an ongoing role for MYC in CIN. MYC reprograms mitotic gene expression, and we identify TPX2 to be permissive for spindle assembly in MYC-high cells. TPX2 depletion blocks mitotic progression, induces cell death, and prevents tumor growth. Further elevating TPX2 expression reduces mitotic defects in MYC-high cells. MYC and TPX2 expression may be useful biomarkers to stratify patients for anti-mitotic therapies. Our studies implicate MYC as a regulator of mitosis and suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution. : Rohrberg et al. identify a reversible role of the MYC oncogene for inducing chromosomal instability by inducing error-prone mitosis. MYC-high tumor cells rely on the mitotic regulator TPX2 to survive the altered mitotic program, revealing a synthetic-lethal interaction between MYC overexpression and TPX2 loss as a potential therapeutic strategy. Keywords: MYC, TPX2, mitotic spindle assembly, chromosomal instability, CIN, mitosis, microtubules, synthetic-lethality, receptor triple-negative breast cancer, TNBC

The MYC oncogene promotes tumorigenesis in part by facilitating cell cycle entry thus driving cellular proliferation. Tumors that overexpress MYC frequently demonstrate aneuploidy, numerical chromosome alterations associated with highly aggressive cancers, rapid tumor evolution, and poor patient outcome. While the role of MYC in overcoming the G1/S checkpoint is well established, it remains poorly understood whether MYC induces chromosomal instability (CIN). Here, we identify a direct influence of MYC on mitotic progression. MYC overexpression induces defects in microtubule nucleation and spindle assembly promoting chromosome segregation defects, micronuclei and CIN. We examined which mitotic regulators are required for the survival of MYC-overexpressing cells and found a reliance on high TPX2 expression. TPX2, a master microtubule regulator, is overexpressed together with MYC in multiple cell lines, in mouse tumor models and in aggressive human breast cancers. High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in cells with deregulated MYC, whereas TPX2 depletion blocks mitotic progression, induces cell death and prevents tumor growth. Importantly, attenuation of MYC expression reverses the mitotic defects observed, even in established tumor cell lines, implicating an ongoing role for high MYC in the persistence of a CIN phenotype in tumors. Here, we implicate the MYC oncogene as a regulator of spindle assembly and dynamics and identify a new MYC-TPX2 synthetic-lethal interaction that could represent a future therapeutic strategy in MYC-overexpressing cancers. Our studies suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution.

Tumors that overexpress the MYC oncogene frequently demonstrate aneuploidy, numerical chromosome alterations associated with highly aggressive cancers, rapid tumor evolution, and poor patient outcome. Here, we identify that MYC overexpression induces reversible defects in microtubule nucleation and mitotic spindle assembly, promoting chromosome segregation defects, micronuclei and chromosomal instability (CIN). High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in cells with MYC overexpression; whereas TPX2 depletion blocks mitotic progression, induces cell death and prevents tumor growth. Attenuating MYC expression reverses mitotic defects, even in established tumor cell lines, implicating an ongoing role for high MYC in the persistence of CIN in tumors. Our studies implicate the MYC oncogene as a regulator of spindle assembly and identify a new MYC-TPX2 synthetic-lethal interaction that could represent a future therapeutic strategy in MYC-overexpressing cancers. Moreover, our studies suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution.

Purpose Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is often resistant to conventional therapies. The MYC oncogene is commonly overexpressed in AML but has remained an elusive target. We aimed to examine the consequences of targeting MYC both directly and indirectly in AML overexpressing MYC/Myc due to trisomy 8/15 (human/mouse), FLT3-ITD mutation, or gene amplification. Methods We performed in vivo knockdown of Myc (shRNAs) and both in vitro and in vivo experiments using four drugs with indirect anti-MYC activity: VX-680, GDC-0941, artemisinin, and JQ1. Results shRNA knockdown of Myc in mice prolonged survival, regardless of the mechanism underlying MYC overexpression. VX-680, an aurora kinase inhibitor, demonstrated in vitro efficacy against human MYC-overexpressing AMLs regardless of the mechanism of MYC overexpression, but was weakest against a MYC-amplified cell line. GDC-0941, a PI3-kinase inhibitor, demonstrated efficacy against several MYC-overexpressing AMLs, although only in vitro. Artemisinin, an antimalarial, did not demonstrate consistent efficacy against any of the human AMLs tested. JQ1, a bromodomain and extra-terminal bromodomain inhibitor, demonstrated both in vitro and in vivo efficacy against several MYC-overexpressing AMLs. We also confirmed a decrease in MYC levels at growth inhibitory doses for JQ1, and importantly, sensitivity of AML cell lines to JQ1 appeared independent of the mechanism of MYC overexpression. Conclusions Our data support growing evidence that JQ1 and related compounds may have clinical efficacy in AML treatment regardless of the genetic abnormalities underlying MYC deregulation. Electronic supplementary material The online version of this article (doi:10.1007/s00280-015-2766-z) contains supplementary material, which is available to authorized users.

Genomic analyses of patient tumors have unearthed an overwhelming number of recurrent somatic alterations in genes that undoubtedly have dramatic effects on breast tumor biology, clinical outcomes and responses to therapeutics. In triple negative breast cancer (TNBC), there is an urgent need to translate this emerging patient genomic data into new therapeutic paradigms. To develop genotype-specific therapies, we have established an isogenic cell-line drug screen that measures the impact of gene activation on a panel of emerging, clinically relevant compounds targeting a variety of cancer pathways. We seek to uncover genotype-dependent sensitivity to drugs, synthetic lethal relationships, and validate in breast cancer cells and in vivo mouse models. We focused on emerging compounds that are already approved or in testing for human use and we expect that this work will serve as a prelude to one or more clinical trials in TNBC. RESULTS: We have developed a synthetic-lethal interaction mapping strategy to uncover the impact of gene activation on responses to a panel of emerging therapeutics. We believe that this approach can identify core synthetic lethal interactions which underlie drug sensitivity and can be used as a foundation to identify patient populations that will selectively respond to drug treatments. We have developed a high-throughput screening platform that can rapidly and accurately assess the proliferative capacity of cell lines in response to a panel of inhibitors. We have generated a panel of isogenic cell lines which closely mimic the impact of recurrent gene mutations and overexpression common in breast cancer. We have currently generated 23 cell lines expressing wild-type and/or mutant forms of these genes. To date we have screened 12 known oncogenes against our panel of 94 emerging therapeutic compounds. Using this systems approach, the resulting interaction map highlights both known and novel connections between oncogene activation and drug responses and provides a modular roadmap for the exploration of synthetic lethal relationships. Based on the coordinate activity of multiple drugs targeting the same pathway, we identified oncogenes that can drive resistance to EGFR inhibitors and PI3K/AKT inhibitors. We also recapitulated a known synthetic lethal relationship between MYC and CDK inhibition which we have previously described and we are now evaluating in a clinical trial. Therefore, our approach identified both resistance and synthetic lethal relationships that have been independently described, indicating that this platform is a good model for predicting synthetic lethality in vivo. Several novel and previously uncharacterized connections were also uncovered, including novel connections between MYC activity and several promising drugs in clinical development. Synthetic-lethal interactions discovered using this chemical-genetic systems approach and their mechanistic validation will be described in the presentation. Furthermore, the important role of patient advocacy in developing this project will be described. CONCLUSION: A novel systems biology approach that uses chemical-genetic maps of oncogenes and emerging therapeutics can define synthetic-lethal interactions and actionable therapeutics for breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-10-01.

INTRODUCTION: Metastatic prostate cancer is a lethal disease. Many patients develop a form of the disease resistant to Androgen Deprivation Therapy (ADT) called castration resistant prostate cancer (CRPC), and androgen refractory tumors emerge. A subset of these involve gaining small cell and neuroendocrine (NE) features, resulting in neuroendocrine prostate cancer (NEPC). The identification of key drivers and the mechanism of emergence is critical, as NEPC is associated with poor outcomes. Of interest is the neuron lineage differentiation gene achaete-scute complex homologue 1 (ASCL1). Mouse models of small cell lung cancer (SCLC) show ASCL1 as a key promoter of tumor growth in vivo. While there is overlap between SCLC and NEPC (inactivation of RB1 and TP53, ASCL1 overexpression (OE)), it's unclear if ASCL1 contributes to transdifferentiation of prostate epithelium into NEPC. In addition to genetic drivers, defined soluble factors can promote reprogramming of fibroblasts into neurons. We thus hypothesize that ASCL1 in combination with soluble factors may promote transdifferentiation of prostate epithelium to NEPC. METHODS: CRISPR technology is used to knockout (KO) RB1 and TP53 in LNCaP cells. Lentiviral vector is used to OE for ASCL1 or RFP in LNCaP or LNCap p53/RB1 KO cells. Gene expression for AR, AR target genes, neuronal, and NE genes is measured via qRT-PCR and Western. Regular FBS or neuronal media containing either FGF2/BDNF/NTF3 or all soluble factors is used for cell culture. RESULTS: RNAseq data from metastatic AR+ CRPC, AR- NEPC, and SCLC tumors and cell lines shows OE of ASCL1 in AR- NEPC and SCLC. LNCaP ASCL1 OE cells show no change in cellular growth in FBS, CSS, or NM + supplements (BDNF, FGF2, NT3) compared to control. However, LNCaP p53/RB1 KO ASCL1 OE cells + 10μM Enzalutamide (ENZ) proliferated similar to the cells without any drug treatment when cultured in NM + sup (96h, p=0.999). Both LNCaP ± ASCL1 and LNCaP p53/RB1 KO ± ASCL1 cells grown in NM + sup show significantly decreased AR and AR target gene expression (~10 fold, p< 0.05). FGF8 (~5 fold), SOX2 (>300 fold), and SYP (>10 fold) gene expression increased significantly (p CONCLUSION: Loss of TP53 and RB1 are not sufficient to induce transdifferentiation to a NEPC state, but ASCL1 OE induced the expression of SOX2 and SYP that may trigger the process. Microenvironment effects operating through paracrine factors induced the expression of a NE gene program and reduced AR expression. Future studies will investigate the role of ASCL1 in promoting resistance to ENZ in prostate adenocarcinoma cells as well as its role in in vivo tumor growth. ASCL1, NEUROD1, and MYTL1 that are OE in NEPC and SCLC in combination with individual soluble factors will be used to delineate the mechanism required for the transdifferentiation process. Citation Format: Seema Sinha, Michael D. Nyquist, Alexandra Corella, Ilsa Coleman, Peter S. Nelson. Role of ASCL1 in neuroendocrine prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-199.

More than 70% of common solid tumors are aneuploid. Aneuploidy has been implicated in tumorigenesis for decades. A direct cause of aneuploidy is chromosomal instability (CIN) that fuels tumor evolution by providing a heterogeneous subpopulation of cells that contributes to the lethal outcome of cancer, metastasis, therapeutic failure, and drug resistance. The oncogene c-MYC is overexpressed in many cancers and has been shown to induce structural and numerical chromosomal aberrations and aneuploidy. While the role of MYC in overcoming the G1/S checkpoint is well established, the role of MYC in altering mitotic dynamics and eliciting CIN remains poorly understood. Here, we identify a direct influence of increased MYC expression on mitotic progression and spindle assembly. We observe MYC-dependent microtubule nucleation and polymerization defects in mitosis. MYC overexpression induces altered spindle morphology promoting chromosome segregation defects resulting in micronuclei formation, CIN and aneuploidy. Importantly, attenuation of MYC expression reverses the mitotic defects observed, even in established tumor cell lines, implicating an ongoing role for MYC in the persistence of a CIN phenotype in tumors. Gene expression data reveal that MYC regulates numerous genes involved in mitosis, kinetochore function and microtubule behavior. We performed a screen to identify microtubule regulators required for the survival of MYC overexpressing cells. MYC overexpressing cells are reliant on high TPX2 expression, a master microtubule regulator. High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in MYC high cells, while TPX2 depletion attenuates cell viability and tumor growth in vivo. Our data suggest that TPX2 is a transcriptional target of MYC. We postulate that high levels of TPX2 are required to tolerate mitotic stress induced by MYC. TPX2 compensates for impaired mitotic progression and prevents the development of physiologically intolerable levels of CIN. High levels of TPX2 might be required for tumors with oncogene-induced mitotic stress to faithfully proceed through mitosis providing an explanation for the abundant upregulation of TPX2 in cancer. Thus, we describe a conditional role for MYC in altering spindle assembly and dynamics, resulting in a dependency on TPX2 for tumor survival. Citation Format: Julia Rohrberg, Alexandra Corella, Moufida Taileb, Marie-Lena Jokisch, Roman Camarda, Sanjeev Balakrishnan, Alicia Zhou, Andrei Goga. The oncogene MYC induces chromosomal instability through dysregulating mitotic progression evoking a dependency on TPX2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1462.

There is an urgent need in oncology to link molecular aberrations in tumors with therapeutics that can be administered in a personalized fashion. One approach identifies synthetic–lethal genetic interactions or dependencies that cancer cells acquire in the presence of specific mutations. Using engineered isogenic cells, we generated a systematic and quantitative chemical–genetic interaction map that charts the influence of 51 aberrant cancer genes on 90 drug responses. The dataset strongly predicts drug responses found in cancer cell line collections, indicating that isogenic cells can model complex cellular contexts. Applying this dataset to triple-negative breast cancer, we report clinically actionable interactions with the MYC oncogene, including resistance to AKT–PI3K pathway inhibitors and an unexpected sensitivity to dasatinib through LYN inhibition in a synthetic lethal manner, providing new drug and biomarker pairs for clinical investigation. This scalable approach enables the prediction of drug responses from patient data and can accelerate the development of new genotype-directed therapies. Significance: Determining how the plethora of genomic abnormalities that exist within a given tumor cell affects drug responses remains a major challenge in oncology. Here, we develop a new mapping approach to connect cancer genotypes to drug responses using engineered isogenic cell lines and demonstrate how the resulting dataset can guide clinical interrogation. Cancer Discov; 5(2); 154–67. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 97

Summary Androgen receptor (AR) signaling is a distinctive feature of prostate carcinoma (PC) and represents the major therapeutic target for treating metastatic prostate cancer (mPC). Though highly effective, AR antagonism can produce tumors that bypass a functional requirement for AR, often through neuroendocrine (NE) transdifferentiation. Through the molecular assessment of mPCs over two decades, we find a phenotypic shift has occurred in mPC with the emergence of an AR-null NE-null phenotype. These "double-negative" PCs are notable for elevated FGF and MAPK pathway activity, which can bypass AR dependence. Pharmacological inhibitors of MAPK or FGFR repressed the growth of double-negative PCs in vitro and in vivo . Our results indicate that FGF/MAPK blockade may be particularly efficacious against mPCs with an AR-null phenotype.

202 Background: Recent clinical studies have taken advantage of the dichotomous ability of androgen receptor signaling to elicit growth-suppressive and differentiating effects by giving hyper-physiological levels of testosterone. This approach has demonstrated safety, improved quality of life, and met primary endpoints for efficacy in clinical trials; however, novel strategies to enhance and optimize the anti-tumor efficacy of this approach are needed. Methods: We performed high-throughput drug screening in PCa cell lines to identify agents showing synergy with high-dose androgen treatment. Results: The Survivin inhibitor YM155, demonstrated a potent synergy with high-dose androgen. YM155 IC50 values shifted from 24 nM without R1881 to 3.85 nM with 160 pM R1881.Using qRT-PCT and ChIP-qPCR we establish the mechanism of this synergy was due to the direct upregulation of YM155 transporter SLC35F2 by androgen receptor transcriptional activity. Knockdown and overexpression of SLC35F2 dramatically modulated sensitivity to YM155. In two xenograft models SLC35F2 and tumor DHT levels are highly correlated and castration-induced decreases in tumor androgens cause an identical decline in levels of SLC35F2. In CRPC metastases SLC35F2 expression significantly correlated with AR activity score (r = 0.62, p < 0.001). However, ~1/3 of tumors examined had transcripts for AR and SLC35F2, 1/3 AR+ but SLC35F2 negative, and 1/3 are negative for both. Conclusions: We describe a novel synergy between YM155 and high-dose androgen therapy mediated by the direct upregulation of YM155 transporter SLC35F2 by androgen receptor and implicate SLC35F2 expression as a potential biomarker for YM155 sensitivity. Whether ligand independent AR variants (AR-Vs) can constitutively mediate SLC35F2 expression is unknown, but if so would also provide a means of stratifying identifying men likely to respond to YM155.This study demonstrates a novel paradigm for precision medicine in the pharmacologic induction of drug sensitivity in a tissue restricted manner. Hormonal induction of carrier-mediated drug transport is a completely novel and unexplored concept that is poised to exploit androgen-based treatment strategies.

Background: Collaborative team science provides a starting point for comprehensive change, and advocates have a unique and important role developing and engaging in transdisciplinary collaboratives that focus on new questions and new possibilities to advance the science of ethnic and medically underserved health care disparities. Participating in four areas : 1) research and programmatic support, 2) education and outreach, 3) policy and strategy, and 4) representation and advisory, the UCSF Breast Science Advocacy Core (BSAC) Program, a volunteer affiliate of the Breast Oncology Program (BOP), one of ten multidisciplinary research programs under the umbrella of the UCSF Helen Diller Comprehensive Cancer Center promotes a transformative, transdisciplinary, integrated environment to study the biological basis of the diseases that comprise breast cancer; to define the risk of developing or progressing with specific types of breast cancer; to develop novel interventions that work locally and globally to reduce morbidity and mortality from breast cancer and its treatment; and to leverage new collaborative research, education, and mentoring/training opportunities that address cancer outcome disparities. Advocates involved in KOMEN, DOD, PCORI, AND CBCRP funded research and training grants apply four core principles that forge synergy with NCI Advocacy Research Working Group Recommendations: 1)strategic innovation, 2)collaborative execution, 3)evidence based decision-making, and 4) ethical codes of conduct. Embracing transdisciplinary professionalism, researchers and advocates build on their track record as shared value partners committed to furthering the collective impact of science advocacy exchange (SAE). Study Objectives: Genomic analyses of patient tumors have unearthed an overwhelming number of recurrent somatic alterations in genes that have dramatic effects on tumor biology, patient drug responses, and clinical outcomes. In one study, high grade triple negative breast cancer (TNBC) accounts for 34% of breast cancers in African American women versus 21% in white women. A growing body of evidence has shown that African American women have biologically more aggressive disease, independent of social determinants, and suffer the highest mortality rates. While biological breakthroughs of the last decade have greatly advanced our understanding of cancer, in advanced TNBC, a poor prognosis subtype, there is an urgent need to translate this evolving patient genomic data into new therapeutic paradigms. Our study focuses on the intersection of synthetic lethal approaches, MYC driven human cancers, and immunotherapy as an “innovation agenda”. A distinct MYC vision highlights how overexpression is associated with aggressive outcomes and poor patient outcomes, and synthetic lethal strategies to target MYC (CDK inhibitors, PIM2, as well as the PDI immune pathways) have potential for addressing outcome disparities In African American Women with Triple Negative Breast Cancer (TNBC). Key Findings: We have developed a screening technique that can be used to rapidly and accurately identify potential synthetic lethal interactions in TNBC. This platform utilizes an isogenic cell line system that we have developed to model oncogene activation in TNBC. A growing body of evidence has shown that: 1) Quantitative approach maps genotype-specific drug responses in isogenic cells 2) Systematic discovery of biomarkers for cancer drugs under clinical investigation 3) Clinically actionable synthetic lethal interaction between MYC and dasatinib is discovered 4) Mechanism of dasatinib action through inhibition of LYN kinase is described Key Take-Away Message: The inclusion of advocates in convergent science settings remind academic stakeholders that research is there to benefit the patient as they attempt to spark innovation, democratize science, and support smarter interventions that expedite the incredible potential of future investments in bioscience within disparities arenas. Citation Format: Susan Samson, Alicia Y. Zhou, Maria Martins, Alexandra Corella, Dai Horiuchi, Christina Yau, Taha Rakshandehroo, John Gordan, Rebecca Levin, Jeff Johnson, John Jascur, Mike Shales, Antonio Sorrentino, Jaime Cheah, Paul Clemens, Alykhan Shamji, Stuart Schreiber, Nevan Krogan, Kevan Shokat, Frank McCormick, Sourav Bandyopadhyay, Andrei Goga. Genomics, advocacy, and emerging therapeutics to address triple-negative breast cancer (TNBC) outcome disparities. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C88.

The greatest clinical challenge in treating breast cancer occurs in those patients whose tumors lack expression of the estrogen and progesterone receptors and that of the HER2 oncoprotein. No targeted therapeutic strategies currently exist against this aggressive type of “triple negative” breast cancer (TNBC) due to lack of validated targets. We previously found that MYC mRNA, protein, and its signaling were disproportionally elevated in TN compared to receptor positive (RP) breast cancer. We sought to take advantage of the unique molecular feature found in this tumor type to identify potent and effective treatment strategies. Since MYC is an oncogenic transcription factor, rationally designed small molecule inhibitors that can directly inhibit its activity are not available for clinical use. An alternative approach to selectively kill MYC-driven tumors is to inhibit those proteins that are indispensable for the viability of such tumors, but are not essential in non-tumorigenic cells. This form of “indirect” treatment strategy has become known as the “synthetic-lethal” approach. To identify novel targets that are readily druggable for treating MYC-driven TNBC, we conducted a kinome MYC synthetic lethal shRNA screen in non-immortalized human mammary epithelial cells expressing a 4-hydroxytamoxifen (TAM)-activatable MycER transgene (HMEC-MycER). Of 600 human kinases targeted by 2,000 individual shRNA clones, 9 kinases were identified as hits as they were essential specifically for the MYC-activated HMEC cells. Among these hits, we focused on PIM1, a non-essential kinase, the knock-down of which had the greatest efficacy in causing cell death in the MYC-activated cells and had minimum inhibitory effect on the growth of the control cells. We determined that PIM1 expression was elevated in TN tumors and was associated with poor prognosis specifically in patients with hormone receptor-negative tumors. Small molecule PIM kinase inhibitors halted the growth of human TN tumors with elevated MYC expression in a patient-derived tumor xenograft (PDX) mouse model by inhibiting oncogenic transcriptional activity of MYC while simultaneously restoring the function of the endogenous cell cycle inhibitor p27. Thus, our findings warrant clinical evaluation of small molecule PIM kinase inhibitors in patients with TN tumors that exhibit elevated MYC expression. Note: This abstract was not presented at the conference. Citation Format: Dai Horiuchi, Alicia Y. Zhou, Alexandra N. Corella, Christina Yau, Sanjeev Balakrishnan, Kai Kessenbrock, Devon A. Lawson, Roman Camarda, Brittany N. Anderton, Alexey V. Bazarov, Henok Eyob, Julia Rohrberg, Paul Yaswen, Michael T. McManus, Hope S. Rugo, Zena Werb, Andrei Goga. PIM kinase as a novel therapeutic target for triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B34.

There is an urgent need in oncology to link molecular aberrations in tumors with altered cellular behaviors, such as metabolic derangements, and to identify novel therapeutics for cancer treatment. We have sought to identify synthetic-lethal genetic interactions that cancer cells acquire in the presence of specific mutations. Using engineered isogenic cells, we generated an unbiased and quantitative chemical-genetic interaction map that measures the influence of 51 aberrant cancer genes on 90 drug responses. The dataset strongly predicts drug responses found in cancer cell line collections, indicating that isogenic cells can model more complex cellular contexts. Applied to triple-negative breast cancer, we report clinically actionable interactions with the MYC oncogene including resistance to PI3K/AKT pathway inhibitors and an unexpected sensitivity to dasatinib through LYN inhibition in a synthetic-lethal manner. These studies provide new drug and biomarker pairs for clinical investigation. We have also performed global metabolomics analysis in a subset of the isogenic cell lines demonstrating alterations in metabolic pathways that are shared across multiple oncogenes, as well as those that are distinct to specific oncogenic drivers. This scalable approach enables the prediction of drug responses from patient data and can be used to accelerate the development of new genotype-directed therapies. This abstract is also presented as a poster at the Translation of the Cancer Genome conference. Citation Format: Maria M. Martins, Alicia Y. Zhou, Alexandra Corella, Dai Horiuchi, Christina Yau, Taha Rakshandehroo, John D. Gordan, Rebecca S. Levin, Jeff Johnson, John Jascur, Mike Shales, Antonio Sorrentino, Jaime Cheah, Paul A. Clemons, Alykhan Shamji, Stuart Schreiber, Stuart Schreiber, Nevan J. Krogan, Kevan M. Shokat, Kevan M. Shokat, Frank McCormick, Daniel Nomura, Sourav Bandyopadhyay, Andrei Goga. Functional analysis of diverse oncogenic driver mutations using an isogenic cell line library identifies novel drug responses and alterations in metabolism. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR07.

There is an urgent need in oncology to link molecular aberrations in tumors with therapeutics that can be administered in a personalized fashion. One approach identifies synthetic-lethal genetic interactions or emergent dependencies that cancer cells acquire in the presence of specific mutations. Using engineered isogenic cells, we generated an unbiased and quantitative chemical-genetic interaction map that measures the influence of 51 aberrant cancer genes on 90 drug responses. The dataset strongly predicts drug responses found in cancer cell line collections, indicating that isogenic cells can model more complex cellular contexts. Applied to triple-negative breast cancer, we report clinically actionable interactions with the MYC oncogene including resistance to AKT/PI3K pathway inhibitors and an unexpected sensitivity to dasatinib through LYN inhibition in a synthetic-lethal manner, providing new drug and biomarker pairs for clinical investigation. This scalable approach enables the prediction of drug responses from patient data and can be used to accelerate the development of new genotype-directed therapies. Citation Format: Alicia Y. Zhou, Maria M. Martins, Alexandra Corella, Dai Horiuchi, Christina Yau, Taha Rakshandehroo, John D. Gordan, Rebecca S. Levin, Jeff Johnson, John Jascur, Mike Shales, Antonio Sorrentino, Jaime Cheah, Paul A. Clemons, Alykhan Shamji, Stuart L. Schreiber, Nevan J. Krogan, Kevan M. Shokat, Frank McCormick, Andrei Goga, Sourav Bandyopadhyay. Identification of novel drug interactions with MYC via a quantitative chemical-genetic interaction map. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B48.

Background: Genomic analyses of patient tumors have unearthed an overwhelming number of recurrent somatic alterations in genes that have dramatic effects on tumor biology, patient drug responses, and clinical outcomes. In one study, high-grade triple negative breast cancer (TNBC) accounts for 34% of breast cancers in African American women versus 21% in white women. African American women have biologically more aggressive disease, independent of social determinants, and suffer the highest mortality rates. In advanced TNBC, a poor prognosis subtype, there is an urgent need to translate this emerging patient genomic data into new therapeutic paradigms. Objectives: Our study focuses on emerging compounds that are already approved (i.e., Dasatinib) or in testing for human use and we expect that this work will serve as a prelude to one or more clinical trials in TNBC. We seek to determine if the treatment of metastatic TNBC recurrence with more targeted genotype-specific agents could improve the outcomes/survival of all women in this particularly aggressive poor prognosis subset, including African American women. Methods: To guide the development of genotype-specific therapies in TNBC, we have established an isogenic cell-line drug screen that measures the impact of gene activation on a panel of emerging, clinically relevant compounds targeting a variety of cancer pathways. Using engineered isogenic cells, we generated an unbiased and quantitative chemical-genetic interaction map that measures the influence of 51 aberrant cancer genes on 90 drug responses. We believe that this approach can identify core synthetic lethal interactions, which underlie drug sensitivity and can be used as a foundation to identify patient populations that will selectively respond to drug treatments. Results: Using our systems approach, our interaction map highlights both known and novel connections between oncogene activation and drug responses and provides a modular roadmap for the exploration of synthetic lethal relationships. Applied to triple-negative breast cancer, we report clinically actionable interactions with the MYC oncogene including resistance to AKT/PI3K pathway inhibitors and an unexpected sensitivity to dasatinib through LYN inhibition in a synthetic-lethal manner. Ensuring that the voice of the patient is represented in our scientific inquiry, advocacy has played a significant role in the development and realization of this project. Aligning experiential and professionalized expertise, trained advocates explore relentless challenges and opportunities for moving the science forward. Conclusion: A novel systems biology approach that uses module maps of oncogenes and emerging therapeutics can define synthetic-lethal interactions and actionable therapeutics to help decrease TNBC outcomes/survival disparities in African American women. Citation Format: Alicia Y. Zhou, Maria M. Martins, Alexandra Corella, Dai Horiuchi, Christina Yau, Taha Rakshandehroo, John D. Gordan, Rebecca S. Levin, Jeff Johnson, John Jascur, Mike Shales, Antonio Sorrentino, Jaime Cheah, Paul A. Clemons, Alykhan Shamji, Stuart Schreiber, Nevan J. Krogan, Kevan M. Shokat, Frank McCormick, Susan Samson, Andrei Goga, Sourav Bandyopadhyay. A systems approach combining genomics, advocacy, and emerging novel therapeutics to address triple-negative breast cancer (TNBC) outcomes disparities. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B44.

The proto-oncogene MYC affects diverse cellular processes ranging from cell proliferation, metabolism, to pluripotency of embryonic stem cells. While de-regulated MYC signaling has been commonly identified in a variety of human malignancies, no therapeutic strategies have been clinically established to specifically target tumors that present elevated MYC expression. We have previously reported that triple-negative (TN) breast cancers, which lack the expression of predictive biomarkers of response (i.e., estrogen/progesterone receptors, human epidermal growth factor receptor 2), exhibit significantly elevated MYC expression as well as MYC pathway activation (Horiuchi, D., et al. 2012 JEM). To understand the vulnerabilities of TN tumors, we performed a kinome synthetic-lethal shRNA screen in human mammary epithelial cells in which MYC activity can be controlled (i.e., HMEC-MycER cells). The screen yielded 10+ potential synthetic lethal partners of MYC, including recently described AMPK-related kinase 5 (Liu, L., et al. 2012. Nature). One of our top hits was the proto-oncogene PIM1, a non-essential serine/threonine kinase previously shown to be a genetic enhancer of MYC in transgenic mouse models of lymphomas and prostate cancer. Our bioinformatics studies performed on four independent clinical cohorts (n: 146 ∼ 683 patients each) showed that in all cohorts both MYC and PIM1 were significantly enriched in the TN populations. PIM1 expression was correlated with that of MYC in three out of the four cohorts. Furthermore, we found that PIM1 overexpression alone was a poor prognostic factor associated with diminished recurrence-free survival. Our in vivo efficacy studies using conventional as well as novel “human-in-mouse,” patient derived orthotopic xenograft (PDX) models demonstrated that PIM was essential for the growth of MYC-overexpressing human TN tumors. In the PDX tumors, PIM inhibition nearly completely arrested tumor growth, which was associated with significant up-regulation of an endogenous cell cycle inhibitor p27, a known PIM1 substrate, down-regulation of protein synthesis machinery, and inhibition of MYC activity itself. These observations held true in a panel of breast cancer cell lines, and elevating the p27 expression alone was sufficient to inhibit cancer cell proliferation. Thus, our findings warrant the use of PIM kinase inhibitors in treating TN tumors that exhibit the elevated expression of MYC and/or PIM. Citation Format: Dai Horiuchi, Alicia Y. Zhou, Alexandra N. Corella, Christina Yau, Devon A. Lawson, Alexey V. Bazarov, Paul Yaswen, Michael T. McManus, Zena Werb, Alana L. Welm, Andrei Goga. PIM1 kinase inhibition halts the growth of MYC-overexpressing triple-negative breast tumors. [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 LB-122. doi:10.1158/1538-7445.AM2014-LB-122

While de-regulated MYC signaling has been identified in a wide variety of human malignancies, no targeted therapeutic strategies have been established to target tumors with elevated MYC expression. We have previously reported that receptor “triple negative” (TN) breast cancers, which lack predictive biomarkers of response (i.e., estrogen/progesterone receptors, human epidermal growth factor receptor 2), exhibit significantly elevated MYC expression as well as MYC pathway activation (Horiuchi, D., et al. 2012 JEM). In an effort to uncover the potential Achilles’ heels of TN tumors, we performed a kinome, synthetic-lethal shRNA screen in human mammary epithelial cells in which MYC activity can be controlled (i.e., HMEC-MycER cells). The screen yielded 13 potential synthetic lethal partners of MYC, including recently described AMPK-related kinase 5 (Liu, L., et al. 2012. Nature). One of our top hits is the proto-oncogene PIM1, previously shown by others to be a genetic enhancer of MYC in transgenic mouse models of lymphomas and prostate cancer. Our bioinformatics studies performed on four independent clinical cohorts (n: 146 ∼ 683 patients each) show that in all the cohorts both MYC and PIM1 are significantly enriched in the TN populations. MYC and PIM1 expression are correlated in three out of the four cohorts. Furthermore, we find that PIM1 overexpression is a poor prognostic factor associated with diminished recurrence-free survival. Our in vivo efficacy studies using conventional as well as novel “human-in-mouse” orthotopic xenograft models demonstrate that PIM1 is required for the growth of MYC-overexpressing human TN tumors. We are currently investigating the cellular mechanisms by which PIM kinase inhibition causes such growth defects. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B232. Citation Format: Dai Horiuchi, Alicia Y. Zhou, Alexandra N. Corella, Christina Yau, Devon A. Lawson, Alexey V. Bazarov, Paul Yaswen, Michael T. McManus, Alana L. Welm, Zena Werb, Andrei Goga. PIM1 kinase is essential for the growth of MYC-overexpressing triple-negative breast tumors and is an efficacious therapeutic target. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B232.