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Supplementary Figures 1-6

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Myeloid cell receptor tyrosine kinases TYRO3, AXL, and MERTK and their ligands, GAS6 and PROTEIN S, physiologically suppress innate immune responses, including in the tumor microenvironment. Here, we showed that myeloid-derived suppressor cells (MDSC) dramatically upregulated TYRO3, AXL, and MERTK and their ligands [monocytic MDSCs (M-MDSC)>20-fold, polymorphonuclear MDSCs (PMN-MDSC)>15-fold] in tumor-bearing mice. MDSCs from tumor-bearing Mertk−/−, Axl−/−, and Tyro3−/− mice exhibited diminished suppressive enzymatic capabilities, displayed deficits in T-cell suppression, and migrated poorly to tumor-draining lymph nodes. In coimplantation experiments using TYRO3−/−, AXL−/−, and MERTK−/− MDSCs, we showed the absence of these RTKs reversed the protumorigenic properties of MDSCs in vivo. Consistent with these findings, in vivo pharmacologic TYRO3, AXL, and MERTK inhibition diminished MDSC suppressive capability, slowed tumor growth, increased CD8+ T-cell infiltration, and augmented anti–PD-1 checkpoint inhibitor immunotherapy. Mechanistically, MERTK regulated MDSC suppression and differentiation in part through regulation of STAT3 serine phosphorylation and nuclear localization. Analysis of metastatic melanoma patients demonstrated an enrichment of circulating MERTK+ and TYRO3+ M-MDSCs, PMN-MDSCs, and early-stage MDSCs (e-MDSC) relative to these MDSC populations in healthy controls. These studies demonstrated that TYRO3, AXL, and MERTK control MDSC functionality and serve as promising pharmacologic targets for regulating MDSC-mediated immune suppression in cancer patients.

Supplemental cell lines list

Evaluation of AXL inhibitor R428.

Supplemental Figure 2 shows impact of UNC2025 on colony forming potential.

Supplemental Figure 4 shows TAM-family kinase expression in melanoma cell lines.

PDF - 164KB, Jurkat and 697 cell cultures were treated with UNC569 (500 nM) for 1 hour. Pervanadate was added to cell cultures for 3 minutes to stabilize the phosphorylated form of Mer. Cells were harvested after 24 and after 48 hours. Mer was immunoprecipitated from cell lysates and total Mer protein and Mer phosphoprotein (p-Mer) were detected by western blot.

Supplemental Figure 7 shows combined UNC2025 and vemurafenib treatment is tolerated in mice.

Supplemental Table 1: Concentration of UNC2025 in Mouse Serum and Tumors.

PDF - 172KB, Jurkat cell cultures were treated with the indicated concentrations of UNC569 for 1 hour. Pervanadate was added to cell cultures for 3 minutes to stabilize the phosphorylated form of Tyro3. Tyro3 was immunoprecipitated from cell lysates and total Tyro3 protein and Tyro3 phosphoprotein (p-Tyro3) were detected by western blot. Numbers on the right indicate the location of molecular weight (kD) markers. Immunoblots are representative of three independent experiments.

PDF - 294KB, TAM receptor expression in Jurkat cell line was detected by western blot analysis. Whole cell lysates were prepared and phosphorylated (denoted by p-) and total proteins were detected. Western blots representative of three independent experiments are shown. Blots were stripped and reprobed with anti-tubulin antibody to confirm similar protein loading.

PDF - 634KB, Eighteen fish (Myc1-Myc18) were treated with UNC569 for 2 weeks. Animals were imaged pre- and post-treatment, with GFP intensities quantified as described in the text. Ten of 18 fish (55.6%; Myc1-Myc8, Myc10, Myc16) showed >50% regressions. Six fish (33.3%; Myc9, Myc11-15) had 25-50% responses, and 2/18 (11.1%; Myc17, Myc18) progressed on treatment. Average diminution in tumor burden across the entire cohort was 47.8%. Four images are shown for each animal: Day 0 columns shows the raw image and GFP intensity plot for each fish prior to UNC treatment. Day 14 columns show post-treatment images and intensity plots. GFP Score day 14 columns show final responses, depicted as % of original cancer remaining.

Supplemental Figures 6 shows efficacy of UNC2025 in a melanoma wild-type PDX mode.

The TAM (TYRO3, AXL, MERTK) family receptor tyrosine kinases (RTK) play an important role in promoting growth, survival, and metastatic spread of several tumor types. AXL and MERTK are overexpressed in head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and non–small cell lung cancer (NSCLC), malignancies that are highly metastatic and lethal. AXL is the most well-characterized TAM receptor and mediates resistance to both conventional and targeted cancer therapies. AXL is highly expressed in aggressive tumor types, and patients with cancer are currently being enrolled in clinical trials testing AXL inhibitors. In this study, we analyzed the effects of AXL inhibition using a small-molecule AXL inhibitor, a monoclonal antibody (mAb), and siRNA in HNSCC, TNBC, and NSCLC preclinical models. Anti-AXL–targeting strategies had limited efficacy across these different models that, our data suggest, could be attributed to upregulation of MERTK. MERTK expression was increased in cell lines and patient-derived xenografts treated with AXL inhibitors and inhibition of MERTK sensitized HNSCC, TNBC, and NSCLC preclinical models to AXL inhibition. Dual targeting of AXL and MERTK led to a more potent blockade of downstream signaling, synergistic inhibition of tumor cell expansion in culture, and reduced tumor growth in vivo. Furthermore, ectopic overexpression of MERTK in AXL inhibitor–sensitive models resulted in resistance to AXL-targeting strategies. These observations suggest that therapeutic strategies cotargeting both AXL and MERTK could be highly beneficial in a variety of tumor types where both receptors are expressed, leading to improved survival for patients with lethal malignancies. Mol Cancer Ther; 17(11); 2297–308. ©2018 AACR.

Supplemental Figure 3 shows impact of UNC2025 on cell viability.

Treatment of non–small cell lung cancer (NSCLC) has been transformed by targeted therapies directed against molecular aberrations specifically activated within an individual patient's tumor. However, such therapies are currently only available against a small number of such aberrations, and new targets and therapeutics are needed. Our laboratory has previously identified the MERTK receptor tyrosine kinase (RTK) as a potential drug target in multiple cancer types, including NSCLC. We have recently developed UNC2025—the first-in-class small molecule inhibitor targeting MERTK with pharmacokinetic properties sufficient for clinical translation. Here, we utilize this compound to further validate the important emerging biologic functions of MERTK in lung cancer pathogenesis, to establish that MERTK can be effectively targeted by a clinically translatable agent, and to demonstrate that inhibition of MERTK is a valid treatment strategy in a wide variety of NSCLC lines independent of their driver oncogene status, including in lines with an EGFR mutation, a KRAS/NRAS mutation, an RTK fusion, or another or unknown driver oncogene. Biochemically, we report the selectivity of UNC2025 for MERTK, and its inhibition of oncogenic downstream signaling. Functionally, we demonstrate that UNC2025 induces apoptosis of MERTK-dependent NSCLC cell lines, while decreasing colony formation in vitro and tumor xenograft growth in vivo in murine models. These findings provide further evidence for the importance of MERTK in NSCLC, and demonstrate that MERTK inhibition by UNC2025 is a feasible, clinically relevant treatment strategy in a wide variety of NSCLC subtypes, which warrants further investigation in clinical trials. Mol Cancer Ther; 14(9); 2014–22. ©2015 AACR.

Supplemental Figure 3: pMERTK and MERTK are not Predictive of Sensitivity to UNC2025.

Supplemental Figure 1: Lack of Expression of Relevant Potential Off-Target Kinases.Sub-confluent cultures of the H2228, A549, Colo699, and H1299 NSCLC cell lines were lysed.

PDF - 358KB, Cells were plated in methylcellulose in the presence of the indicated concentrations of UNC1653 or vehicle only. Colonies were counted after eight days. Long-term colony growth of Jurkat cells in methylcellulose is shown. Mean values and standard errors were derived from 3 independent experiments.

Supplemental Figure 2: UNC2025 Inhibits Survivin Expression in Various Cancer Types.

1uM CO-1686

Purpose:In this era of precision-based medicine, for optimal patient care, results reported from commercial next-generation sequencing (NGS) assays should adequately reflect the burden of somatic mutations in the tumor being sequenced. Here, we sought to determine the prevalence of clonal hematopoiesis leading to possible misattribution of tumor mutation calls on unpaired Foundation Medicine NGS assays.Experimental Design:This was a retrospective cohort study of individuals undergoing NGS of solid tumors from two large cancer centers. We identified and quantified mutations in genes known to be frequently altered in clonal hematopoiesis (DNMT3A, TET2, ASXL1, TP53, ATM, CHEK2, SF3B1, CBL, JAK2) that were returned to physicians on clinical Foundation Medicine reports. For a subset of patients, we explored the frequency of true clonal hematopoiesis by comparing mutations on Foundation Medicine reports with matched blood sequencing.Results:Mutations in genes that are frequently altered in clonal hematopoiesis were identified in 65% (1,139/1,757) of patients undergoing NGS. When excluding TP53, which is often mutated in solid tumors, these events were still seen in 35% (619/1,757) of patients. Utilizing paired blood specimens, we were able to confirm that 8% (18/226) of mutations reported in these genes were true clonal hematopoiesis events. The majority of DNMT3A mutations (64%, 7/11) and minority of TP53 mutations (4%, 2/50) were clonal hematopoiesis.Conclusions:Clonal hematopoiesis mutations are commonly reported on unpaired NGS testing. It is important to recognize clonal hematopoiesis as a possible cause of misattribution of mutation origin when applying NGS findings to a patient's care.See related commentary by Pollyea, p. 5790

Purpose:Lung cancer is the leading cause of cancer-related death. Non–small cell lung cancer (NSCLC) accounts for 85% of all lung cancers and over 60% express wild-type EGFR (wtEGFR); however, EGFR tyrosine kinase inhibitors (TKIs) have limited effect in most patients with wtEGFR tumors. We previously identified MERTK tyrosine kinase as a potential therapeutic target in NSCLC and developed MRX-2843, a novel MERTK-selective inhibitor with favorable properties for clinical translation. The goal of this study was to determine whether MERTK and EGFR inhibitor combination therapy could provide antitumor efficacy against wtEGFR NSCLC.Experimental Design: An unbiased screen of 378 kinase inhibitors was conducted to identify synergistic interactions with MRX-2843 and biochemical and therapeutic effects were determined in vitro and in vivo.Results:Numerous irreversible EGFR TKIs, including CO-1686 and osimertinib, synergized with MRX-2843 to inhibit wtEGFR NSCLC cell expansion, irrespective of driver oncogene status. CO-1686 and MRX-2843 combination therapy inhibited MERTK, wtEGFR, and ERBB2/ERBB3 and decreased downstream PI3K-AKT, MAPK-ERK, and AURORA kinase (AURK) signaling more effectively than single agents. Inhibition of PI3K, AKT or AURK, but not MEK, synergized with CO-1686 to inhibit tumor cell expansion, suggesting their roles as key redundant resistance pathways. Treatment with MRX-2843 and CO-1686 or osimertinib prevented xenograft growth while single agents had limited effect. Tumor growth inhibition was durable even after treatment with combination therapy was stopped.Conclusions:Our data support the application of MRX-2843 in combination with an irreversible EGFR TKI as a novel strategy for treatment of patients with wtEGFR NSCLC.

Purpose: MERTK tyrosine kinase is ectopically expressed in 30% to 50% of acute lymphoblastic leukemias (ALL) and more than 80% of acute myeloid leukemias (AML) and is a potential therapeutic target. Here, we evaluated the utility of UNC2025, a MERTK tyrosine kinase inhibitor, for treatment of acute leukemia.Experimental Design: Preclinical in vitro and in vivo assays using cell lines and primary leukemia patient samples were used to evaluate antileukemic effects of UNC2025.Results: UNC2025 potently inhibited prosurvival signaling, induced apoptosis, and reduced proliferation and colony formation in MERTK-expressing ALL and AML cell lines and patient samples. Approximately 30% of primary leukemia patient samples (78 of 261 total) were sensitive to UNC2025. Sensitive samples were most prevalent in the AML, T-ALL, and minimally differentiated (M0) AML subsets. UNC2025 inhibited MERTK in bone marrow leukemia cells and had significant therapeutic effects in xenograft models, with dose-dependent decreases in tumor burden and consistent two-fold increases in median survival, irrespective of starting disease burden. In a patient-derived AML xenograft model, treatment with UNC2025 induced disease regression. In addition, UNC2025 increased sensitivity to methotrexate in vivo, suggesting that addition of MERTK-targeted therapy to current cytotoxic regimens may be particularly effective and/or allow for chemotherapy dose reduction.Conclusions: The broad-spectrum activity mediated by UNC2025 in leukemia patient samples and xenograft models, alone or in combination with cytotoxic chemotherapy, supports continued development of MERTK inhibitors for treatment of leukemia. Clin Cancer Res; 23(6); 1481–92. ©2016 AACR.

Supplemental Table 1: Antibody Suppliers and Catalog Numbers Supplemental Table 2: Cell Cycle Profiles in Acute Leukemia Cell Lines Treated with UNC2025. Mean values, standard errors, and p values compared to vehicle treatment from 3 independent experiments are shown. (NS=Not Significant, *p

combined

Supplementary data file

Supplementary Figure 2 from Activated Tyrosine Kinase Ack1 Promotes Prostate Tumorigenesis: Role of Ack1 in Polyubiquitination of Tumor Suppressor Wwox

Aberrant activation of tyrosine kinases is linked causally to human cancers. Activated Cdc42-associated kinase (Ack1), an intracellular tyrosine kinase, has primarily been studied for its signaling properties but has not been linked to specific pathologic conditions. Herein, we report that expression of activated Ack1 in LNCaP cells, while minimally increasing growth in culture, enhanced anchorage-independent growth in vitro and dramatically accelerated tumorigenesis in nude mice. Molecular chaperone heat shock protein 90β (Hsp90β)–bound Ack1 and treatment of cells with geldanamycin, a Hsp90 inhibitor, inhibited Ack1 kinase activity and suppressed tumorigenesis. Further, we identify the tumor suppressor WW domain containing oxidoreductase (Wwox) as an Ack1-interacting protein. Activated Ack1 tyrosine phosphorylated Wwox, leading to rapid dissociation of the Ack1-Wwox complex and concomitant Wwox polyubiquitination followed by degradation. Tyrosine phosphorylation of Wwox was critical for its degradation, as splice variant WwoxΔ5-8 that was not phosphorylated by Ack1 failed to undergo polyubiquitination and degradation. It has been reported that phosphorylation of Wwox at Tyr33 stimulated its proapoptotic activity. We observed that Y33F Wwox mutant was still tyrosine phosphorylated and polyubiquitinated by Ack1 action. Site-directed mutagenesis revealed that activated Ack1 primarily phosphorylated Wwox at Tyr287, suggesting that phosphorylation of distinct tyrosine residues activate or degrade Wwox. Primary androgen-independent prostate tumors but not benign prostate showed increased tyrosine-phosphorylated Ack1 and decreased Wwox. Taken together, these data indicate that Ack1 stimulated prostate tumorigenesis in part by negatively regulating the proapoptotic tumor suppressor, Wwox. Further, these findings suggest that Ack1 could be a novel therapeutic target for prostate cancer.

Supplementary Figure Legends from Activated Tyrosine Kinase Ack1 Promotes Prostate Tumorigenesis: Role of Ack1 in Polyubiquitination of Tumor Suppressor Wwox

Supplementary Figure 1 from Activated Tyrosine Kinase Ack1 Promotes Prostate Tumorigenesis: Role of Ack1 in Polyubiquitination of Tumor Suppressor Wwox

Introduction: Translational research is essential to the success of every cancer center. Increasingly doctors ask patients to donate tissue to study all aspects of the disease to improve cancer treatments and quality of life. Patients are key stakeholders and partners in the success of the translational research process. The University of North Carolina (UNC), Lineberger Comprehensive Cancer Center (LCCC) researchers would like to increase the number of patients that donate tissue to support research and value patients who donate. Securing the right tissues at the right time, and processing and storing them properly is essential for research that leads to breakthrough discoveries. Purpose: LCCC supports the inclusion of patient advocates in all cancer center research. Therefore, the UNC Breast SPORE patient advocates initiated a program called Patients and Researchers Together (PART) to ensure patients work closely with researchers to drive the best research forward. The PART-Tissue program aims to develop a process to increase the donation and usage of patient donated tissues. This program is built on significant patient engagement that leads to a bi-directional patient and researcher PARTnership. We believe patients will be more willing to donate tissue if they are informed about the research process, understand the impact research has to improve patient’s lives, and are valued as PARTners of LCCC. Objectives: The first objective is to promote the excellence of LCCC translational research to all stakeholders, including patients and the community. Second; create a patient-centered tissue donation program that will meet researcher needs across LCCC, and Third; ensure the tissue collection process is primarily focused on recognizing patient’s voluntary contributions while minimizing patient burden. Methods: We first created a multi-stakeholder committee to ensure the participation of key stakeholders in all activities. We developed a process to identify relevant materials to assist in tissue donation at LCCC. The project focused on a Lineberger breast cancer tissue collection protocol (LCCC9819). We discussed patient, researcher and clinician/surgeon needs that reflect the vision of the PART program. We identified the need for several types of informational materials for patients, developed them with multi-stakeholder involvement and evaluated their accuracy, readability, understandability, and health literacy. We assessed the current and future needs of LCCC researchers by conducting interviews and surveys. We also engaged patient advocates as PARTners throughout this project. Results: We initiated the PART-Tissue program in August 2020 and we have engaged multiple stakeholders throughout the project. We evaluated LCCC9819 from the patient, research and clinical perspectives. We identified a series of informational materials needed for patients to introduce them to the value of tissue donation and to introduce them to the LCCC9819 protocol. We developed two introductory materials and a study summary. We conducted interviews and developed a survey to collect information about researcher needs at LCCC. In pilot interviews, we noticed a high need for fresh tissue, which requires real-time coordination between clinicians, clinical research coordinators, surgeons, pathologists and researchers. We also developed a website to connect directly with patients and the community about the importance of tissue donation that can lead to discoveries in cancer risk reduction and treatment. The goal is to develop a process where patients feel encouraged to donate tissue samples and are appreciated as partners with researchers who use the tissue to improve the lives of cancer patients. Citation Format: Patricia A. Spears, Vernal Branch, Jennifer A. Potter, Missy Van Lokeren, Nasrin H. Babadi, Adriana S. Beltran, Laurie Betts, Coleen Crespo, Barbara Dean, Amy L. Garrett, Laura T. Jensen, Gary L. Johnson, Hayley Morris, Philip Spanheimer, Charles M. Perou, Lisa A. Carey, H. Shelton Earp. Patients and Researchers Together (PART); a patient-centered tumor tissue collection PARTnership between patients and researchers to increase tissue donations for breast cancer research [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-15-01.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of childhood ALL. The early T-precursor (ETP-ALL) subset is characterized by an immature T-cell phenotype, chemoresistance, and high rates of induction failure. MERTK receptor tyrosine kinase is ectopically expressed in half of T-ALLs, particularly those with an immature T-cell phenotype, suggesting a role in ETP-ALL. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is essential for ETP-ALL cell survival. Here, we show that MERTK and BCL-2 mRNA and protein are preferentially expressed in ETP-ALL patient samples. The dual MERTK/FLT3 inhibitor MRX-2843 decreased MERTK activation and downstream signaling, inhibited cell expansion, and induced cell death in ETP-ALL cell lines. Further, 54% (21/39) of primary T-ALL patient samples were sensitive to MERTK inhibition. Treatment with MRX-2843 significantly reduced leukemia burden and prolonged survival in cell-line-derived T-ALL and ETP-ALL xenograft models. In a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 markedly reduced peripheral blood leukemia and spleen weight compared to vehicle-treated mice and prolonged survival. MRX-2843 also synergized with venetoclax to provide enhanced anti-leukemia activity in ETP-ALL cell cultures, with a dose ratio of 1:20 MRX-2843:venetoclax providing optimal synergy. These data demonstrate the therapeutic potential of MRX-2843 in patients with T-ALL and provide rationale for clinical development. MRX-2843 monotherapy is currently being tested in patients with relapsed leukemia (NCT04872478). Further, our data indicate that combined MERTK and BCL-2 inhibition may be particularly effective for treatment of ETP-ALL.

Enhancer of Zeste Homolog 2 (EZH2) and androgen receptor (AR) are crucial chromatin regulators involved in the development and/or progression of prostate tumor, including advanced castration-resistant prostate cancer (CRPC). To sustain prostate tumorigenicity, EZH2 establishes noncanonical biochemical interaction with AR for mediating oncogene activation, in addition to its canonical role as a transcriptional repressor and enzymatic subunit of Polycomb Repressive Complex 2 (PRC2). However, the molecular basis underlying non-canonical activities of EZH2 in prostate cancer remains elusive and therapeutic strategies for targeting EZH2:AR-mediated oncogene activation activities are also lacking. Here, we report that a cryptic transactivation domain of EZH2 (EZH2TAD) binds both AR and AR spliced variant 7 (AR-V7, an AR variant enriched in CRPC), mediating assembly and/or recruitment of transactivation-related machineries at genomic sites that lack PRC2 binding. Such noncanonical targets of EZH2:AR/AR-V7:(co)activators are enriched for the clinically-relevant oncogenes. We also show that EZH2TAD is required for the chromatin recruitment of EZH2, for EZH2-mediated oncogene activation, and for CRPC growth in vitro and in vivo. To completely block EZH2’s multifaceted oncogenic activities in prostate cancer, we employed MS177, a recently developed proteolysis targeting chimera (PROTAC) of EZH2. Strikingly, MS177 achieved on-target depletion of both EZH2’s canonical (EZH2:PRC2) and noncanonical (EZH2TAD:AR/AR-V7:coactivators) complexes in prostate tumor, eliciting much more potent antitumor effects than the catalytic inhibitors of EZH2. Overall, this study reports previously unappreciated requirements of EZH2TAD for mediating EZH2’s noncanonical (co)activator recruitment and gene-activation functions in prostate tumor and suggests EZH2-targeting PROTACs as potentially attractive therapeutics for the treatment of aggressive prostate tumors that rely on the circuits wired by EZH2 and AR.

The challenge of rapid macromolecular synthesis enforces the energy-hungry cancer cell mitochondria to switch their metabolic phenotypes, accomplished by activation of oncogenic tyrosine kinases. Precisely how kinase activity is directly exploited by cancer cell mitochondria to meet high-energy demand, remains to be deciphered. Here we show that a non-receptor tyrosine kinase, TNK2/ACK1 (tyrosine kinase non receptor 2), phosphorylated ATP5F1A (ATP synthase F1 subunit alpha) at Tyr243 and Tyr246 (Tyr200 and 203 in the mature protein, respectively) that not only increased the stability of complex V, but also increased mitochondrial energy output in cancer cells. Further, phospho-ATP5F1A (p-Y-ATP5F1A) prevented its binding to its physiological inhibitor, ATP5IF1 (ATP synthase inhibitory factor subunit 1), causing sustained mitochondrial activity to promote cancer cell growth. TNK2 inhibitor, (

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.

PURPOSE: Understanding breast cancer mortality disparities by race and age is complex due to disease heterogeneity, comorbid disease, and the range of factors influencing access to care. It is important to understand how these factors group together within patients. METHODS: We compared socioeconomic status (SES) and comorbidity factors in the Carolina Breast Cancer Study Phase 3 (CBCS3, 2008-2013) to those for North Carolina using the 2010 Behavioral Risk Factor Surveillance Study. In addition, we used latent class analysis of CBCS3 data to identify covariate patterns by SES/comorbidities, barriers to care, and tumor characteristics and examined their associations with race and age using multinomial logistic regression. RESULTS: Major SES and comorbidity patterns in CBCS3 participants were generally similar to patterns in the state. Latent classes were identified for SES/comorbidities, barriers to care, and tumor characteristics that varied by race and age. Compared to white women, black women had lower SES (odds ratio (OR)=6.3, 95% confidence interval (CI); 5.2, 7.8), more barriers to care (OR=5.6, 95% CI; 3.9, 8.1) and several aggregated tumor aggressiveness features. Compared to older women, younger women had higher SES (OR=0.5, 95% CI; 0.4, 0.6), more barriers to care (OR=2.1, 95% CI; 1.6, 2.9) and aggregated tumor aggressiveness features. CONCLUSIONS: CBCS3 is representative of North Carolina on comparable factors. Patterns of access to care and tumor characteristics are intertwined with race and age, suggesting that interventions to address disparities will need to target both access and biology.

Aberrant activation of the androgen receptor (AR) may play a critical role in castration resistant prostate cancer. After ligand binding, AR is recruited to the androgen responsive element (ARE) sequences on the DNA where AR interaction with coactivators and corepressors modulates transcription. We demonstrated that phosphorylation of AR at Tyr-267 by Ack1/TNK2 tyrosine kinase results in nuclear translocation, DNA binding, and androgen-dependent gene transcription in a low androgen environment. In order to dissect downstream mechanisms, we searched for proteins whose interaction with AR was regulated by Ack1. SLIRP (SRA stem-loop interacting RNA binding protein) was identified as a candidate protein. Interaction between AR and SLIRP was disrupted by Ack1 kinase activity as well as androgen or heregulin treatment. The noncoding RNA, SRA, was required for AR-SLIRP interaction. SLIRP was bound to ARE’s of AR target genes in the absence of androgen. Treatment with androgen or heregulin led to dissociation of SLIRP from the ARE. Whole transcriptome analysis of SLIRP knockdown in androgen responsive LNCaP cells showed that SLIRP affects a significant subset of androgen-regulated genes. Our data suggest that Ack1 kinase and androgen regulate interaction between AR and SLIRP and that SLIRP functions as a coregulator of AR with properties of a corepressor in a context-dependent manner.