Wang, Liewei

Abstract Background: Cyclin dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in hormone-receptor (HR)-positive metastatic breast cancer (MBC), but progression of disease is inevitable. Serum thymidine kinase-1 (TK1) is a secreted marker of proliferation that is prognostic in patients (pts) with HR-positive, HER2-negative MBC and may be predictive of ET and CDK 4/6i response. PROMISE (NCT0281902) is a prospective study enrolling women with HR-positive MBC starting palbociclib (Pb) + letrozole (L) (1st line) or Pb + fulvestrant (2nd line). We undertook a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome in order to identify novel genomic variants and pathways associated with an early decline in TK1 (measured after 2 months) and PFS. Additionally, patient derived xenografts/organoids were generated at baseline and progression to test new therapeutic approaches to overcome resistance to CDK4/6i and ET. We report the initial association between the baseline genomic landscape and baseline TK1 levels. Methods: FFPE tumor biopsies were obtained for DNA/RNA sequencing (TempusTM) and blood samples for TK1 (Divitum® assay, Biovica) were collected pretreatment (pre-Pb) and after 2 cycles of Pb + ET (post-Pb2). Both whole-exome (exome capture) sequencing (WES) and RNA-Seq used the Integrated DNA Technologies xGen Exome Research Panel v1.0 capture kit. TK1+ disease was defined as > 200 Du/L and TK1- disease as below limit of detection up to 200 Du/L. We tested the association between genomic and transcriptomic characteristics with baseline TK1 data in pretreatment samples where both WES and RNA-seq and TK1 was available. The data were analyzed using bioinformatics pipelines for somatic and germline mutations and copy number alterations. The current analysis focuses on baseline 1st-line pre-Pb omics data in conjunction with baseline TK1 levels. The database was locked for analysis on 5/29/2020. Results: Thirty-three pts (median age: 59 yrs.) were evaluable, with paired samples for TK1 in 32. Six pts had TK1+ disease pre-Pb and post-Pb2. Twenty-two pts had TK1- disease pre-Pb and post-Pb2. Four pts had a decrease in TK1 after 2 cycles of treatment that altered the classification from TK1+ to TK1-. Both baseline RNA seq and serum TK1 (n=16) were available for 4 TK1+ and 12 TK1- pts. In this group, 476 genes were differentially regulated (398 upregulated; 78 downregulated). Pathway analysis demonstrated enrichment in complement and coagulation cascade pathway, PPAR signaling pathway, and metabolism-related pathways related to up-regulation of CYP and UGT gene families. Further testing for the association of WES data with baseline TK1+ (n=8) and TK1- (n=16) disease demonstrated somatic copy number variations on chromosomes 6, 11, 12 and 15. CDK4 copy number gains were observed in 3/8 TK1+ pts and 0/16 TK1- pts. We also observed that somatic mutations (LOH, copy number and/or SNV/INDELs) were more prevalent in the TK1+ compared to the TK1- pre-Pb group in several cancer-associated genes (FAS [p=0.06] PTEN, PIK3CB, NAB2, SOX9 and FAT1 [p=0.08], TP53, and MAP2K4 [p=0.22]). Conversely, we also noted that 6/7 pts with GATA3 mutations had TK1- disease (p=0.23). Conclusions: Using a comprehensive “omics” approach, our data suggest that a secreted biomarker of proliferation (TK1) obtained prior to initiating CDK4/6i and ET for the first line treatment of HR+ MBC is associated with established and novel genes and pathways associated with prognosis of pts receiving ET and CDK 4/6i. Analysis of on-treatment (after 2 cycles) tumor RNA seq and its association with change in TK1 as well as data from the 2nd-line cohort will be presented at the meeting. Citation Format: Ciara C O Sullivan, Krishna R Kalari, Vera J Suman, Peter T Vedell, Ann Moyer, Abraham D Eyman Casey, Jason Sinnwell, Xiaojia Tang, Kevin Thompson, Alvaro Moreno-Aspitia, Donald W Northfelt, Minetta C Liu, Tufia C Haddad, Saranya Chumsri, Prema Peethambaram, Kathryn J Ruddy, Karthik V Giridhar, Roberto A Leon-Ferre, Adrian Nordmark, Mattias Bergqvist, Brendan P McMenomy, Richard M Weinshilboum, Liewei Wang, Matthew P Goetz. Novel genomic variants and pathways associated with baseline serum thymidine kinase 1 levels in HR-positive HER2-negative MBC patients commencing palbociclib and letrozole [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 PS5-24.

Abstract Background: The tamoxifen (TAM) metabolite, ENDX, demonstrated promising antitumor activity in endocrine resistant breast cancer (BC) in both phase I and phase II settings. Furthermore, ENDX resulted in superior in vivo antitumor activity compared to TAM and letrozole in aromatase-expressing aromatase inhibitor-sensitive and resistant MCF7AC1 models. Recently, we identified protein kinase C beta 1 (PKCβ1), which regulates cell proliferation and tumorigenic transformation, as a novel target of ENDX. ENDX-bound PKCβ1 at concentrations achieved in phase I/II ENDX studies (100-300 nM). In contrast, TAM binding to PKCβ1 occurred at concentrations 7-10 folds higher (2 μM) than achievable with TAM 20 mg/day dosing. However, the clinical relevance of targeting PKCβ1 kinase activity is unclear, since drugs that target PKCβ1 (enzastaurin) have been ineffective in BC and other solid tumors. Therefore, we sought to understand how ENDX altered PKCβ1 and to further compare and contrast ENDXs effects to that of PKCβ1 kinase inhibition in ERα+ BC. Methods: The effects of PKCβ1 silencing and ENDX treatment on gene expression was analyzed by RNAseq in MCF7AC1 cells. The impact of PKCβ1-silencing on cell cycle was evaluated by flow cytometry. Protein expression of cell cycle regulators in PKCβ1 and ENDX-treated MCF7AC1 and T47D cells were compared to TAM and enzastaurin in vitro and to letrozole, TAM or control in vivo. The effects of PKCβ1 and drugs on growth were analyzed by cell proliferation assays. PRKCB gene amplification was assessed in primary tumors using TCGA data and in metastatic tumors using whole-exome sequencing data from patients enrolled in the PROMISE study (NCT 03281902). Results: RNAseq analysis revealed E2F targets and G2M checkpoints as the top hallmark genesets significantly downregulated in both PKCβ1-silenced and ENDX-treated MCF7AC1 cells. Flow cytometry demonstrated that PKCβ1 silencing increased G1 and reduced S phases of the cell cycle. Western blot analyses of PKCβ1-silenced MCF7AC1 and T47D cells displayed reduced protein levels of the cell cycle regulators Cyclin D1, Retinoblastoma (Rb), phospho-RbS807/811, CDK4, Chk1 and E2F1 that regulate G1/S transition. While short term ENDX (48 hours) treatment did not alter PKCβ1 levels, prolonged in vitro ENDX treatment profoundly reduced PKCβ1 protein levels and the aforementioned cell cycle regulators, faithfully replicating PKCβ1 silencing effects. In contrast, enzastaurin had no impact on proliferation or cell cycle proteins in either model. Consistent with this finding, ENDX, but not TAM or letrozole, reduced protein levels of ERα and cell cycle regulators in vivo. Overexpression of PKCβ1 induced TAM, but not ENDX, resistance and had little impact on responsiveness to enzastaurin. While PRKCB gene amplification was uncommon in newly diagnosed ERα+/HER2- BC (5%, TCGA), PRKCB was amplified in 40% of metastatic ERα+/HER2- BC (PROMISE study). Conclusion: We have confirmed the relevance of a new ENDX target, PKCβ1, in ERα+/HER2- BC. While targeting PKCβ1 kinase activity elicited no anticancer effects in ERα+ cells, PKCβ1 downregulation, either by siRNA or ENDX, resulted in profound ERα turnover, reduced protein levels of essential cell cycle mediators and profoundly inhibited cell proliferation. Furthermore, PKCβ1 protein expression is associated with TAM, but not ENDX, resistance, a finding whose clinical relevance is further magnified by identification of PRKCB amplification in metastatic ERα+ BC, confirming its potential importance in progression. Efforts are currently underway to elucidate the mechanistic basis for ENDX-induced PKCβ1 and ERα degradation and the contribution of these effects to the superior antitumor activity of ENDX in ERα+ BC. Citation Format: Swaathi Jayaraman, Mary J Kuffel, Krishna R Kalari, Kevin J Thompson, Xiaojia Tang, Vera J Suman, Elizabeth S Bruinsma, Ciara C O'Sullivan, Liewei Wang, Richard Weinshilboum, James N Ingle, John R Hawse, Matthew P Goetz. Antitumor activity of Z-endoxifen (ENDX) is mediated via PKCβ1-dependent ERα loss and cell cycle arrest in ERα-positive breast cancer [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 PD8-04.

Abstract Introduction: The immune microenvironment of high-risk HR+ breast cancer (BC) is poorly understood. BEAUTY is a prospective neoadjuvant chemotherapy (NAC) study of stage I-III BC patients treated with neoadjuvant weekly taxane followed by anthracycline-based chemotherapy. Among clinical luminal B BC (St. Gallen criteria) from BEAUTY, we used high-plex digital spatial profiling (DSP) to characterize the intra-epithelial tumor and stromal immune landscapes to 1) assess the impact of NAC on these landscapes and 2) identify immune biomarkers predictive of response to NAC. Methods: The tissue set included FFPE sections of resected tumors from 35 patients (median 51y; range: 21-71y) with clinically-defined luminal B BC (ER > 10%/grade 2/Ki67 ≥ 15% or ER > 10%/grade 3), and 16 paired pre-NAC biopsies. Nanostring GeoMX DSP was used to quantitate 58 immune and BC biomarker proteins in intra-epithelial, cytokeratin-positive tumor segments and adjacent stromal (cytokeratin-negative/SYTO13 (nuclear stain)-positive) segments. Data were normalized using the geometric mean of two negative controls (RbIgG and MsIgG1). A general linear model with negative binomial identified differentially-expressed (DE) proteins in pre/post-NAC tumors. Based on DE protein data and biologic function, a targeted protein subset (N=19) was evaluated in pre-NAC biopsies for associations between protein abundance and residual cancer burden (RCB) class (0-II vs. III) (Wilcoxon-rank sum test: p-value < 0.025 considered significant) or RCB ‘breast only’ index (Spearman correlation). Results: Comparing tumor segments in pre-NAC specimens, intra-epithelial segments were predictably enriched in cytokeratin, ER, PR and Ki-67, but also CD127 and NY-ESO-1; whereas stromal segments were enriched in proteins associated with T cell subsets (e.g. CD3, CD4, CD8), macrophages (CD68 and CD163), antigen presentation/dendritic cells (CD11c, HLA-DR) and immune checkpoint proteins (PD-L1, PD-L2, B7-H3, TIM-3, VISTA) among others. While NAC did not alter the spatial distribution of proteins (intra-epithelial vs. stromal segments), it markedly attenuated the immune landscape, with decreased abundance of functionally-diverse immune proteins (e.g. CD45, CD3, CD4, CD127, granzyme B, CTLA4; STING, B7-H3, CD11c, and CD68, log2FC: 0.27-1.52 p < 0.05), including low abundance proteins PD-1, PD-L1, PD-L2, CD20, and OX40L (log 2FC: 0.15-1.16, p < 0.05). Both PR and Ki-67 decreased in post-NAC tumors whereas ER was not significantly altered (p < 0.05). CD8, CD14, CD163, HLA-DR, IDO-1 and TIM-3 were not significantly altered by NAC. In the pre-NAC biopsies (which had subsequent residual tumor burdens of RCB class 0 or I (n=1 each), II (n=6), and III (n=8), and median RCB ‘breast only’ index of 3.39 (range: 0.00-38.02), 19 proteins were used for RCB analysis (CD3, CD4, CD8, CD14, CD34, CD44, CD45, CD68, CD127, CD163, CTLA4, granzyme B, STING, B7-H3, fibronectin, Ki-67, HLA-DR, SMA and TIM3). Among them, stromal CD127 was significantly higher in RCB class III than RCB class 0-II (p=0.021) and RCB ‘breast only’ index was positively correlated with intra-epithelial granzyme B (rho = 0.61; p=0.012), and negatively correlated with intra-epithelial Ki-67 (rho= -0.71; p=0.0022). Conclusion: In this series of clinical luminal B BC, NAC markedly attenuated the tumoral immune landscapes with a small set of “NAC-resistant” immune proteins. Among a targeted set, stromal CD127 was significantly higher in RCB III, and RCB breast-only index was positively correlated with intra-epithelial granzyme B. These data provide insight into the impact of NAC on HR+ BC, and identify potential immune biomarkers to predict response to neoadjuvant chemotherapy. Citation Format: Jodi M Carter, Judy C Boughey, Jun He, Vera J Suman, Xue Wang, Jennifer M Kachergus, Krishna R Kalari, Liewei Wang, Richard Weinshilboum, Ann M Moyer, Sarah A McLaughlin, Alvaro Moreno-Aspitia, Donald W Northfelt, Richard J Gray, James N Ingle, E. Aubrey Thompson, Matthew P Goetz. Neoadjuvant chemotherapy selectively alters spatially-defined immune landscapes in clinical luminal B HR+/HER2- breast cancers: Analysis of the breast cancer genome guided therapy study (BEAUTY) [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 PD7-05.

Abstract Background: Patient derived xenografts are a standard tool used to study cancer biology and evaluate drug response phenotypes. It is well known that PDX take rate is associated with more aggressive clinical features [higher grade, biologic subtype (e.g. TNBC vs luminal)]. Previous data from a small retrospective cohort of patients (pts) with no prior chemo (n=24) suggested that primary breast tumor PDX take rate was associated with reduced overall survival. (Derose Nat Med. 2011) However, whether take rate is prognostic in newly diagnosed pts receiving standard of care neoadjuvant chemotherapy (NAC) is unknown. From the prospective BEAUTY clinical trial of pts treated with NAC in which PDX was attempted from pre-NAC as well as after chemo (post-NAC), we recently reported no difference in take rate comparing pts with or without a pathologic complete response (pCR) (Yu Breast Canc Res 2018). Herein we report the association between PDX take rate and recurrence from tumor samples implanted pre-NAC as well as post-NAC (time of surgery). Methods: The BEAUTY study is a prospective NAC study which enrolled breast cancer pts (Stage I-III; n=140) treated with neoadjuvant weekly taxane +/-trastuzumab followed by anthracycline-based chemotherapy. Percutaneous tumor biopsies were obtained prior to NAC and tumor samples from residual disease at surgery were additionally used to establish PDXs. Tumor take rate was defined as percent of pts with the development of at least one stably transplantable (passed at least for four generations) xenograft that was pathologically confirmed as breast cancer. Time to breast cancer recurrence was defined as the time from surgery to documentation of a local, regional, or distant recurrence. Gray’s test was used to assess whether the cumulative incidence (CI) of a breast cancer recurrence differs with respect to either pre-NAC PDX take or post-NAC PDX take. Results: Of 140 pts enrolled in the BEAUTY study, tumor tissue for PDX from the pre-NAC tumor was available for implantation in 113. As previously published, PDX take rate from pre-NAC tumor was 27.4% (31/113), and varied according to tumor clinical subtype [51.3% (20/39) in triple negative breast cancer (TNBC), 26.5% (9/34) in HER2+, 5.0% (2/40) in Luminal]. With median follow up of 5.7 years (range: 3 months to 6.75 years), 17 pts developed local, regional or distant disease relapse (4 of whom had a PDX established pre-NAC). The cumulative incidence of breast cancer relapse after surgery was not found to differ according to pre-NAC PDX take (5 yr CI rate: 13.6% no take; 13.4% take; p=0.8911). In pts with TNBC, the group with the highest take rate, there was also no significant difference in incidence of BC recurrence between those without PDX take and those with PDX take (5 yr CI rate: 21.4% vs 16.2% p=0.7314). PDXs were established from residual tissue from surgery (post-NAC) in 6 of 34 pts (17.6%), [specifically, TNBC: 5/9; Her2+: 1/8; and Luminal: 0/17]. Nine of these 34 pts developed a local, regional or distant recurrence. There was a tendency for pts who had a PDX established from their residual disease to have a higher incidence of a breast event (p=0.1092). The 5 year cumulative incidence of a breast event was 19.6% for pts whose post-NAC PDX did not take and 50.0% for pts whose post-NAC PDX took. Discussion: In pts receiving NAC for breast cancer, we observed no significant difference between establishment of a pre-NAC PDX and breast cancer relapse. In contrast, post-NAC PDX take rate (from residual tumor obtained from the breast at surgery) was associated with a tendency to have a higher incidence of a breast cancer event. PDXs remain a valuable tool for the evaluation of tumor biology and development of new therapeutics, especially those models established from pts with chemotherapy resistance. Citation Format: Judy C. Boughey, Vera J. Suman, Jia Yu, Katelyn Santo, Jason P. Sinnwell, Jodi M. Carter, Krishna R. Kalari, Xiaojia Tang, Sarah A. McLaughlin, Alvaro Moreno Aspitia, John A. Copland, III, Donald W. Northfelt, Richard J. Gray, Katie N. Hunt, Amy Lynn Conners, Richard Weinshilboum, Liewei Wang, Matthew P. Goetz. Association between patient derived xenograft (PDX) take rate and breast cancer recurrence in the prospective breast cancer genome guided therapy study (BEAUTY) [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 PD7-04.

Abstract Background: With few targeted therapies available for triple-negative breast cancer (TNBC), there is a critical need to identify biomarkers that can predict chemotherapy-response. Copy number amplifications (CNAmps) have been implicated in tumorigenesis, progression, and treatment response. Previously, we performed a prospective neoadjuvant chemotherapy study (BEAUTY) and identified a 5 Mb CNAmp in TNBC patients associated with pathological complete response rates to taxane- and anthracycline-based chemotherapy. Here we further interrogated this amplicon and its role in chemotherapy response. Methods: Using siRNA screening of genes identified in the 5 Mb CNAmp, followed by cytotoxicity assays, we identified RDH10 significantly associated with chemo-sensitivity in TNBC cell lines. Functional studies were performed using RDH10 knockdown or overexpression followed by cytotoxicity, cell proliferation, luciferase reporter assays, HPLC or western blot. Results: RDH10 is a rate-limiting step involved in the synthesis of all-trans retinoic acid (ATRA) - the active metabolite of retinoid metabolism that is frequently aberrant in cancer. Previous studies demonstrated ATRA to be a potent inhibitor of PIN1, a key regulator of several oncogenic signaling pathways often amplified in TNBC. We discovered that genetic manipulation of RDH10 affected ATRA intracellular concentrations, contributing to PIN1 degradation and taxane-response. Furthermore, co-treatment of TNBC cells with ATRA significantly increased taxane-sensitivity. Conclusion: RDH10 amplification increases endogenous levels of ATRA and degrades PIN1 in TNBC. ATRA-mediated degradation of PIN1 sensitizes TNBC to chemotherapy, suggesting that RDH10 may be a novel biomarker of taxane response and the addition of ATRA to standard chemotherapy may improve chemo-sensitivity.

Abstract Triggering receptor expressed on myeloid cell 2 (TREM2) is a surface receptor that, in the central nervous system, is exclusively expressed on microglia. TREM2 variants have been linked to increased risk for neurodegenerative diseases, but the functional effects of microglial TREM2 remain largely unknown. To this end, we investigated TAR-DNA binding protein 43 kDa (TDP-43)-related neurodegenerative disease via viral-mediated expression of human TDP-43 protein (hTDP-43) in mice or inducible expression of hTDP43 with defective nuclear localization signals in transgenic mice. We found that TREM2 deficiency impaired microglia phagocytic clearance of pathological TDP-43, and enhanced neuronal damage and motor function impairments. Mass cytometry analysis revealed that hTDP-43 induced a TREM2-dependent subpopulation of microglia with high CD11c expression and higher phagocytic ability. Using mass spectrometry, we further demonstrated an interaction between TDP-43 and TREM2, in vitro and in vivo, in hTDP-43-expressing transgenic mouse brains. We computationally identified the region within hTDP-43 that interacts with TREM2 and observed the potential interaction in ALS patient tissues. Our data reveal the novel interaction between TREM2 and TDP-43, highlighting that TDP-43 is a potential ligand for microglial TREM2 and the interaction mediates neuroprotection of microglial TREM2 in TDP-43-related neurodegeneration.

Abstract Cell division cycle 25 (CDC25) dual specificity phosphatases positively regulate the cell cycle by activating cyclin-dependent kinase/cyclin complexes. Here, we demonstrate that in addition to its role in cell cycle regulation, CDC25B functions as a regulator of protein phosphatase 2A (PP2A), a major cellular Ser/Thr phosphatase, through its direct interaction with PP2A catalytic subunit. Importantly, CDC25B alters the regulation of AMP-activated protein kinase signaling (AMPK) by PP2A, increasing AMPK activity by inhibiting PP2A to dephosphorylate AMPK. CDC25B depletion leads to metformin resistance by inhibiting metformin-induced AMPK activation. Furthermore, dual inhibition of CDC25B and PP2A further inhibits growth of 3D organoids isolated from patient derived xenograft model of breast cancer compared to CDC25B inhibition alone. Our study identifies CDC25B as a regulator of PP2A, and uncovers a mechanism of controlling the activity of a key energy metabolism marker, AMPK.

Abstract Chromosome region maintenance protein 1 (CRM1) mediates protein export from the nucleus and is a new target for anticancer therapeutics. Broader application of KPT-330 (selinexor), a first-in-class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the antitumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid-organ cancers ex vivo and in vivo. The K+CS combination was not toxic to nonmalignant cells as compared with malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared with KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51, and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell-cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of poly (ADP-ribose) polymerase inhibitors further potentiates the K+CS antitumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.

Abstract Background: With few targeted therapies available for triple-negative breast cancer (TNBC), there is a critical need to identify biomarkers that can predict chemotherapy-response. Copy number amplifications (CNAmps) have been implicated in tumorigenesis, progression, and treatment response. Previously, we performed a prospective neoadjuvant chemotherapy study (BEAUTY) and identified a 5 Mb CNAmp in TNBC patients associated with pathological complete response rates to taxane- and anthracycline-based chemotherapy. Here we further interrogated this amplicon and its role in chemotherapy response. Methods: Using siRNA screening of genes identified in the 5 Mb CNAmp, followed by cytotoxicity assays, we identified RDH10 significantly associated with chemo-sensitivity in TNBC cell lines. Functional studies were performed using RDH10 knockdown or overexpression followed by cytotoxicity, cell proliferation, luciferase reporter assays, HPLC or western blot.Results: RDH10 is a rate-limiting step involved in the synthesis of all-trans retinoic acid (ATRA) - the active metabolite of retinoid metabolism that is frequently aberrant in cancer. Previous studies demonstrated ATRA to be a potent inhibitor of PIN1, a key regulator of several oncogenic signaling pathways often amplified in TNBC. We discovered that genetic manipulation of RDH10 affected ATRA intracellular concentrations, contributing to PIN1 degradation and taxane-response. Furthermore, co-treatment of TNBC cells with ATRA significantly increased taxane-sensitivity.Conclusion: RDH10 amplification increases endogenous levels of ATRA and degrades PIN1 in TNBC. ATRA-mediated degradation of PIN1 sensitizes TNBC to chemotherapy, suggesting that RDH10 may be a novel biomarker of taxane response and the addition of ATRA to standard chemotherapy may improve chemo-sensitivity.

Abstract Purpose: To determine genetic variants that could differentiate aromatase inhibitors (AIs) efficacy in early stage breast cancer. Experimental Design: We performed a stratified cox-proportional hazards analysis utilizing stratification factors and other covariates examining Breast Cancer Free Interval (BCFI) SNP-treatment interaction in Caucasian patients entered on the MA.27 trial. This analysis involved 4465 patients (2226 on anastrozole and 2239 on exemestane arm) including 245 (121 on anastrozole and 124 events on exemestane arm) with a breast event. Preclinical laboratory studies included luciferase activity, chromatin immunoprecipitation (ChIP) assay, and cell migration assays. Results: We identified 887 SNPs with a p-value <1E-4 that could differentiate anastrozole from exemestane efficacy. We next used GTEx databases to determine whether these SNPs might be cis-eQTLs with nearby genes, and found that 95 SNPs were eQTL with 14 genes. Functional validation of SNP effect in these 14 genes on response to anastrozole and exemestane revealed that 3 SNPs showed genotype-dependent differences between anastrozole and exemestane. Two of the three SNPs, rs1877193 and rs6735923 located upstream of LY75 gene, were associated with higher LY75 gene expression. Our drug-SNP interaction GWAS showed that both SNPs were associated with better BCFI for exemestane compared to anastrozole (HR= 0.447, 0.458). The SNP rs62293499 located downstream of the GPR160 gene was also associated with better BCFI for exemestane compared to anastrozole (HR=0.433). Interestingly, all 3 SNPs were associated with worse outcome (shorter BCFI) in anastrozole treated patients (HR=1.39~1.58), but with longer BCFI in the exemestane treated patients (HR=0.58~0.71) based on our GWAS results in MA.27 trial. However, all 3 SNPs showed no association with BCFI if the two treatment arms, anastrozole and exemestane, were combined (HR=0.92~1.08). Consistently, cells with Ly75 or GPR160 SNP variant genotypes were more sensitive to exemestane compared to anastrozole. Mechanistically, the 3 SNPs regulated estrogen receptor-dependant LY75 and GPR160 expression. LY75 suppression induced epithelial-to-mesenchymal transition (EMT) in breast cancer cell lines, accompanied by increased migratory capacity in vitro. LY75 knockdown also resulted in predominant downregulation of functional pathways such as cell proliferation, while pathways associated with mesenchymal stimulation were generally increased. GPR160 knockdown also resulted in downregulation of cell proliferation pathways. Conclusions: This SNP genotype and AI treatment interaction clinical study revealed unique genetic variants that differentiate anastrozole and exemestane efficacy. The signals were lost in the GWAS analysis when anastrozole and exemestane were combined. Preclinical laboratory studies revealed novel functions of LY75 and GPR160 in breast cancer. These findings represent potential steps towards individualized AI therapy. Citation Format: Junmei Cairns, James N. Ingle, Krishna R. Kalari, Lois E. Shepherd, Matthew J. Ellis, Paul E. Goss, Poulami Barman, Erin E. Carlson, Matthew P. Goetz, Richard M. Weinshilboum, Liewei Wang. The interaction between SNP genotype and aromatase inhibitor treatment response in early breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-103.