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supplementary methods and figure legends

Supplementary Figure S1. ER, PR, and HER2 mRNA and protein levels across endocrine-sensitive (parental) and endocrine-resistant BC cell lines. Supplementary Figure S2. Reduced sensitivity to CDK4/6 inhibition is associated with IFN-signaling in ER+ and endocrine-resistant derivative BC cell lines. Supplementary Figure S3. The cyclin D-CDK4/6-Rb axis is altered in ER+ cell lines with acquired resistance to palbociclib. Supplementary Figure S4. Effect of acute IFN-signaling activation on sensitivity to palbociclib in ER+ BC cell lines. Supplementary Figure S5. Proteomic profiles of MCF7 P, MCF7 EDR, and their PalboR derivatives. Supplementary Figure S6. Relationship between IFN-signaling and Rb status in ER+/HER2- BC. Supplementary Figure S7. DNMT1 mRNA levels in palbociclib-sensitive and PalboR MCF7 and T47D cell lines. Supplementary Figure S8. Mutations of DNA damage response genes are not associated with IFN-signaling in ER+ BC. Supplementary Figure S9. Levels of ER and ER signalling in palbociclib-sensitive and PalboR MCF7 and T47D cell lines. Supplementary Figure S10. HALLMARK gene sets significantly up-regulated in ER+/HER2- BC patients stratified by the status (+/-) of RBsig and IRPS. Supplementary Figure S11. Impact of selected IRPS genes on relapse-free survival of ER+ BC patients.

Purpose:Tumor-infiltrating lymphocytes (TIL) are associated with benefit to trastuzumab and chemotherapy in patients with early-stage HER2+ breast cancer. The predictive value of TILs, TIL subsets, and other immune cells in patients receiving chemotherapy-sparing lapatinib plus trastuzumab treatment is unclear.Experimental Design: Hematoxylin and eosin–stained slides (n = 59) were used to score stromal (s-)TILs from pretreatment biopsies of patients enrolled in the neoadjuvant TBCRC006 trial of 12-week lapatinib plus trastuzumab therapy (plus endocrine therapy for ER+ tumors). A 60% threshold was used to define lymphocyte-predominant breast cancer (LPBC). Multiplexed immunofluorescence (m-IF) staining (CD4, CD8, CD20, CD68, and FoxP3) and multispectral imaging were performed to characterize immune infiltrates in single formalin-fixed paraffin-embedded slides (n = 33).Results:The pathologic complete response (pCR) rate was numerically higher in patients with LPBC compared with patients with non-LPBC (50% vs. 19%, P = 0.057). Unsupervised hierarchical clustering of the five immune markers identified two patient clusters with different responses to lapatinib plus trastuzumab treatment (pCR = 7% vs. 50%, for cluster 1 vs. 2 respectively; P = 0.01). In multivariable analysis, cluster 2, characterized by high CD4+, CD8+, CD20+ s-TILs, and high CD20+ intratumoral TILs, was independently associated with a higher pCR rate (P = 0.03). Analysis of single immune subpopulations revealed a significant association of pCR with higher baseline infiltration by s-CD4, intratumoral (i-) CD4, and i-CD20+ TILs.Conclusions:LPBC was marginally associated with higher pCR rate than non-LPBC in patients with lapatinib plus trastuzumab treated HER2+ breast cancer. Quantitative assessment of the immune infiltrate by m-IF is feasible and may help correlate individual immune cell subpopulations and immune cell profiles with treatment response.

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies.Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo.Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

Supplementary tables

Supplementary Table S1. Details of antibodies used in this study. Supplementary Table S2. Q-PCR primers. Supplementary Table S3. HALLMARK gene-set enrichment analysis (GSEA) of baseline tumor samples from the NeoPalANA trial. Supplementary Table S4. Palbociclib IC50 values in MCF7 P, MCF7 EDR, T47D P-LM, and T47D EDR palbociclib-resistant (PalboR)-LM cell lines. Supplementary Table S5. Top-20 Gene Ontology (GO) terms significantly enriched in PalboR cell lines. Supplementary Table S6. HALLMARK gene sets significantly up-regulated in cell lines with acquired resistance to palbociclib. Supplementary Table S7. RPPA of MCF7 P, MCF7 EDR, and their PalboR derivatives.

Supplemental Materials and Methods and Materials present a complete description of methods, reagents, and statistics required to reproduce the experiments in the paper.

Supplementary Figures S1-S8

Purpose:Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor–positive (ER+)/HER2− breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes.Experimental Design:Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation–resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes.Results:Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an “IFN-related palbociclib-resistance Signature” (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2− tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis.Conclusions:Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

Supplementary tables S1-S4

Background. We recently reported that the biosynthetic mevalonate (MVA) pathway that produces cholesterol and isoprenoid intermediates, regulates the YAP/TAZ (Y/T) transcriptional co-activators to promote resistance to treatment regimens that effectively inhibit HER2 signaling in HER2+ BC. We further showed that the mTORC1 complex and survivin protein, which contribute to HER2-elicited oncogenic signaling in HER2-driven BC cells, are alternatively activated by the MVA pathway-Y/T axis in anti-HER2 therapy resistant cell models. Of note, other recent reports also showed that increased Y/T activity enables resistant cells to proliferate when other oncogenic pathways (e.g., RAS, EGFR, and FGFR) were effectively inhibited. Here, we sought to further determine the molecular underpinnings of MVA-Y/T axis-driven anti-HER2 therapy resistance to discover novel therapeutic targets and predictive biomarkers for HER2+ BC.Methods. SKBR3 HER2+ BC parental (P) cells and their lapatinib plus trastuzumab (LT) resistant (LTR) derivatives with sustained HER2 inhibition were treated with the MVA pathway inhibitor simvastatin (Sim), with or without the MVA metabolite to rescue Sim’s inhibitory effects. P and LTR cells were also transfected with control or combined Y/T siRNAs. The transcriptomes of all treatment groups were assessed by RNA-seq. Integrative bioinformatics analyses were used to identify differentially expressed (DE) genes and gene sets with functional annotations in LTR vs. P cells upon different interventions.Results. We found that cell cycle and cell proliferation processes were among the top common DE molecular signatures preferentially downregulated (DN) in LTR vs. P cells by both Sim and Y/T knockdown (KD). The top common genes preferentially DN in LTR vs. P cells include the cell cycle regulatory genes CDCA3 and ERCC6L, and the nucleotide metabolism genes TYMS and RRM2. Interestingly, 20% of the genes preferentially DN in LTR vs. P cells by Sim or Y/T KD were predicted to be direct Y/T transcriptional targets based on previously reported ChIP-seq data (PMID: 26258633). Of the Y/T-dependent genes, a significant enrichment of Sim-repressed genes was observed in both P and LTR cells (P = 1.2e-115 and P = 5.5e-138, respectively). The proportion of these enriched genes was higher in LTR vs. P cells (61% vs. 29%). Of note, we found that the global inhibited genes in LTR cells upon Sim or Y/T KD were significantly enriched for the genes DN by short-term LT treatment in P cells (P < 2.2e-16). Likewise, of the genes nominated as putative molecular players in the MVA pathway-Y/T-mediated resistance, BIRC5 (survivin), CDC6, KIF2C, RRM2, and TYMS were recently also reported to be DN in HER2+ tumors treated with neo-adjuvant LT in the PAMELA trial (NCT01973660), a finding that is in line with what we observed in our P cells treated with short-term LT. Conclusions. Upon acquisition of resistance to sustained HER2 inhibition, the MVA pathway-Y/T axis takes over the regulation of pro-proliferative transcriptional programs that are generally downstream of HER2 signaling in treatment-naïve HER2+ BC. The MVA pathway-Y/T axis leads to Y/T-driven transcriptional reprogramming, an emerging mechanism of therapy resistance to anti-HER2 and other targeted therapies that warrants further investigation. The identification of multiple cell cycle related processes as putative targets of the MVA pathway-Y/T axis presents additional targetable vulnerabilities and implies that inhibitors of Y/T and cell cycle checkpoints may help circumvent anti-HER2 resistance in the clinical setting. Citation Format: Vidyalakshmi Sethunath, Xiaoyong Fu, Pamela L Luna, Sarmistha Nanda, Martin Shea, Jamunarani Veeraraghavan, Carmine De Angelis, Huizhong Hu, Chad Shaw, Mothaffar Rimawi, C. Kent Osborne, Rachel Schiff. Insights into the molecular underpinnings of the mevalonate pathway-YAP/TAZ-driven anti-HER2 therapy resistance in HER2+ breast cancer (BC) [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 PS5-29.

Purpose: Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor–positive (ER+)/HER2− breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. Experimental Design: Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation–resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes. Results: Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an “IFN-related palbociclib-resistance Signature” (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2− tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. Conclusions: Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

Background: Adhesion G protein-coupled receptors (GPCRs) form the second largest GPCR subfamily and control many cellular processes involved in cancer. ADGRF1 is an adhesion GPCR overexpressed and gene amplified in HER2+ and triple-negative breast cancer (BC). We have previously found a role of ADGRF1 in promoting tumorigenicity and metastatic processes in cell-based studies in HER2+ BC. The goal of this study was to evaluate the effects of ADGRF1 overexpression in vivo in HER2+ BC. Methods: BT474 stable cell lines were generated using lentiviral system containing ADGRF1 cDNA using PHAGE system, overexpressing ADGRF1 in Doxycycline (Dox)-inducible manner. Mice (N=8/group) were injected with various numbers of cells with (+) or without (-) Dox (10K, 30K, 100K, 300K, 1 million) to assess for tumor initiation and growth. Transcriptomic and proteomic analysis of ADGRF1-overexpressing BT474 cells was also performed using RNAseq and RPPA analysis, respectively. The candidates were validated using western blotting and IHC staining. Cell cycle analysis was conducted by measuring DNA content with propidium iodide using flow cytometry. Results: Dox-induced overexpression of ADGRF1 in mice led to inhibition of tumor growth compared to no Dox treated tumors. Furthermore, fewer mice injected with Dox-treated cells formed tumors. Our RNA-Seq data revealed downregulation of genes involved in cell cycle regulation, which included E2F targets, G2M checkpoint pathways, and MYC targets. Likewise in RPPA analysis, several proteins in the cell cycle pathway (FOXO1, AuroraA, Ki67, Ezh2, CHAF1, DEPTOR, Rb1, Phospho-Rb (S780), and Hexokinase II) were downregulated by ADGRF1 overexpression. Validation of RNAseq and RPPA candidates demonstrated lower number of Ki67+ cells and reduced NF-kB expression, and inhibition of STAT3 phosphorylation. Furthermore, we found that ADGRF1 overexpression by Dox also led to cell cycle arrest at GO/G1 phase. Conclusion: Our results suggest that ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer. These results are in contrast to the in vitro data demonstrating a role of ADGRF1 in promoting tumorigenicity and metastatic processes in HER2+ BC. Understanding the mechanism of this difference is critical in investigating ADGRF1 as a drug target in breast cancer. Citation Format: Raksha Bhat, Noor Mazin Abdulkareem, Lanfang Qin, Carmine De Angelis, Hala Yasser, Hariprasad Thangavel, Jennifer Torres, Vidyalakshmi Sethunath, Tamika Mitchell, Sarmistha Nanda, Resel Pereira, Martin Shea, Rachel Schiff, Meghana Trivedi. ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-07.

Purpose: Tumor-infiltrating lymphocytes (TIL) are associated with benefit to trastuzumab and chemotherapy in patients with early-stage HER2+ breast cancer. The predictive value of TILs, TIL subsets, and other immune cells in patients receiving chemotherapy-sparing lapatinib plus trastuzumab treatment is unclear. Experimental Design: Hematoxylin and eosin–stained slides (n = 59) were used to score stromal (s-)TILs from pretreatment biopsies of patients enrolled in the neoadjuvant TBCRC006 trial of 12-week lapatinib plus trastuzumab therapy (plus endocrine therapy for ER+ tumors). A 60% threshold was used to define lymphocyte-predominant breast cancer (LPBC). Multiplexed immunofluorescence (m-IF) staining (CD4, CD8, CD20, CD68, and FoxP3) and multispectral imaging were performed to characterize immune infiltrates in single formalin-fixed paraffin-embedded slides (n = 33). Results: The pathologic complete response (pCR) rate was numerically higher in patients with LPBC compared with patients with non-LPBC (50% vs. 19%, P = 0.057). Unsupervised hierarchical clustering of the five immune markers identified two patient clusters with different responses to lapatinib plus trastuzumab treatment (pCR = 7% vs. 50%, for cluster 1 vs. 2 respectively; P = 0.01). In multivariable analysis, cluster 2, characterized by high CD4+, CD8+, CD20+ s-TILs, and high CD20+ intratumoral TILs, was independently associated with a higher pCR rate (P = 0.03). Analysis of single immune subpopulations revealed a significant association of pCR with higher baseline infiltration by s-CD4, intratumoral (i-) CD4, and i-CD20+ TILs. Conclusions: LPBC was marginally associated with higher pCR rate than non-LPBC in patients with lapatinib plus trastuzumab treated HER2+ breast cancer. Quantitative assessment of the immune infiltrate by m-IF is feasible and may help correlate individual immune cell subpopulations and immune cell profiles with treatment response.

Forkhead box A1 (FOXA1) is a pioneer factor that facilitates chromatin binding and function of lineage-specific and oncogenic transcription factors. Hyperactive FOXA1 signaling due to gene amplification or overexpression has been reported in estrogen receptor-positive (ER + ) endocrine-resistant metastatic breast cancer. However, the molecular mechanisms by which FOXA1 up-regulation promotes these processes and the key downstream targets of the FOXA1 oncogenic network remain elusive. Here, we demonstrate that FOXA1 overexpression in ER + breast cancer cells drives genome-wide enhancer reprogramming to activate prometastatic transcriptional programs. Up-regulated FOXA1 employs superenhancers (SEs) to synchronize transcriptional reprogramming in endocrine-resistant breast cancer cells, reflecting an early embryonic development process. We identify the hypoxia-inducible transcription factor hypoxia-inducible factor-2α (HIF-2α) as the top high FOXA1-induced SE target, mediating the impact of high FOXA1 in activating prometastatic gene sets and pathways associated with poor clinical outcome. Using clinical ER + /HER2 − metastatic breast cancer datasets, we show that the aberrant FOXA1/HIF-2α transcriptional axis is largely nonconcurrent with the ESR1 mutations, suggesting different mechanisms of endocrine resistance and treatment strategies. We further demonstrate the selective efficacy of an HIF-2α antagonist, currently in clinical trials for advanced kidney cancer and recurrent glioblastoma, in reducing the clonogenicity, migration, and invasion of endocrine-resistant breast cancer cells expressing high FOXA1. Our study has uncovered high FOXA1-induced enhancer reprogramming and HIF-2α–dependent transcriptional programs as vulnerable targets for treating endocrine-resistant and metastatic breast cancer.

Despite effective strategies, resistance in HER2+ breast cancer remains a challenge. While the mevalonate pathway (MVA) is suggested to promote cell growth and survival, including in HER2+ models, its potential role in resistance to HER2-targeted therapy is unknown. Parental HER2+ breast cancer cells and their lapatinib-resistant and lapatinib + trastuzumab–resistant derivatives were used for this study. MVA activity was found to be increased in lapatinib-resistant and lapatinib + trastuzumab–resistant cells. Specific blockade of this pathway with lipophilic but not hydrophilic statins and with the N-bisphosphonate zoledronic acid led to apoptosis and substantial growth inhibition of R cells. Inhibition was rescued by mevalonate or the intermediate metabolites farnesyl pyrophosphate or geranylgeranyl pyrophosphate, but not cholesterol. Activated Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) and mTORC1 signaling, and their downstream target gene product Survivin, were inhibited by MVA blockade, especially in the lapatinib-resistant/lapatinib + trastuzumab–resistant models. Overexpression of constitutively active YAP rescued Survivin and phosphorylated-S6 levels, despite blockade of the MVA. These results suggest that the MVA provides alternative signaling leading to cell survival and resistance by activating YAP/TAZ–mTORC1–Survivin signaling when HER2 is blocked, suggesting novel therapeutic targets. MVA inhibitors including lipophilic statins and N-bisphosphonates may circumvent resistance to anti-HER2 therapy warranting further clinical investigation. Implications: The MVA was found to constitute an escape mechanism of survival and growth in HER2+ breast cancer models resistant to anti-HER2 therapies. MVA inhibitors such as simvastatin and zoledronic acid are potential therapeutic agents to resensitize the tumors that depend on the MVA to progress on anti-HER2 therapies.

Translocation renal cell carcinoma (tRCC) is an aggressive and poorly-characterized subtype of kidney cancer driven by MiT/TFE gene fusions. Here, we define the landmarks of tRCC through an integrative analysis of 152 tRCC patients identified across multiple genomic, clinical trial, and retrospective cohorts. Most tRCCs harbor few somatic alterations apart from MiT/TFE fusions and homozygous deletions at chromosome 9p21.3 (19.2% of cases). Transcriptionally, tRCCs display a heightened NRF2-driven antioxidant response that is associated with resistance to many targeted therapies. Consistently, we find that outcomes for tRCC patients treated with vascular endothelial growth factor receptor inhibitors (VEGFR-TKI) are worse than those treated with immune checkpoint inhibition (ICI). Multiparametric immunofluorescence confirmed the presence of CD8 + tumor-infiltrating T cells compatible with a clinical benefit from ICI and revealed an exhaustion immunophenotype distinct from clear cell RCC. Our findings comprehensively define the clinical and molecular features of tRCC and may inspire new therapeutic hypotheses.

Lapatinib (L) plus trastuzumab (T), with endocrine therapy for estrogen receptor (ER)+ tumors, but without chemotherapy, yielded meaningful response in HER2+ breast cancer (BC) neoadjuvant trials. The irreversible/pan-HER inhibitor neratinib (N) has proven more potent than L. However, the efficacy of N+T in comparison to pertuzumab (P) + T or L + T (without chemotherapy) remains less studied. To address this, mice bearing HER2+ BT474-AZ (ER+) cell and BCM-3963 patient-derived BC xenografts were randomized to vehicle, N, T, P, N+T, or P+T, with simultaneous estrogen deprivation for BT474-AZ. Time to tumor regression/progression and incidence/time to complete response (CR) were determined. Changes in key HER pathway and proliferative markers were assessed by immunohistochemistry and western blot of short-term-treated tumors. In the BT474-AZ model, while all N, P, T, N + T, and P + T treated tumors regressed, N + T-treated tumors regressed faster than P, T, and P + T. Further, N + T was superior to N and T alone in accelerating CR. In the BCM-3963 model, which was refractory to T, P, and P + T, while N and N + T yielded 100% CR, N + T accelerated the CR compared to N. Ki67, phosphorylated (p) AKT, pS6, and pERK levels were largely inhibited by N and N + T, but not by T, P, or P + T. Phosphorylated HER receptor levels were also markedly inhibited by N and N + T, but not by P + T or L + T. Our findings establish the efficacy of combining N with T and support clinical testing to investigate the efficacy of N + T with or without chemotherapy in the neoadjuvant setting for HER2+ BC.

Androgen receptor (AR) signaling is the central driver of prostate cancer across disease states. While androgen deprivation therapy (ADT) is effective in the initial treatment of prostate cancer, resistance to ADT or to next-generation androgen pathway inhibitors invariably arises, most commonly through the re-activation of the AR axis. Thus, orthogonal approaches to inhibit AR signaling in advanced prostate cancer are essential. Here, via genome-scale CRISPR-Cas9 screening, we identify protein arginine methyltransferase 1 (PRMT1) as a critical mediator of AR expression and signaling. PRMT1 regulates the recruitment of AR to genomic target sites and the inhibition of PRMT1 impairs AR binding at lineage-specific enhancers, leading to decreased expression of key oncogenes, including AR itself. In addition, AR-driven prostate cancer cells are uniquely susceptible to combined AR and PRMT1 inhibition. Our findings implicate PRMT1 as a key regulator of AR output and provide a preclinical framework for co-targeting of AR and PRMT1 in advanced prostate cancer.

Background: Lapatinib (L) plus trastuzumab (T) alone or with endocrine therapy for HER2+/ER+ tumors but without chemotherapy, yielded complete tumor eradication in xenograft models. In neoadjuvant trials (NCT00548184, 00999804, 01973660), a substantial number of patients achieved pathologic complete response with this same strategy. The irreversible pan-HER inhibitor neratinib (N) has been recently approved by the FDA for early stage HER2+ breast cancer and has shown greater potency compared to L in the preclinical setting. However, the therapeutic efficacy of N in combination with T (N+T) and how it compares to pertuzumab (P) +T (without chemotherapy) has not been well studied. We hypothesize that dual HER2 inhibition using N+T will be highly efficacious and more effective than P+T due to more complete blockade of the HER pathway. Here, we evaluate the therapeutic efficacy of N, P, and T, either alone or in combination, with a primary focus on comparing N+T vs. P+T in established cell line- and patient-derived xenograft (PDX) models. Methods: Athymic nude and SCID/Beige mice bearing BT474-AZ cell line (ER+/HER2+), and BCM-3963 PDX tumors (ER-/HER2+, wild-type PIK3CA), respectively were randomized to vehicle, N (20mg/kg, 5 days/week), T (10mg/kg, twice a week), P (6mg/kg, once a week), N+T, or P+T, with simultaneous estrogen (E2) deprivation (ED) in BT474-AZ model. Treatment response was assessed by biweekly tumor measurements. Study endpoints included time to tumor regression (TTR) and progression (TTP) (tumor halving/doubling over baseline, respectively), and the rate and time of complete response (CR and TCR, respectively). Results were analyzed using survival analysis (Kaplan-Meier estimates) and generalized Wilcoxon tests. Results: In the BT474-AZ model, mice treated with E2+vehicle and ED+vehicle showed steady tumor growth, with a median TTP of 8 and 25 days, respectively. While tumor regression was observed in 100% of mice treated with N, P, T, N+T, and P+T, tumors treated with N+T regressed faster compared to P (p Model/TreatmentN of miceMedian TTP (Days)Median TTR (Days)Median TCR (Days)CR (%)BT474-AZ E2+Vehicle98--0ED+Vehicle1025--0ED+N13-214100ED+T12-519100ED+P12-185492ED+N+T13-210100ED+P+T14-414100BCM-3963 Vehicle1511--0N15-417100T1416--0P1319--0N+T19-614100P+T1617--0 Conclusions: Our findings establish the preclinical efficacy of combining N with T for HER2+ breast cancer and warrant further clinical testing to investigate the efficacy of N+T without chemotherapy in the neoadjuvant setting for patients with HER2+ breast cancer. Citation Format: Veeraraghavan J, Sethunath V, Qin L, Shea MJ, Mitchell T, De Angelis C, Nanda S, Diala I, Lalani AS, Hilsenbeck SG, Rimawi MF, Osborne CK, Schiff R. Neratinib in combination with trastuzumab is superior to each alone and to pertuzumab plus trastuzumab in HER2-positive in vivo breast cancer models [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 P6-17-12.

Our long-term goal is to discover adhesion GPCR targets in breast cancer. Our previous studies have found GPR110 to be overexpressed in tumorigenic cell population as well as in anti-HER2 drug-resistant derivatives of HER2+ breast cancer cells. In subsequent studies, we found that GPR110 knockdown inhibited anchorage-independent cell growth, mammosphere formation, and invasion/migration of HER2+ breast cancer cells. Conversely, overexpression of GPR110 by lentiviral delivery of cDNA enhanced anchorage-independent cell growth, mammosphere formation, and invasion/migration in HER2+ breast cancer cells. In addition, GPR110 overexpression led to increase in the % of Aldefluor-positive tumorigenic cell population, further emphasizing the role of GPR110 as a mediator of tumorigenesis in addition to the metastatic processes in HER2+ breast cancer. Among various subtypes of breast cancer, GPR110 expression was higher in HER2+ and basal subtypes, most of which are triple-negative (negative for ER, PR, and HER2), compared to luminal A and B subtypes. GPR110 was either gene amplified or upregulated in 4% of all breast cancers based on the publicly available TCGA dataset. GPR110 overexpression predicted poorer recurrence-free survival in triple-negative breast cancer. Furthermore, GPR110 was overexpressed in brain metastatic lesions compared to mammary tumors in patient-derived xenograft models of triple-negative breast cancer (WHIM2 and WHIM30). Knocking down GPR110 reduced anchorage-dependent and -independent cell growth, mammosphere formation, and invasion/migration of triple-negative breast cancer cells. Overall, our results suggest that GPR110 may be a potential drug target in HER2+ and triple-negative breast cancer. Drug discovery efforts to identify GPR110 antagonists will provide useful pharmacological tools for validating GPR110 as a drug target in breast cancer. Since GPR110 is also overexpressed in various other types of cancer, understanding the mechanism of GPR110 upregulation and signaling in cancer is an important future direction. This work was supported by the Department of Defense Grants W81XWH-14-1-0340 and W81XWH-14-1-0341 to Drs. Trivedi and Schiff, respectively. Citation Format: Bhat R, Qin L, De Angelis C, Sahay D, Bhargava D, Creighton C, Yadav P, Yazdanfard S, Alrawi A, Yadav V, Vasaikar S, Nanda S, Sethunath V, Fu X, Zhang B, Narkar V, Schiff R, Trivedi M. Role of GPR110 in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-10.

522 Background: Our preclinical findings suggest a role for the mevalonate pathway (MVA) in treatment resistance in HER2+ BC by providing alternative growth and survival signaling to bypass potent HER2 blockade, which could be overcome by the MVA inhibitors statins and nitrogen-containing bisphosphonates (NBs). Here we explored the effect of MVA inhibitors’ use on pts’ outcomes in the ALTTO trial (BIG2-06; NCT00490139). Methods: In the ALTTO trial, 8381 pts with HER2+ BC were randomized to 1 year of adjuvant lapatinib (L), trastuzumab (T), L+T, or T→L. All pts with documented treatment start with statins or NBs < 1 year after randomization were considered as MVA inhibitors users. Survival curves, with a median follow-up of 6.9 years, for disease-free survival (DFS), distant relapse-free interval (DRFI), BC-specific survival (BCSS), and overall survival (OS) according to MVA inhibitors use were estimated by the Kaplan Meier method and Log-rank test. All multivariate survival analyses employed a Cox proportional hazards regression model, adjusting for tumor size, nodal status, hormonal receptor (HoR), menopausal status, BMI, timing of chemo, and randomization arm. We considered interactions terms in Cox’s model between MVA inhibitors use and randomization arm, hormonal status, and BMI group. Results: Among the 8381 pts included in this study, 493 and 299 were statins or NBs users, respectively. Table 1 summarizes the significant differences in pts’ characteristics according to MVA inhibitors use ( P

Translocation renal cell carcinoma (tRCC) is a poorly-characterized subtype of kidney cancer driven by MiT/TFE gene fusions. Here, we define the landmarks of tRCC through an integrative analysis of 152 patients with tRCC identified across genomic, clinical trial, and retrospective cohorts. Most tRCCs harbor few somatic alterations apart from MiT/TFE fusions and homozygous deletions at chromosome 9p21.3 (19.2% of cases). Transcriptionally, tRCCs display a heightened NRF2-driven antioxidant response that is associated with resistance to targeted therapies. Consistently, we find that outcomes for patients with tRCC treated with vascular endothelial growth factor receptor inhibitors (VEGFR-TKI) are worse than those treated with immune checkpoint inhibition (ICI). Using multiparametric immunofluorescence, we find that the tumors are infiltrated with CD8(+) T cells, though the T cells harbor an exhaustion immunophenotype distinct from that of clear cell RCC. Our findings comprehensively define the clinical and molecular features of tRCC and may inspire new therapeutic hypotheses.

Advances in HER2-targeted therapies have improved the survival of patients with HER2-positive breast cancer. The standard-of-care treatment for localized disease has been chemotherapy and 1 year of adjuvant HER2-targeted therapy, typically with the anti-HER2 antibody trastuzumab. Despite the effectiveness of this treatment, disease relapse occurs in a subset of patients; thus, focus has been placed on escalating treatment by either combining different HER2-targeted agents or extending the duration of HER2-targeted therapy. Indeed, dual HER2-targeted therapies and extended-duration anti-HER2 therapy, as well as adjuvant therapy with the anti-HER2 antibody–drug conjugate T-DM1, have all been approved for clinical use. Emerging evidence suggests, however, that some patients do not derive sufficient benefit from these additional therapies to offset the associated toxicities and/or costs. Similarly, the universal use of chemotherapy might not benefit all patients, and treatment de-escalation through omission of chemotherapy has shown promise in clinical trials and is currently being explored further. The future of precision medicine should therefore involve tailoring of therapy based on the genetics and biology of each tumour and the clinical characteristics of each patient. Predictive biomarkers that enable the identification of patients who will benefit from either escalated or de-escalated treatment will be crucial to this approach. In this Review, we summarize the available HER2-targeted agents and associated mechanisms of resistance, and describe the current therapeutic landscape of early stage HER2-positive breast cancer, focusing on strategies for treatment escalation or de-escalation. HER2-targeted therapy has greatly improved the outcomes of patients with HER2-positive breast cancer, with a range of agents now approved or in late-stage clinical development. In the era of precision medicine, efforts are being made to further improve patient outcomes by personalizing HER2-targeted treatment regimens, primarily though escalation or de-escalation of therapy according to the disease biology. In this Review, the authors provide an overview of the current landscape of HER2-targeted therapy and discuss the evidence supporting such tailored therapeutic strategies.

PURPOSE: CDK4/6 inhibitors are highly effective against ER+/HER2- breast cancer (BC); however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i’s may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. EXPERIMENTAL DESIGN: Parental BC cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen-deprivation resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes. RESULTS: Parental and EndoR BC lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high interferon (IFN) signaling and reduced CDK4/6i sensitivity; thus an ‘IFN-Related Palbociclib-Resistance Signature’ (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1-signaling compared to their short-term treated or untreated counterparts. In primary ER+/HER2- tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. CONCLUSION: Aberrant IFN-signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN-pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

The role of HER2 and PIK3CA mutations in anti-HER2 resistance is gaining more importance in HER2-positive (+) breast cancer. We recently reported that acquired resistance to lapatinib (Lap)-containing regimens is mediated by HER2 L755S, which could be overcome using the irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (Nrb). However, less is known about the role of L755S in resistance to next-generation TKIs, particularly when co-occurring with PIK3CA mutations. HER2+ BT474 cell models with primary or sequential acquired resistance (R) to Lap (LapR) or Nrb (NrbR) and their parental (P) counterparts were profiled for alterations in signaling and gene expression by RPPA, western blot, and RNA-seq. For drug efficacy studies, change in cell growth was assessed using imaging-based high-throughput system. Proteomic profiling revealed partial restoration of HER2 phosphorylation and downstream signaling in the LapR and NrbR derivatives. RNA-seq analysis showed that the LapR and NrbR models, but not P cells, harbor HER2 L755S mutation. Importantly, the NrbR but not LapR cells also co-acquire a PIK3CA pathogenic mutation. GSEA analysis of RNA-seq data showed significant downregulation of G2/M checkpoint in the R derivatives compared to P cells, suggesting genetic instability. In line with the presence of HER2 and PIK3CA activating mutations and HER pathway reactivation in the R models, GSEA revealed an enrichment of mTORC1 and KRAS signaling in the R cells. Furthermore, enrichment of epithelial mesenchymal transition signature and downregulation of apical surface genes was observed in the R models compared to P cells, suggestive of their aggressive phenotype. Interestingly, the LapR cells remained sensitive to Nrb, though a higher dose (IC50: ~50nM) was required compared to P cells (IC50: ~2nM). The LapR and NrbR cells were cross-resistant to the HER2-selective TKI tucatinib, and trastuzumab. We recently showed that the NrbR cells were either partially or completely sensitive to poziotinib or TDM1, respectively, suggesting their therapeutic promise against HER2- and PIK3CA-mutant tumors. Of note, our studies using small molecule agents targeting HER and its downstream pathway to facilitate treatment of CNS lesions suggest that AKT or mutant PIK3CA inhibitors are effective only when combined with either neratinib or poziotinib, but not tucatinib, findings which we are currently expanding to xenograft-derived organoids. Overall, our findings suggest a complex disease evolution upon resistance to neratinib but indicate their potentially continued efficacy in overcoming resistance through drug combinations. Ongoing integrative omics analysis to determine the genomic and mutational complexity and landscape will uncover additional mechanistic insights and guide the discovery of other actionable targets. Citation Format: Jamunarani Veeraraghavan, Ragini Mistry, Sarmistha Nanda, Sreyashree Bose, Chia Chia Liu, Vidyalakshmi Sethunath, Martin J. Shea, Tamika Mitchell, Meenakshi Anurag, Michael A. Mancini, Irmina Diala, Alshad S. Lalani, Fabio Stossi, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. Acquired neratinib resistance is associated with acquisition of HER2 and PIK3CA mutations and can be overcome using potent drug combinations in HER2-positive breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1077.

Background Lung cancer is the leading cause of cancer-related deaths across the world. In this study, we present therapeutically relevant genetic alterations in lung adenocarcinoma of Indian origin. Materials and methods Forty-five primary lung adenocarcinoma tumors were sequenced for 676 amplicons using RainDance cancer panel at an average coverage of 1500 × (reads per million mapped reads). To validate the findings, 49 mutations across 23 genes were genotyped in an additional set of 363 primary lung adenocarcinoma tumors using mass spectrometry. NIH/3T3 cells over expressing mutant and wild-typeFGFR3 constructs were characterized for anchorage independent growth, constitutive activation, tumor formation and sensitivity to FGFR inhibitors usingin vitro and xenograft mouse models. Results We present the first spectrum of actionable alterations in lung adenocarcinoma tumors of Indian origin, and shows that mutations ofFGFR3 are present in 20 of 363 (5.5%) patients. TheseFGFR3 mutations are constitutively active and oncogenic when ectopically expressed in NIH/3T3 cells and using a xenograft model in NOD/SCID mice. Inhibition ofFGFR3 kinase activity inhibits transformation of NIH/3T3 overexpressingFGFR3 constructs and growth of tumors driven byFGFR3 in the xenograft models. The reduction in tumor size in the mouse is paralleled by a reduction in the amounts of phospho-ERK, validating thein vitro findings. Interestingly, theFGFR3 mutations are significantly higher in a proportion of younger patients and show a trend toward better overall survival, compared with patients lacking actionable alterations or those harboringKRAS mutations. Conclusion We present the first actionable mutation spectrum in Indian lung cancer genome. These findings implicateFGFR3 as a novel therapeutic in lung adenocarcinoma.

// Pawan Upadhyay 1, * , Sudhir Nair 2, * , Ekjot Kaur 3 , Jyotirmoi Aich 1 , Prachi Dani 1 , Vidyalakshmi Sethunath 1 , Nilesh Gardi 1 , Pratik Chandrani 1 , Mukul Godbole 1 , Kavita Sonawane 2 , Ratnam Prasad 1 , Sadhana Kannan 4 , Beamon Agarwal 5 , Shubhada Kane 6 , Sudeep Gupta 7 , Shilpee Dutt 3 , Amit Dutt 1 1 Integrated Genomics Laboratory, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai- 410210, India 2 Division of Head and Neck Oncology, Department of Surgical Oncology, Tata Memorial Hospital, Tata Memorial Centre,Mumbai- 4100012, India 3 Shilpee Laboratory, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai- 410210, India 4 Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai- 410210, India 5 Department of Pathology, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai- 410210, India 6 Department of Pathology, Tata Memorial Hospital, Tata Memorial Centre, Mumbai- 400012, India 7 Department of Medical Oncology, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Mumbai- 400012, India * These authors have contributed equally to this work Correspondence to: Amit Dutt, email: adutt@actrec.gov.in Keywords: early stage tongue cancer, exome and transcriptome sequencing, IHC based expression analysis, cancer stem cell-like feature, Notch pathway inhibitors Received: January 25, 2016 Accepted: June 07, 2016 Published: July 06, 2016 ABSTRACT Background: Notch pathway plays a complex role depending on cellular contexts: promotes stem cell maintenance or induces terminal differentiation in potential cancer-initiating cells; acts as an oncogene in lymphocytes and mammary tissue or plays a growth-suppressive role in leukemia, liver, skin, and head and neck cancer. Here, we present a novel clinical and functional significance of NOTCH1 alterations in early stage tongue squamous cell carcinoma (TSCC). Patients and Methods: We analyzed the Notch signaling pathway in 68 early stage TSCC primary tumor samples by whole exome and transcriptome sequencing, real-time PCR based copy number, expression, immuno-histochemical, followed by cell based biochemical and functional assays. Results: We show, unlike TCGA HNSCC data set, NOTCH1 harbors significantly lower frequency of inactivating mutations (4%); is somatically amplified; and, overexpressed in 31% and 37% of early stage TSCC patients, respectively. HNSCC cell lines over expressing NOTCH1 , when plated in the absence of attachment, are enriched in stem cell markers and form spheroids. Furthermore, we show that inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 inhibits spheroid forming capacity, transformation, survival and migration of the HNSCC cells suggesting an oncogenic role of NOTCH1 in TSCC. Clinically, Notch pathway activation is higher in tumors of non-smokers compared to smokers (50% Vs 18%, respectively, P =0.026) and is also associated with greater nodal positivity compared to its non-activation (93% Vs 64%, respectively, P =0.029). Conclusion: We anticipate that these results could form the basis for therapeutic targeting of NOTCH1 in tongue cancer.

Despite the availability of potent HER-targeted agents, de novo and acquired resistance is common and continues to pose a major challenge, especially in the advanced setting. Amassing evidence point to the importance of HER2 mutations, including the most common HER2 L755S mutation, in mediating anti-HER2 resistance. The HER2 L755S mutation, in particular, is observed to be enriched in metastatic lesions compared to primary breast tumors. The need for effective therapy to treat tumors harboring HER2 mutations prevails. We have previously reported that acquired resistance to lapatinib (L)-containing treatments, mediated by HER2 L755S, could be overcome by the recently FDA-approved irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (N). While N has shown great promise in patients with HER2-mutant metastatic breast cancer, its efficacy is somewhat limited. More recently, tucatinib, a HER2-selective TKI, has been shown to be effective in HER2-positive (+) brain metastases. Its potency in the context of HER2 mutations, however, has not yet been fully studied. In this study, we used the HER2+ BT474-L resistant (LR) cells, harboring endogenous HER2 L755S mutation, and parental (P) cells to first determine whether tucatinib may be effective in overcoming resistance mediated by HER2 mutations. Our results showed that while N effectively inhibited the growth of LR cells, although at a dose higher than that needed to inhibit the growth of naïve P cells, tucatinib failed to inhibit the growth of LR cells. Our results suggest that HER2 L755S mutation may confer cross-resistance to tucatinib. To further study mechanisms of resistance to 2nd generation anti-HER2 agents, we recently developed cell models with acquired resistance to N, through long-term exposure of the BT474-P and LR cells to increasing doses of N. These cells were profiled by reverse phase protein array (RPPA) and western blot analysis, which revealed restoration of HER2 phosphorylation in the NR derivatives, despite being cultured in the continuous presence of N. Interestingly, RNA-seq analysis revealed the presence of HER2 L755S mutation in all the NR derivatives, but not in the P cells, suggesting that the reactivated HER2 signaling observed in NR cells could be attributed to the emergence/acquisition of HER2 L755S mutation. Furthermore, while the P cells were highly sensitive to tucatinib, L, and the monoclonal antibody trastuzumab (T), the NR derivatives were totally resistant to these agents, suggesting that N resistance may also confer cross-resistance to tucatinib, L, and T. Additional molecular characterization to examine differential gene expression and mutational profile of the resistant derivatives, as well as testing of novel anti-HER2 regimens and drug combinations targeting downstream mediators to overcome resistance, both in vitro and in vivo, is ongoing. Citation Format: Jamunarani Veeraraghavan, Ragini Mistry, Sarmistha Nanda, Vidyalakshmi Sethunath, Martin Shea, Tamika Mitchell, Meenakshi Anurag, Michael A. Mancini, Fabio Stossi, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. HER2 L755S mutation is associated with acquired resistance to lapatinib and neratinib, and confers cross-resistance to tucatinib in HER2-positive breast cancer models [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 1911.

PURPOSE: G protein-coupled receptors (GPCRs) represent the largest family of druggable targets in human genome. Although several GPCRs can cross-talk with the human epidermal growth factor receptors (HERs), the expression and function of most GPCRs remain unknown in HER2+ breast cancer (BC). In this study, we aimed to evaluate gene expression of GPCRs in tumorigenic or anti-HER2 drug-resistant cells and to understand the potential role of candidate GPCRs in HER2+ BC. METHODS: Gene expression of 352 GPCRs was profiled in Aldeflur+ tumorigenic versus Aldeflur- population and anti-HER2 therapy-resistant derivatives versus parental cells of HER2+ BT474 cells. The GPCR candidates were confirmed in 7 additional HER2+ BC cell line models and publicly available patient dataset. Anchorage-dependent and -independent cell growth, mammosphere formation, and migration/invasion was evaluated upon GPR110 knockdown by siRNA in BT474 and SKBR3 parental and lapatinib+trastuzumab-resistant (LTR) cells. RESULTS: Adhesion and class A GPCRs were overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population of BT474 cells, respectively. GPR110 was the only GPCR overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population in BT474, SKBR3, HCC1569, MDA-MB-361, AU565, and/or HCC202 cells and in HER2+ BC subtype in patient tumors. Using BT474 and SKBR3 parental and LTR cells, we found that GPR110 knockdown significantly reduced anchorage-dependent/independent cell growth as well as migration/invasion of parental and LTR cells and mammosphere formation in LTR derivatives and not in parental cells. CONCLUSION: Our data suggests a potential role of GPR110 in tumorigenicity and in tumor cell dissemination in HER2+ BC.

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies.Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo.Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

Background: In the PALOMA-3 trial, the combination of the CDK4/6 inhibitor palbociclib (P) with fulvestrant (F) led to a significant improvement in clinical outcomes, compared to F+Placebo, in endocrine-pretreated patients (pts) with ER+/HER2- metastatic breast cancer (BC). Not all pts, however, benefit from F+P, and virtually all ultimately progress. Elucidating the resistance mechanisms would assist in identifying biomarkers to select pts who may most benefit from F+P, as well as novel therapeutic targets for combating resistance. Materials and Methods: The ER+ MCF7 BC cell lines made resistant (R) to long-term estrogen deprivation (EDR) or tamoxifen (TamR), and the 600MPE de novo TamR cells were tested for sensitivity to F, P and F+P. MCF7 EDR and MCF7 TamR cells were exposed to increasing concentrations of F+P to generate R derivatives (FPR, cells grow in the presence of F 100nM + P 100nM). The transcriptomic and proteomic profiles of MCF7 EDR, TamR, EDR/FPR and TamR/FPR were determined by RNA-seq and reverse-phase protein arrays (RPPA), respectively. Pathway analysis was performed by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). mRNA and protein levels of select markers were assessed by RT-PCR and Western blot (WB), respectively. Cell growth, colony formation, and migratory and invasive potential were assessed by methylene blue staining, clonogenic assay, and in vitro migration and invasion assays. Results: Treatment of MCF7 EDR and TamR cells with P resulted in a dose-dependent inhibition of cell growth [IC50 = 149nM and 89nM, respectively] and colony formation [clonogenic inhibition at 100nM >85% and >95%, respectively]. In both MCF7 endocrine-R models, P (100nM) or F (100nM) monotherapy significantly inhibited cell growth and reduced phosphorylated (p)-Rb and the E2F target CDK2 protein levels compared to vehicle control. However, F+P resulted in an enhanced cell growth inhibition (>90%) and a more pronounced reduction in p-Rb and CDK2 expression compared to each agent alone. In the FPR lines, ‘oncogenic signatures’ representative of altered KRAS and MEK signaling were highly enriched compared to their sensitive counterparts, as revealed by GSEA analysis of the transcriptomic profiles. IPA analysis of the proteomic profiles confirmed the activation of the MAPK signaling pathway in FPR cells and identified EGFR as one of the top ranked upstream regulators. Increased EGFR mRNA and protein levels in FPR models were confirmed by RT-PCR and WB analysis. Additionally, gene signatures of cyclin D1 hyperactivation and increased cyclin D1 protein levels were enriched in FPR cell lines. Importantly, the EGFR inhibitor gefitinib (1µM), the pan-HER inhibitor neratinib (250nM), and the MEK1 inhibitor selumetinib (S) (1µM) selectively or more greatly inhibited the cell growth and reduced Cyclin D1 protein levels in FPR cells compared to their sensitive counterparts. Further, we found a significant enrichment of the ‘hallmark’ epithelial-mesenchymal transition gene signature in FPR models, which is in line with their increased migratory and invasive capabilities. Treatment with S reduced the migration and invasion of the FPR cells. Finally, the aggressive 600MPE de novo TamR cells harboring hyperactive MEK/MAPK signaling due to the KRAS and MAP2K4 activating mutations were significantly less sensitive to F, P, and F+P. However, the addition of S to F+P significantly increased the cell growth inhibition in this model. Conclusion: Acquired and intrinsic resistance to F+P is associated with hyperactivation of the EGFR/RAS/MEK signaling pathway, which sustains cell proliferation and promotes an aggressive phenotype. The EGFR/RAS/MEK pathway may serve as a predictive biomarker for F+P resistance and offer therapeutic targets to overcome resistance in pts with ER+/HER2- BC. Citation Format: Carmine De Angelis, Maria Letizia Cataldo, Agostina Nardone, Jamunarani Veeraraghavan, Xiaoyong Fu, Sarmistha Nanda, Lanfang Qin, Vidyalakshmi Sethunath, Resel Pereira, Britta Weigelt, Jorge S. Reis-Filho, Mothaffar F. Rimawi, Rinath Jeselsohn, Kent Osborne, Rachel Schiff. Activation of the EGFR/RAS/p42,44 MAPK axis as a convergent mechanism of resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-02.

Background: The CDK4/6 inhibitors (i) palbociclib (Palbo), ribociclib, and abemaciclib remarkably improved the outcome of patients with metastatic ER+/HER2- breast cancer (BC) and are now under clinical investigation in early BC. Despite high efficacy, de novo and acquired resistance to CDK4/6i is common. Elucidating the molecular basis for sensitivity and resistance to CDK4/6i is crucial to identify predictive biomarkers and novel therapeutic targets to improve patient outcome. Materials and Methods: MCF7, T47D and ZR75-1 parental (P) BC cells and their derivatives made resistant to tamoxifen, estrogen deprivation (EDR), or fulvestrant were used. The P and EDR models of MCF7 and T47D cells were chronically exposed to increasing concentrations of Palbo to generate derivatives with acquired resistance to Palbo (PalboR). The transcriptomic profiles of P, endocrine-resistant (EndoR) and PalboR models were determined by RNA-seq. IC50s were determined by exposing MCF7, T47D, and ZR75-1 P and EndoR lines (n=12) to increasing concentrations of Palbo. Cell growth was assessed by methylene blue staining, and changes in the mRNA and protein levels of key cell cycle molecules were assessed by RT-PCR and Western blot, respectively. Gene expression data from the Cancer Dependency Map (DepMap), baseline tumors from the NeoPalAna (NCT01723774) and neoMONARCH (NCT02441946) neoadjuvant trials, as well as the TCGA and METABRIC datasets were interrogated for correlations of gene signatures and patient outcome (by KMPlot). Results: Palbo treatment resulted in a dose-dependent inhibition of the growth of P and EndoR BC cell lines, with varying degree of sensitivity among the models. GSEA analysis comparing the least sensitive (IC50>350nM) vs. the most sensitive (IC50 Citation Format: Carmine De Angelis, Xiaoyong Fu, Maria Letizia Cataldo, Agostina Nardone, Valerie M. Jansen, Jamunarani Veeraraghavan, Sarmistha Nanda, Lanfang Qin, Vidyalakshmi Sethunath, Resel Pereira, Matteo Benelli, Ilenia Migliaccio, Luca Malorni, Joshua Donaldson, Pier Selenica, David N. Brown, Britta Weigelt, Jorge S. Reis-Filho, Ben H. Park, Sara A. Hurvitz, Dennis J. Slamon, Mothaffar F. Rimawi, Rinath Jeselsohn, Kent Osborne, Rachel Schiff. High levels of interferon-response gene signatures are associated with de novo and acquired resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-01.

Background: Despite the advent of HER2-targeted therapies, including the monoclonal antibody trastuzumab (T) and the HER1/2 inhibitor lapatinib (L), for HER2+ breast cancer (BC), resistance still poses a major challenge. L and L+T resistant (R) HER2+ cells with inhibited HER2 signaling showed upregulation of RNA levels of the mevalonate pathway (MVA) enzymes (which were inhibited by short term treatment of parental (P) cells with L or L+T) and increased sensitivity to MVA inhibition by statins (e.g. simvastatin (Sim)), suggesting MVA’s role as an escape mechanism of resistance. Here we investigated the therapeutic potential of another MVA inhibitor Zoledronate (ZA) and the role of mTOR and YAP/TAZ (Y/T) in mediating this resistance. Lastly, we tested if co-blockade of the MVA or its downstream effectors further sensitizes HER2+ models to anti-HER2 therapies. Methods: SKBR3 and AU565 P cells and their LR and LTR derivatives were used. The effects of MVA perturbations on cell growth and HER2 or MVA signaling were studied by methylene blue staining and Western blot (WB), respectively. Y/T activity was tested by a luciferase reporter assay and functionally validated by siRNA knockdown and dominant-active (DomA) YAP overexpression. YAP target gene expression was assessed by RT PCR. SCID-Beige mice bearing HER2+ BCM-3963 PDX tumors were treated with vehicle, L, Sim, or L+Sim and monitored for time to complete response (CR). Results: ZA, like Sim, showed a selective inhibition of cell growth and mTOR signaling in R vs. P cells, which was rescued only by the downstream metabolite geranyl geranyl pyrophosphate (GGPP), but not by the upstream metabolite mevalonate, indicating the on-target effect of ZA. Increased Y/T activity in R models was confirmed, and both Sim and ZA inhibited TAZ levels and induced phospho-YAP levels, which were rescued by the corresponding downstream metabolites. Y/T knockdown inhibited growth and mTOR signaling in R vs. P cells, and DomA YAP negated the mTOR inhibition by Sim. Sim and ZA also significantly decreased levels of the Y/T target gene survivin in R vs. P cells, and the expression was rescued by the downstream metabolites. Inhibition of MVA by Sim or ZA or its downstream signaling effectors, Y/T (by siRNA) and mTOR (by everolimus), enhanced the L sensitivity in P cells. Conversely, DomA YAP reduced the sensitivity of P cells to L. In the presence of Sim or ZA, L treatment more strongly inhibited levels of phospho-S6, a downstream target of mTORC1, compared to L alone. Preliminary in vivo data showed that treatment with L+Sim vs. L alone shortened the median time to CR and numerically increased CR rates. Conclusions: The MVA pathway mediates anti-HER2 therapy resistance via Y/T, survivin, and mTOR, in some cell models and this resistance can be overcome by Sim and ZA. The potential of MVA pathway inhibition to enhance anti-HER2 therapy efficacy warrants further clinical studies. Citation Format: Vidyalakshmi Sethunath, Huizhong Hu, Carmine DeAngelis, Jamunarani Veeraraghavan, Lanfang Qin, Martin Shea, Tamika Mitchell, Sarmistha Nanda, Resel Pereira, Susan G. Hilsenbeck, Mothaffar F. Rimawi, Kent C. Osborne, Rachel Schiff. Targeting the mevalonate pathway in HER2+breast cancer to overcome resistance and enhance anti-HER2 therapy efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4757.

Background: Despite the advent of HER2-targeted therapies for HER2+ breast cancer (BC), including the monoclonal antibody trastuzumab (T) either alone or in combinations, resistance still poses a major clinical challenge. Using our broad panel of HER2+ cell lines made resistant (R) to T alone (TR), and to lapatinib plus T (LTR), we observed that in resistant models where HER2 remains inhibited, especially the LTR derivative, the mevalonate (MVA) pathway is activated and provides an alternative proliferative signal, including the activation of mTOR, to drive resistance. While these resistant cell models were hypersensitive to the widely-used cholesterol-lowering statins, the role of other MVA pathway inhibitors such as zoledronic acid (ZA), which is in clinical use to treat bone metastasis, in overcoming resistance to HER2-targeted therapy has not been explored. Based on recent reports and our preliminary data using reverse phase protein array (RPPA) analysis, the YAP/TAZ transcription factor (TF) emerged as a potential mediator of MVA pathway signaling to mTOR. Here, we investigated the therapeutic efficacy of additional MVA pathway inhibitors and the role of YAP/TAZ in mediating resistance to HER2-targeted therapy. Methods: HER2+ SKBR3 and AU565 BC cells and their LTR derivatives were used. Changes in cell growth upon genetic and pharmacologic inhibition of the MVA pathway were quantified by methylene blue staining. Luciferase reporter assays and western blots (WB) measured changes in total and phosphorylated (S127 and S381/inactive) YAP protein levels to examine activity of the YAP/TAZ TF complex. To validate the function of YAP/TAZ in resistance, we performed YAP/TAZ knockdown (siRNA), overexpression of dominant-active YAP constructs (S381A, S381/127A), and qRT-PCR assessment of YAP/TAZ target gene expression. Results: ZA, like simvastatin (Sim), selectively inhibited the growth of resistant cells in a dose-dependent manner. This inhibition was rescued by geranyl geranyl pyrophosphate (GGPP), a downstream metabolite, but not by MVA, an upstream metabolite, indicating the on-target effect of ZA. Further, ZA and Sim combination showed a synergistic growth-inhibitory effect in R but not in parental (P) cells. YAP/TAZ luciferase reporter assays and phosphorylated YAP and total TAZ levels by WB, confirmed the increased activity of YAP/TAZ in R models, which was selectively inhibited by Sim or ZA and was rescued by the corresponding downstream metabolites. YAP/TAZ knockdown selectively inhibited resistant cell growth and mTOR signaling in R vs. P cells, and dominant-active YAP/TAZ rescued the mTOR inhibition by Sim. YAP/TAZ inhibition by siRNA or by Sim significantly decreased the expression of YAP/TAZ target gene survivin in R vs. P cells, and the Sim inhibition was rescued by MVA. Conclusions: The MVA pathway plays a critical role in mediating resistance to anti-HER2 therapy, which was overcome by Sim and ZA either alone or in combination. Given the synergistic effect of Sim and ZA, their combination may offer a therapeutic strategy to overcome HER2-targeted therapy resistance. Our results also reveal the role of YAP/TAZ in MVA pathway-mediated HER2-targeted therapy resistance, which could suggest new biomarkers and therapeutic targets. Citation Format: Sethunath V, Hu H, De Angelis C, Veeraraghavan J, Qin L, Rimawi MF, Osborne KC, Schiff R. Targeting the mevalonate pathway in HER2-positive breast cancer to overcome resistance to anti-HER2 therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-04.

Notch pathway play a context-dependent tumor-suppressive or pro-oncogenic role in solid tumors and hematological malignancies. In head and neck cancer, inactivating mutations in NOTCH1 are associated with poor prognosis. Here we perform meta-analysis of DNA copy number and gene expression profiling of NOTCH signaling pathway genes in 35 paired primary T1 and T2 stage tongue tumors, comprising of Notch ligands, receptors and target genes. We find 32% of primary tongue squamous cell carcinoma (TSCC) tumors amplify NOTCH signaling pathway components; and over express activated NOTCH1based on immuno-histochemical staining in an additional series of 50 surgically resected paired TSCC tumors. Interestingly, these alterations: amplification at Notch ligand DLL3 (χ2 = 7.575, P = 0.023); over expression of Notch pathway target gene HEY2 transcript (χ2 = 9.885, P = 0.007); and expression of activated NOTCH1 (χ2 = 7.575, P = 0.023) positively correlate with N+ve and non- smoking status of patients. In a parallel effort, we characterized four HNSCC cell lines– NT8E, OT9, AW13516, and AW8507– established from Indian patients using whole exome, whole transcriptome sequencing and SNP array assays. Significant amplification and over expression of NOTCH signaling pathway components were observed in AW13516 and NT8E cells. Inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 could inhibit transformation, survival, migration and invasion of NT8E cells. Taken together, we present that activation of Notch signaling pathway in a subset of TSCC patients is associated with nodal positivity and non-smoking status among early tongue cancer patients. Citation Format: Pawan Upadhyay, Jyotirmoi Aich, Vidyalakshmi Sethunath, Prachi Dani, Sadhana Kannan, Pratik Chandrani, Kavitha Sonawane, Beamon Agarwal, Shubhada Kane, Sudhir Nair, Amit Dutt. Pro-oncogenic role of NOTCH1 in early tongue squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4811. doi:10.1158/1538-7445.AM2015-4811