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Background: The HER2-specific tyrosine kinase inhibitor (TKI) tucatinib (Tuca) recently approved for advanced HER2+ breast cancer is making a move towards the early setting. Given its growing use, resistance is inevitable, as observed in the HER2CLIMB study, where only one patient with brain metastasis remained progression free after 2 years on Tuca. Driven by the prevailing lack of knowledge about the mechanisms of resistance, in this study, we sought to define these mechanisms and identify treatment strategies to overcome them. We previously reported (SABCS 2021) that our BT474 TucaR models acquired EGFR amplification and showed elevated levels of phosphorylated (p) and total (t) EGFR, pHER2, pHER3, and downstream pAKT and pS6. Since the HER pathway is activated by ligands, here we aim to assess if hyperactivation of EGFR via high levels of its ligands is an alternative mechanism of Tuca resistance. Materials and Methods: Our recently developed HER2+ BT474 (ATCC and AZ) cell models with acquired resistance to Tuca (TucaR) developed through long-term exposure to gradually increasing doses of Tuca and their naïve parental (P) were used. Genomic (DNA-seq), transcriptomic (RNA-seq), and proteomic (western blot) characterization were performed. Changes in cell growth and migration were assessed by methylene blue and Incucyte wound healing assays, respectively. Results: RNA-seq analysis demonstrated that the levels of TGFα was significantly higher in our BT474 TucaR models compared to P cells. Our results now demonstrate that exogenous supplementation of EGF to BT474-P cells rescues the Tuca-mediated inhibition of pEGFR, pHER2, and the downstream pAKT, pERK, and pS6 levels. Exogenous EGF was also found to reduce the levels of apoptosis, as assessed by cleaved PARP, mitigating the Tuca-induced cell death. Exogenous EGF or TGFα rendered naïve BT474 and SKBR3 cells resistant to Tuca while neratinib, a pan-HER TKI, effectively inhibited this ligand-driven cell growth. We previously showed that the HER signaling reactivation observed in our EGFR-amplified TucaR cells was inhibited by the EGFR-specific TKI gefitinib (Gef) (SABCS 2021) and that the TucaR cells displayed enhanced migratory capabilities (AACR 2022). Here, we demonstrate that in addition to curbing the growth of TucaR cells, Gef, either alone or together with Tuca, also markedly reverts the migration of the TucaR cells. Knockdown (KD) of EGFR but not HER2 selectively and substantially inhibited the migration of the TucaR cells. KD of EGFR also had a marked cell killing effect on only the TucaR cells, whereas HER2 KD inhibited the growth of P but not TucaR cells. Our findings are consistent with the notion that while the P cells are functionally dependent on HER2, in TucaR cells the survival dependence could be rewired to rely primarily on the hyperactive EGFR signaling. Genomic analysis further revealed that in addition to EGFR amplification, the AZ TucaR cells also acquired a gain of YES1, a src family receptor tyrosine kinase implicated in cancer cell growth, invasion, and metastasis. Functional studies using 2 siRNAs, however, showed that YES1 KD had no effect on the growth of TucaR cells, and the migration of both TucaR and P cells was equally affected by YES1 KD, precluding the potential role of YES1 in driving the resistant and enhanced migratory phenotypes. Conclusions: Hyperactivation of the EGFR pathway via amplification of EGFR or increased expression of its ligands confers resistance to Tuca, which may be overcome using dual/pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Given the rapidly evolving treatment landscape of HER2+ breast cancer and biomarkers of resistance, our novel findings have potentially crucial therapeutic implications and suggest that rationally sequencing the currently available TKIs may be clinically important. Citation Format: Fu-Tien Liao, Tia Gordon, Chia Chia Liu, Pier Selenica, Yingjie Zhu, Juber Patel, Sarmistha Nanda, Lanfang Qin, Xiaoyong Fu, Andrea Gazzo, Antonio Marra, Juan Blanco-Heredia, Britta Weigelt, Jorge Reis-Filho, C. Kent Osborne, Mothaffar Rimawi, Rachel Schiff, Jamunarani Veeraraghavan. Hyperactivation of the EGFR pathway is associated with resistance to tucatinib in HER2-positive breast cancer models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-17.

Background: Clinical trials reported 25-30% pathologic complete response (pCR) rates in HER2+ breast cancer (BC) patients treated with anti-HER2 therapies without chemotherapy. We hypothesize that a multiparameter classifier can identify patients with HER2 “addicted” tumors who may benefit from a chemotherapy-sparing strategy. Patients and Methods: Baseline HER2+ BC specimens from TBCRC023 and PAMELA trials of neoadjuvant lapatinib+trastuzumab (plus endocrine therapy in ER+ tumors) were used. HER2 protein and gene amplification (ratio), HER2-enriched (HER2-E), and PIK3CA mutation status were assessed by dual gene protein assay (GPA), research-based PAM50, and targeted DNA-sequencing. GPA cutoffs and classifier of response were constructed in TBCRC023 using a decision tree algorithm, then validated in PAMELA. Results: In TBCRC023, 72 BCs had GPA, PAM50, and sequencing data, of which 15 had pCR. Recursive partitioning identified cutoffs of HER2 ratio≥4.6 and %3+ IHC-staining≥97.5%. With PAM50 and sequencing data, the model added HER2-E and PIK3CA wild-type (wt). For clinical implementation, the classifier was locked as HER2 ratio≥4.5 and %3+ IHC-staining≥90% and PIK3CA-wt and HER2-E, yielding 55% and 94% positive (PPV) and negative (NPV) predictive values, respectively. Independent validation using 44 PAMELA cases with all three biomarkers yielded 47% PPV and 82% NPV. Importantly, our classifier’s high NPV signifies its strength in accurately identifying patients who may not be good candidates for treatment de-escalation. Conclusions: Our multiparameter classifier differentially identifies patients who may benefit from HER2-targeted therapy alone from those who need chemotherapy and predicts pCR to anti-HER2 therapy alone comparable to chemotherapy plus dual anti-HER2 therapy in unselected patients.

Figure S1. Correlation of the genomic instability index and copy number breakpoint index with AURKA or PLK1 expression (RNAseq) in 1083 invasive breast cancers based on Spearman's correlation statistics. Figure S2. Association of TLK2, AURKA and PLK1 overexpression with chromosome instability (CIN) in breast cancer. Figure S3. Representative microscope images of γ-H2AX foci formation assay showing delayed DSB repair in T47D or MCF10A cells inducibly overexpressing TLK2. Figure S4. In response to irradiation (IR), TLK2 forms foci that partially co-localize with γ-H2AX foci. Figure S5. TLK2 overexpression led to reduced accumulation of G2/M phase cells in response to IR. Figure S6. The mechanisms of G2/M checkpoint signaling in response to irradiation.

supplementary methods and figure legends

Auxiliary Supplementary File

Purpose:Luminal B breast tumors are more aggressive estrogen receptor–positive (ER+) breast cancers characterized by aggressive clinical behavior and a high risk of metastatic dissemination. The underlying pathologic molecular events remain poorly understood with a paucity of actionable genetic drivers, which hinders the development of new treatment strategies.Experimental Design:We performed large-scale RNA sequencing analysis to identify chimerical transcripts preferentially expressed in luminal B breast cancer. The lead candidate was validated by reverse transcription PCR in breast cancer tissues. The effects of inducible ectopic expression or genetic silencing were assessed by phenotypic assays such as MTS, transwell, and transendothelial migration assays, and by clonogenic assays to assess MEK inhibitor sensitivity. Subcellular fractionation, Western blots, and immunoprecipitation were performed to characterize the protein products and elucidate the engaged mechanisms.Results:Here we report a novel tumor-specific chimeric transcript RAD51AP1-DYRK4 preferentially expressed in luminal B tumors. Analysis of 200 ER+ breast tumors detected RAD51AP1-DYRK4 overexpression in 19 tumors (9.5%), which is markedly enriched in the luminal B tumors (17.5%). Ectopic expression of RAD51AP1-DYRK4, but not wild-type RAD51AP1, leads to marked activation of MEK/ERK signaling, and endows increased cell motility and transendothelial migration. More importantly, RAD51AP1-DYRK4 appears to endow increased sensitivity to the MEK inhibitor trametinib through attenuating compensatory activation of HER2/PI3K/AKT under MEK inhibition.Conclusions:This discovery sheds light on a new area of molecular pathobiology of luminal B tumors and implies potential new therapeutic opportunities for more aggressive breast tumors overexpressing this fusion.

Supplementary Methods

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.

Characteristics of TBCRC023 Patients with disease progression

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:Prior neoadjuvant trials with 12 weeks of dual anti-HER2 therapy without chemotherapy demonstrated a meaningful pathologic complete response (pCR) in patients with HER2-positive breast cancer. In this trial, we sought to determine whether longer treatment would increase the rate of pCR.Patients and Methods:TBCRC023 (NCT00999804) is a randomized phase II trial combining a Simon phase II design in the experimental arm with a pick-the-winner design, not powered for direct comparison. Women with HER2-positive breast tumors measuring ≥2 cm (median = 5 cm) were randomized in a 1:2 ratio to 12 versus 24 weeks of lapatinib and trastuzumab. Letrozole (along with ovarian suppression if premenopausal) was administered in patients whose tumors were also estrogen receptor (ER) positive. All evaluable patients were assessed for in-breast pCR.Results:Ninety-seven patients were enrolled (33 in 12-week arm and 64 in 24-week arm), of whom 94 were evaluable. Median age was 51 years, and 55% were postmenopausal. Median tumor size was 5 cm, and 65% were ER-positive. The rate of pCR in the 24-week arm was 28% and numerically superior to the 12-week arm (12%). This was driven by increased pCR in the ER-positive subgroup (33% vs. 9%). Study treatment was well tolerated, with grade 1–2 diarrhea and acneiform rash being the most common toxicities.Conclusions:Treatment with dual anti-HER2 therapy for 24 weeks led to a numeric increase in pCR rate in women with HER2-positive breast cancer, without using chemotherapy. If validated, this approach may help identify patients who may benefit from deescalation of therapy.

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 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.

Purpose:Endocrine resistance remains a major clinical challenge in estrogen receptor (ER)–positive breast cancer. Despite the encouraging results from clinical trials for the drugs targeting known survival signaling, relapse is still inevitable. There is an unmet need to discover new drug targets in the unknown escape pathways. Here, we report Nemo-like kinase (NLK) as a new actionable kinase target that endows previously uncharacterized survival signaling in endocrine-resistant breast cancer.Experimental Design:The effects of NLK inhibition on the viability of endocrine-resistant breast cancer cell lines were examined by MTS assay. The effect of VX-702 on NLK activity was verified by kinase assay. The modulation of ER and its coactivator, SRC-3, by NLK was examined by immunoprecipitation, kinase assay, luciferase assay, and RNA sequencing. The therapeutic effects of VX-702 and everolimus were tested on cell line- and patient-derived xenograft (PDX) tumor models.Results:NLK overexpression endows reduced endocrine responsiveness and is associated with worse outcome of patients treated with tamoxifen. Mechanistically, NLK may function, at least in part, via enhancing the phosphorylation of ERα and its key coactivator, SRC-3, to modulate ERα transcriptional activity. Through interrogation of a kinase profiling database, we uncovered and verified a highly selective dual p38/NLK inhibitor, VX-702. Coadministration of VX-702 with the mTOR inhibitor, everolimus, demonstrated a significant therapeutic effect in cell line-derived xenograft and PDX tumor models of acquired or de novo endocrine resistance.Conclusions:Together, this study reveals the potential of therapeutic modulation of NLK for the management of the endocrine-resistant breast cancers with active NLK signaling.

Supplementary Figures S1-S8

Supplementary Figures 1-15

Supplementary file containing supplementary figures and tables.

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

Treatment discontinuation in TBCRC023 Patients

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: Endocrine resistance remains a major clinical challenge in estrogen receptor (ER)–positive breast cancer. Despite the encouraging results from clinical trials for the drugs targeting known survival signaling, relapse is still inevitable. There is an unmet need to discover new drug targets in the unknown escape pathways. Here, we report Nemo-like kinase (NLK) as a new actionable kinase target that endows previously uncharacterized survival signaling in endocrine-resistant breast cancer. Experimental Design: The effects of NLK inhibition on the viability of endocrine-resistant breast cancer cell lines were examined by MTS assay. The effect of VX-702 on NLK activity was verified by kinase assay. The modulation of ER and its coactivator, SRC-3, by NLK was examined by immunoprecipitation, kinase assay, luciferase assay, and RNA sequencing. The therapeutic effects of VX-702 and everolimus were tested on cell line- and patient-derived xenograft (PDX) tumor models. Results: NLK overexpression endows reduced endocrine responsiveness and is associated with worse outcome of patients treated with tamoxifen. Mechanistically, NLK may function, at least in part, via enhancing the phosphorylation of ERα and its key coactivator, SRC-3, to modulate ERα transcriptional activity. Through interrogation of a kinase profiling database, we uncovered and verified a highly selective dual p38/NLK inhibitor, VX-702. Coadministration of VX-702 with the mTOR inhibitor, everolimus, demonstrated a significant therapeutic effect in cell line-derived xenograft and PDX tumor models of acquired or de novo endocrine resistance. Conclusions: Together, this study reveals the potential of therapeutic modulation of NLK for the management of the endocrine-resistant breast cancers with active NLK signaling.

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.

Purpose: Luminal B breast tumors are more aggressive estrogen receptor–positive (ER+) breast cancers characterized by aggressive clinical behavior and a high risk of metastatic dissemination. The underlying pathologic molecular events remain poorly understood with a paucity of actionable genetic drivers, which hinders the development of new treatment strategies. Experimental Design: We performed large-scale RNA sequencing analysis to identify chimerical transcripts preferentially expressed in luminal B breast cancer. The lead candidate was validated by reverse transcription PCR in breast cancer tissues. The effects of inducible ectopic expression or genetic silencing were assessed by phenotypic assays such as MTS, transwell, and transendothelial migration assays, and by clonogenic assays to assess MEK inhibitor sensitivity. Subcellular fractionation, Western blots, and immunoprecipitation were performed to characterize the protein products and elucidate the engaged mechanisms. Results: Here we report a novel tumor-specific chimeric transcript RAD51AP1-DYRK4 preferentially expressed in luminal B tumors. Analysis of 200 ER+ breast tumors detected RAD51AP1-DYRK4 overexpression in 19 tumors (9.5%), which is markedly enriched in the luminal B tumors (17.5%). Ectopic expression of RAD51AP1-DYRK4, but not wild-type RAD51AP1, leads to marked activation of MEK/ERK signaling, and endows increased cell motility and transendothelial migration. More importantly, RAD51AP1-DYRK4 appears to endow increased sensitivity to the MEK inhibitor trametinib through attenuating compensatory activation of HER2/PI3K/AKT under MEK inhibition. Conclusions: This discovery sheds light on a new area of molecular pathobiology of luminal B tumors and implies potential new therapeutic opportunities for more aggressive breast tumors overexpressing this fusion.

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.

BACKGROUND: HER2-positive (+) breast cancers, defined by HER2 overexpression and/or amplification, are often addicted to HER2 to maintain their malignant phenotype. Yet, some HER2+ tumors do not benefit from anti-HER2 therapy. We hypothesize that HER2 amplification levels and PI3K pathway activation are key determinants of response to HER2-targeted treatments without chemotherapy. PATIENTS AND METHODS: Baseline HER2+ tumors from patients treated with neoadjuvant lapatinib plus trastuzumab [with endocrine therapy for estrogen receptor (ER)+ tumors] in TBCRC006 (NCT00548184) were evaluated in a central laboratory for HER2 amplification by fluorescence in situ hybridization (FISH) (n = 56). HER2 copy number (CN) and FISH ratios, and PI3K pathway status, defined by PIK3CA mutations or PTEN levels by immunohistochemistry were available for 41 tumors. Results were correlated with pathologic complete response (pCR; no residual invasive tumor in breast). RESULTS: Thirteen of the 56 patients (23%) achieved pCR. None of the 11 patients with HER2 ratio

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.

PURPOSE: Luminal B breast tumors are more aggressive estrogen receptor positive breast cancers characterized by aggressive clinical behavior and a high risk of metastatic dissemination. The underlying pathological molecular events remain poorly understood with a paucity of actionable genetic drivers, which hinders the development of new treatment strategies. EXPERIMENTAL DESIGN: We performed large-scale RNAseq analysis to identify chimerical transcripts preferentially expressed in luminal B breast cancer. The lead candidate was validated by Reverse Transcription PCR in breast cancer tissues. The effects of inducible ectopic expression or genetic silencing were assessed by phenotypic assays such as MTS, transwell and transendothelial migration assays, and by clonogenic assays to assess MEK inhibitor sensitivity. Subcellular fractionation, western blots, and immunoprecipitation were performed to characterize the protein products and elucidate the engaged mechanisms. RESULTS: Here we report a novel tumor-specific chimeric transcript RAD51AP1-DYRK4 preferentially expressed in luminal B tumors. Analysis of 200 ER-positive breast tumors detected RAD51AP1-DYRK4 overexpression in 19 tumors (9.5%), which is markedly enriched in the luminal B tumors (17.5%). Ectopic expression of RAD51AP1-DYRK4, but not wild-type RAD51AP1, leads to marked activation of MEK/ERK signaling, and endows increased cell motility and transendothelial migration. More importantly, RAD51AP1-DYRK4 appears to endow increased sensitivity to the MEK inhibitor Trametinib through attenuating compensatory activation of HER2/PI3K/AKT under MEK inhibition. CONCLUSIONS: This discovery sheds light on a new area of molecular pathobiology of luminal B tumors and implies potential new therapeutic opportunities for more aggressive breast tumors overexpressing this fusion.

Background Endocrine therapy is the most common treatment for estrogen receptor (ER)-positive breast cancer, but its effectiveness is limited by high rates of primary and acquired resistance. There are likely many genetic causes, and recent studies suggest the important role of ESR1 mutations and fusions in endocrine resistance. Previously, we reported a recurrent ESR1 fusion called ESR1-CCDC170 in 6–8% of the luminal B breast cancers that has a worse clinical outcome after endocrine therapy. Despite being the most frequent ESR1 fusion, its functional role in endocrine resistance has not been studied in vivo, and the engaged mechanism and therapeutic relevance remain uncharacterized. Methods The endocrine sensitivities of HCC1428 or T47D breast cancer cells following genetic perturbations of ESR1-CCDC170 were assessed using clonogenic assays and/or xenograft mouse models. The underlying mechanisms were investigated by reverse phase protein array, western blotting, immunoprecipitation, and bimolecular fluorescence complementation assays. The sensitivity of ESR1-CCDC170 expressing breast cancer cells to concomitant treatments of tamoxifen and HER/SRC inhibitors was assessed by clonogenic assays. Results Our results suggested that different ESR1-CCDC170 fusions endow different levels of reduced endocrine sensitivity in vivo, resulting in significant survival disadvantages. Further investigation revealed a novel mechanism that ESR1-CCDC170 binds to HER2/HER3/SRC and activates SRC/PI3K/AKT signaling. Silencing of ESR1-CCDC170 in the fusion-positive cell line, HCC1428, downregulates HER2/HER3, represses pSRC/pAKT, and improves endocrine sensitivity. More important, breast cancer cells expressing ectopic or endogenous ESR1-CCDC170 are highly sensitive to treatment regimens combining endocrine agents with the HER2 inhibitor lapatinib and/or the SRC inhibitor dasatinib. Conclusion ESR1-CCDC170 may endow breast cancer cell survival under endocrine therapy via maintaining/activating HER2/HER3/SRC/AKT signaling which implies a potential therapeutic strategy for managing these fusion positive tumors.

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.

Tucatinib (Tuc) was recently approved for metastatic disease and is moving towards the early setting in HER2+ breast cancer (BC). Given the increasing clinical use of Tuc, resistance will likely soon emerge as a challenge. Here, we explore the yet unknown mechanisms of resistance to Tuc and identify treatment strategies to overcome it. Our recently developed models of BT474 (AZ and ATCC) with acquired resistance to Tuc (TucR) and their sensitive parental (P) were used. DNA-seq, RNA-seq, and RPPA/western blot were performed. Knockdown studies were performed using EGFR siRNA. Drug efficacy studies involved cell growth assays by imaging-based or methylene blue assays. We recently reported (SABCS 2021) that our BT474 TucR models acquired EGFR amplification. The TucR cells displayed elevated levels of phosphorylated (p) and total (t) EGFR, pHER2, pHER3, and downstream pAKT and pS6, which were substantially suppressed by the EGFR-specific tyrosine kinase inhibitor (TKI) gefitinib (Gef) or even further when combined with Tuc. Our new results demonstrate that EGFR knockdown selectively inhibits the growth and pHER2 levels in TucR vs P cells, supporting our hypothesis that heterodimerization of amplified EGFR with HER2 leads to higher pHER2 levels in TucR cells. We have recently also shown that TucR models were hypersensitive to Gef and this inhibition was further enhanced with Gef+Tuc, implying their survival dependence on EGFR. Here, we demonstrate that the TucR cells made resistant to 200nM Tuc maintain their resistant growth and elevated EGFR-dependent signaling even when exposed to 500nM, and can begrown as xenografts in the presence of clinically relevant dose of Tuc, emphasizing their true resistance via amplified EGFR. Importantly, both TucR models vs P cells were cross-resistant to trastuzumab but maintain partial sensitivity to TDM1. While the EGFR-specific antibody cetuximab (Cet) was partially effective as a single agent only in the ATCC model, it potently inhibited growth and induced cell killing in combination with Tuc in both models. A significantly greater inhibition in cell growth and survival was also observed when trastuzumab or TDM1 was combined with either Gef or Cet. Taken together, our results suggest that the activation of HER2 and the resistant growth and survival in the TucR models is completely dependent on the amplified EGFR, which we are currently further corroborating by additional mechanistic and xenograft studies. Whilst we have previously reported that resistance to lapatinib and neratinib confer cross-resistance to Tuc, our recent findings show that resistance to Tuc may be overcome using dual/pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Overall, our novel findings hold crucial implications in light of the current treatment landscape of HER2+ BC and biomarkers of resistance, and places a particular emphasis on considerations to sequence currently available TKIs. Citation Format: Jamunarani Veeraraghavan, Fu-Tien Liao, Tia Gordon, Pier Selenica, Sarmistha Nanda, Lanfang Qin, Yingjie Zhu, Juber A. Patel, Andrea Gazzo, Fabio Stossi, Michael A. Mancini, Carolina Gutierrez, Britta Weigelt, Jorge S. Reis-Filho, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. The role of EGFR in resistance to tucatinib and its therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB517A.

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

Background: We recently reported that acquired resistance to the dual HER1/2 TKI lapatinib (Lap) was mediated by HER2 L755S, while resistance to the pan-HER TKI neratinib (Nrb) was associated with co-acquisition of an additional pathogenic PIK3CA mutation. Though the role of HER2 mutations is gaining attention in HER2-positive (+) BC, less is known about their role and clinical implications in next generation TKI resistance, particularly when co-occurring with PIK3CA mutations in HER2+ BC. Investigating optimal treatment combinations and the development of new clinically relevant 3D models are warranted.Materials and Methods: HER2+ BT474 parental (P) cells and models with acquired resistance to Lap (LapR) and Nrb (NrbR) (SABCS20-PD3-09) were used. Xenografts established in mice using P, LapR, and NrbR cells and 3D organoids derived from these xenografts using the Hans Clevers (HC, PMID 29224780) or Mark Burkard (MB, PMID 31175091) method were characterized by qRT-PCR and western blot. Drug efficacy was assessed by growth changes in 2D and 3D models using the IncuCyte system or by microscopy-based analysis. Results: We previously showed that Lap and Nrb resistance confers cross-resistance to tucatinib (Tuca) and trastuzumab, and that targeting the HER and downstream PI3K pathway, especially using small molecule agents that are key for treatment of CNS lesions, is effective only in combination with Nrb or poziotinib (Pozio), but not Tuca. Our new studies revealed that the MEK inhibitor (i) AZD6244 (selumetinib; Sel), mTORi everolimus (Eve), and selective estrogen receptor degrader fulvestrant (Ful) were not effective as single agents in inhibiting the growth of either LapR or NrbR models. Whilst the LapR cells were highly sensitive to the irreversible HER1/2 TKI afatinib (Afa) and the irreversible dual/pan-HER TKI pyrotinib (Pyro) as single agents, the NrbR models were cross-resistant to both TKIs, highlighting the importance of the co-occurring PIK3CA mutation in resistance. Interestingly, Afa and Pyro were only partly effective when combined with Eve+Ful, Sel+Eve, or Sel+the PIK3CAi alpelisib in inhibiting NrbR growth. Consistent with our previously reported findings for Nrb and Pozio, Pyro was highly effective with TDM1. As opposed to the P xenografts, the LapR and NrbR tumors grew in the presence of the respective TKI, confirming their resistant phenotype in vivo. P and resistant xenograft-derived organoids (XDOs) were successfully established using the HC but not MB method, but the HC-derived XDOs were subsequently grown in MB condition and used for molecular and functional studies. Preliminary characterization showed that the LapR tumors and XDOs harbor HER2 L755S, whereas the NrbR tumors and XDOs also have a concomitant PIK3CA E542V mutation, findings that are in line with our 2D results, suggesting that the xenografts and XDOs retain and recapitulate the molecular profile of their 2D or tumor counterparts. Early drug efficacy studies indicate that, akin to the 2D models, the LapR XDOs are highly sensitive to Nrb, whereas both the LapR and NrbR XDOs exhibit cross-resistance to Tuca but remain sensitive to Pozio.Conclusions: Our data suggest that the potency of next generation irreversible HER TKIs in HER2+ BC may be challenged by the emergence of mutations in HER2, together with other co-occurring downstream mutations, such as PIK3CA. Our findings present a clear roadmap for the development of combinatorial therapies that should be individualized for patients with HER2+ BC. Our newly developed XDO strategy may offer a new platform to confirm and prioritize optimal drug combinations to overcome this resistance and may facilitate the near future development of patient-derived organoids for precision medicine of resistant HER2+ BC. Citation Format: Sreyashree Bose, Ragini Mistry, Chia Chia Liu, Sarmistha Nanda, Lanfang Qin, Pier Selenica, Andrea Gazzo, Yingjie Zhu, Michael A. Mancini, Fabio Stossi, Irmina Diala, Lisa D. Eli, Britta Weigelt, Jorge S. Reis-Filho, Mothaffar F. Rimawi, C. Kent Osborne, Rachel Schiff, Jamunarani Veeraraghavan. Resistance to next generation tyrosine kinase inhibitors (TKIs) in HER2-positive breast cancer (BC): Role of HER and PIK3CA mutations and development of new treatment strategies and study models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-01.

Background: With recent approval of the irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (N) and the HER2-specific TKI tucatinib (T) in the advanced setting and their edging towards the early setting in HER2+ BC, resistance will likely emerge as a challenge, as illustrated by the HER2CLIMB study, where only one patient with brain metastasis remained progression free after 2 years on T. We set out to define the mechanisms of resistance to T and treatment strategies to overcome it. Materials and Methods: Our previously characterized HER2+ BT474 models with acquired resistance to lapatinib (L; LapR) or N (NrbR) (SABCS20-PD3-09), and our recently developed models of BT474 and SKBR3 with acquired resistance to T (TucaR) developed through long-term exposure to increasing doses of T (up to 200nM) and their naïve parental (P) were used. Genomic (DNA-seq), transcriptomic (RNA-seq), and proteomic (RPPA, western blot) characterization were performed. Drug efficacy studies involved cell growth assays by the imaging-based IncuCyte system. Results: We recently reported that while LapR is associated with acquisition of HER2 L755S mutation, which partially reactivates the HER pathway, NrbR is associated with the additional co-acquisition of a pathogenic PIK3CA mutation. Preliminary analysis of 2 BT474 TucaR models (ATCC and AZ) showed highly elevated levels of phosphorylated (p) and total (t) EGFR, suggesting EGFR signaling activation. Levels of pHER2, pHER3, and downstream pAKT and pS6 were also markedly higher in the TucaR models compared to P or short-term T. The TucaR but not LapR or NrbR models exhibited EGFR amplification, explaining the higher EGFR levels and signaling. Further, the elevated pEGFR, pHER2, pHER3, pAKT, and pS6 levels in TucaR models were substantially suppressed by the EGFR-specific TKI gefitinib (G) (50, 500nM) or even further when combined with T (500 nM G+200nM T). These results suggest that the higher pHER2 levels in TucaR models is probably due to heterodimerization of the amplified EGFR with HER2 and subsequent HER2 phosphorylation. In contrast to the P cells where the apoptotic marker cleaved (c)-PARP was not induced with G alone (50, 500nM), but with T (200nM) or 500nM G+T, in the TucaR model, 500nM G alone was enough to induce c-PARP, which was further enhanced when combined with T, implying the survival dependence of TucaR cells on EGFR signaling. The TucaR models were hypersensitive to G compared to P cells, and this growth inhibition was further enhanced with G+T. Whilst we previously reported that the LapR and NrbR cells were cross-resistant to T, the TucaR cells remained highly sensitive to the pan-HER TKIs N, poziotinib, and pyrotinib. Finally, in a second HER2+ model SKBR3, at 200nM TucaR, we observed elevated pEGFR, pHER2, pHER3, and pAKT levels, the underlying mechanism of which is under investigation by genomic and molecular analysis. In-depth characterization of our TucaR models to determine the differential gene expression and signatures is ongoing to gain additional mechanistic insights. Conclusions: Our findings suggest that whilst complete blockade of the HER layer using N is evaded by acquisition of HER and PIK3CA mutations, resistance to the HER2 TKI T is associated with EGFR amplification, a finding that underscores the HER signaling pathway redundancy and cross-talk between HER receptors to compensate for partial blockade of the pathway. Further, while resistance to L and N confers cross-resistance to T, resistance to T may be overcome using pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Together, our findings hold crucial implications in light of the current treatment landscape of HER2+ BC, with a particular emphasis on the considerations to strategize the treatment sequence of currently available TKIs. Citation Format: Jamunarani Veeraraghavan, Sreyashree Bose, Ragini Mistry, Pier Selenica, Sarmistha Nanda, Lanfang Qin, Andrea Gazzo, Yingjie Zhu, Michael A Mancini, Fabio Stossi, Britta Weigelt, Jorge S Reis-Filho, C. Kent Osborne, Mothaffar F Rimawi, Rachel Schiff. Acquired resistance to tucatinib is associated with EGFR amplification in HER2+ breast cancer (BC) models and can be overcome by a more complete blockade of HER receptor layer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD8-06.

Background: HER2-positive (HER2+) breast cancer (BC) comprises all the four PAM50 molecular subtypes. Among these, the HER2-Enriched (HER2-E) appear to be associated with higher pathological complete response (pCR) rates following anti-HER2-based regimens. Here, we present a meta-analysis to validate the association of the HER2-E subtype with pCR following anti-HER2-based neoadjuvant treatments with or without chemotherapy (CT). Methods: A systematic literature search was performed in February 2019. The primary objective was to compare the association between HER2-E subtype (versus others) and pCR. Selected secondary objectives were to compare the association between 1) HER2-E subtype and pCR in CT-free studies, 2) HER2-E subtype within hormone receptor (HR)-negative and HR+ disease and 3) HR-negative disease (versus HR+) and pCR in all patients and within HER2-E subtype. A random-effect model was applied. The Higgins��� I2 was used to quantify heterogeneity. Results: Sixteen studies were included, 5 of which tested CT-free regimens. HER2-E subtype was significantly associated with pCR in all patients (odds ratio [OR] = 3.50, p < 0.001, I2 = 33%), in HR+ (OR = 3.61, p < 0.001, I2 = 1%) and HR-negative tumors (OR = 2.28, p = 0.01, I2 = 47%). In CT-free studies, HER2-E subtype was associated with pCR in all patients (OR = 5.52, p < 0.001, I2 = 0%) and in HR + disease (OR = 4.08, p = 0.001, I2 = 0%). HR-negative status was significantly associated with pCR compared to HR + status in all patients (OR = 2.41, p < 0.001, I2 = 30%) and within the HER2-E subtype (OR = 1.76, p < 0.001, I2 = 0%). Conclusions: The HER2-E biomarker identifies patients with a higher likelihood of achieving a pCR following neoadjuvant anti-HER2-based therapy beyond HR status and CT use. Future trial designs to escalate or de-escalate systemic therapy in HER2+ disease should consider this genomic biomarker.

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.

Additional file 8: Figure S8. The effect of SRC and HER2 inhibitors on the endocrine response of ZR-75-1 cells expressing endogenous ESR1–CCDC170 fusion.

Additional file 1: Figure S1. Schematic of the ESR1-CCDC170 fusion variants and the encoded proteins.

Additional file 9: Figure S9. Western blots detecting HER2, HER3, and SRC protein expression in the cell models used in this study.