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Background: CDK4/6 inhibitors (CDK4/6i) in combination with antiestrogens have prolonged survival of patients with ER+ metastatic breast cancer. However, this combination is not curative mainly due to acquired drug resistance. Knowledge about mechanisms of such resistance remains quite incomplete. We report herein a forward-genetics screen to discover a broad spectrum of novel somatic mutations causal to CDK4/6i resistance. Methods: We used CRISPR/Cas9 to delete the DNA mismatch repair (MMR) gene MSH2 in MCF7 and T47D ER+ breast cancer cells. Deficiency of DNA MMR proteins such as MSH2 results in a high nucleotide substitution rate which, in turn, predisposes cells to acquire drug resistance-associated mutations. MSH2-/- MCF7 and T47D cells were infected with a lentiviral barcode library containing ~1000 unique DNA barcodes. MSH2-/- barcoded cells were expanded for ~25 doublings to allow the accumulation of random mutations. Clones resistant to CDK4/6i were selected in the presence of IC90 of palbociclib (200 nM) or abemaciclib (500 nM) for 4-6 weeks. CDK4/6i resistant clones with unique barcode IDs were subjected to whole exome sequencing (WES). Results: Following drug selection, ~73 uniquely barcoded resistant colonies emerged from MCF7 and T47D MSH2-/- clonal lines. As expected, MCF7 and T47D MSH2-/- clones harbored a high mutation burden compared to parental cells. Candidate variants were distilled based on (a) functionality prediction and (b) mutation frequency in Project GENIE. We observed RB1 (5/73 clones; 6.8%) mutations in CDK4/6i resistant clones, providing proof-of-principle that this approach can identify clinically-relevant drug resistant alterations. Overall, we identified non-synonymous alterations in 2,206 genes in T47D palbociclib-resistant, 2,195 genes in T47D abemaciclib-resistant, and 1,312 genes in MCF7 palbociclib-resistant lines. A secondary screen of the 10 genes recurrently mutated in all three CDK4/6i resistant groups identified loss of ASXL1 as top hit. ASXL1 encodes a polycomb repressive complex protein that regulates chromatin accessibility. Loss of ASXL1 has been implicated in myeloid transformation through epigenetic reprogramming. WES of 76 CDK4/6i resistant tumor biopsies (DFCI/MBCproject cohort) identified ASXL1 alterations in two and four patients with acquired and primary resistance, respectively (6/76=7.9%). One of the tumors that progressed after an initial response to palbociclib had acquired the same ASXL1 R549C mutation that was identified in our screen. Among 1,769 tumors from patients treated with CDK4/6i (TEMPUS database), 37 exhibited ASXL1 alterations (4 frameshift, 6 truncating, 3 in-frame del, 24 missense mutations). DNAseq of patient-derived organoids established from post-CDK4/6i metastases identified ASXL1 mutations in 2/7 organoids (29%). ASXL1-/- MCF7 and T47D cells were cross-resistant to fulvestrant. GSEA analysis of RNA-seq data showed upregulation of E2F targets in palbociclib-treated cells stably transduced with ASXL1 shRNA but not control shRNA (Enrichment score=0.75, q=1.00E-09). This was associated with maintenance of RB phosphorylation in the presence of CDK4/6i, markedly higher levels of CDK2, CDK6, cyclins E and A, and downregulation of p21 and p27. Finally, siRNAs targeting CDK2 or cyclin A reduced the viability of ASXL1-deficient T47D cells by 50% and 90%, respectively. Conclusions: An accelerated mutagenesis approach using MMR-deficient ER+ breast cancer cells identified loss of ASXL1 as a novel mechanism of resistance to CDK4/6i. ASXL1 alterations were found in ~8% of tumors from patients with de novo or acquired resistance to CDK4/6i. Knockdown of CDK2 and cyclin A restored sensitivity to CDK4/6i and reduced viability of ASXL1 deficient cells, suggesting CDK2 inhibitors are a treatment approach against these drug-resistant tumors. Citation Format: Dhivya R. Sudhan, Sumanta Chatterjee, Jiwoong Kim, Yunguan Wang, Vishal Kandagatla, Dan Ye, Chang-Ching Lin, Jorge Gomez Tejeda Zanudo, Esha Jain, Arnaldo Marin, Alberto Servetto, Kyung-min Lee, Juan Manuel Povedano, David McFadden, Alex Barrett, Nikhil Wagle, Ariella B. Hanker, Carlos L. Arteaga. Loss of ASXL1 tumor suppressor promotes resistance to CDK4/6 inhibitors in ER+ breast cancer [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 GS3-09.

Background: CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with advanced estrogen receptor-positive (ER+) breast cancer. However, this benefit is transient as virtually all these tumors eventually develop drug resistance and recur. Clinical studies have reported an association of RB1 loss-of-function genomic alterations with acquired resistance to CDK4/6i. Given the enrichment of RB1 alterations post CDK4/6i treatment, ER+/RB1-deficient breast cancer will become a rising patient population in need of discovery of novel treatment strategies. In this study, we sought to identify actionable vulnerabilities for this refractory breast cancer subtype using a genome-wide CRISPR screen. Methods: RB1 was knocked out in ER+ MCF-7 and T47D breast cancer cells using CRISPR-Cas9; complete gene knockout was confirmed by PCR-based genotyping, Sanger sequencing, and immunoblot analysis. Isogenic RB1 knockout (RBKO) and wild-type (WT) T47D cells were used for the genome-wide CRISPR screen. MAGeCKFlute was used to identify differentially essential genes in T47D RBKO vs WT cells; Gene Ontology (GO) analysis was used to prioritize hits. MCF-7 and T47D RBKO cells were used for validating and studying the function of the identified genes. Results: Knockout of RB1 in MCF-7 and T47D cells increased IC50 of abemaciclib, palbociclib, and ribociclib 10-200 fold compared to WT cells. RNA-seq analysis showed upregulation of E2F target gene expression in RBKO vs WT cells. The CRISPR screen revealed that CCND1 and CDK4 lost their essentiality in T47D RBKO cells, suggesting that loss of RB1 uncouples the CDK4/Cyclin D1 complex from E2F-regulated transcription. GO analysis of the top 50 differentially essential hits of RBKO vs WT cells showed an enrichment of protein arginine methyltransferase activity, primarily PRMT5, which post-translationally mono-methylates and symmetrically di-methylates protein arginine. In agreement with this finding, PRMT5 knockout by three individual sgRNAs resulted in more potent growth inhibition of MCF-7 and T47D RBKO cells than WT cells. Further, transfection of PRMT5 siRNA or treatment with the PRMT5 small molecule inhibitor GSK3326595 - currently in clinical trials - resulted in G1 arrest of MCF-7 and T47D RBKO cells as assayed by propidium iodide staining but did not induce caspase 3/7 or PARP cleavage (apoptosis). RNA-seq of PRMT5 siRNA vs control siRNA in MCF-7 and T47D RBKO cells exhibited significant downregulation of E2F Hallmark gene signature, further suggesting PRMT5 inhibition as a strategy to suppress E2F-regulated gene expression when cells lose Rb. The CRISPR screen also revealed that transcription factors that drive ER signaling, such as FOXA1, GATA3, MYC, SPDEF, and ESR1 (the gene encoding ERα), were commonly essential in both T47D WT and RBKO cells. Estrogen deprivation or treatment with fulvestrant inhibited estrogen responsive element (ERE) luciferase reporter activity, expression of putative E2F target genes, and proliferation of both WT and RBKO cells, suggesting that ER+ cells still rely on ERα irrespective of RB1 status. Treatment of MCF-7 and T47D RBKO cells with fulvestrant and GSK3326595 resulted in more potent growth inhibition than each drug alone, suggesting a novel approach to treat ER+/RB1-deficient breast cancer. We are currently testing the antitumor activity of fulvestrant plus GSK3326595 against RBKO xenografts as well as the requirement of arginine methyltransferase activity associated with PRMT5 for growth of ER+/RB1-deficient breast cancer cells. Conclusion: PRMT5 is essential for proliferation of ER+/RB1-deficient breast cancer cells. Targeting PRMT5 in combination with anti-estrogens is a novel and testable strategy to suppress E2F-regulated cell cycle progression of this CDK4/6 inhibitor-resistant breast cancer subtype. Citation Format: Chang-Ching Lin, Tsung-Cheng Chang, Alberto Servetto, Kyung-min Lee, He Zhang, Yunguan Wang, Dan Ye, Sumanta Chatterjee, Dhivya R Sudhan, Hiroaki Akamatsu, Yang Xie, Joshua T Mendell, Ariella B Hanker, Carlos L Arteaga. A genome-wide CRISPR screen identifies PRMT5 as a novel therapeutic target in ER+/RB1-deficient breast cancer [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 P5-17-09.

CONSORT diagram

Supplemental Table S1: The Preoperative Endocrine Prognostic Index (PEPI score) Supplemental Table S2:* Baseline Ki67: 7 patients had missing values Supplemental Table S3a: Driver Mutations Supplemental Table S3b: Driver Copy Number Alterations Supplemental Table S3c: Mutations clinically actionable. Drug prescription Supplemental Table S4: Actionable CNAs. Drug prescription Supplemental Table S5: Overlap between genes upregulated in resistant tumors and transcription factor targets genes Suplemental Table S6: Change from baseline to surgery in gene expressions of the 47 most upregulated genes in the tumors resistant to prolonged neoadjuvant letrozole from 60 HR-positive/HER2-negative breast cancers in POP trial. A decreased gene expression translates into a geometric mean change < 1 . An increased or stable gene expression translates into a geometric mean change {greater than or equal to} 1. Supplemental Table S7: Mutations detected in this study(post-filtering) Supplemental Table S8: Gene expression values (FPKM)

Supplementary data information

Purpose:While FGFR1 amplification has been described in breast cancer, the optimal treatment approach for FGFR1-amplified (FGFR1+) metastatic breast cancer (MBC) remains undefined.Experimental Design: We evaluated clinical response to endocrine and targeted therapies in a cohort of patients with hormone receptor–positive (HR+)/HER2− MBC and validated the functional role of FGFR1-amplification in mediating response/resistance to hormone therapy in vitro.Results:In the clinical cohort (N = 110), we identified that patients with FGFR1+ tumors were more likely to have progesterone receptor (PR)-negative disease (47% vs. 20%; P = 0.005), coexisting TP53 mutations (41% vs. 21%; P = 0.05), and exhibited shorter time to progression with endocrine therapy alone and in combination with CDK4/6 inhibitor, but not with a mTOR inhibitor (everolimus), adjusting for key prognostic variables in multivariate analysis. Furthermore, mTOR-based therapy resulted in a sustained radiological and molecular response in an index case of FGFR1+ HR+/HER2− MBC. In preclinical models, estrogen receptor–positive (ER+)/FGFR1-amplified CAMA1 human breast cancer cells were only partially sensitive to fulvestrant, palbociclib, and alpelisib, but highly sensitive to everolimus. In addition, transduction of an FGFR1 expression vector into ER+ T47D cells induced resistance to fulvestrant that could be overcome by added TORC1 inhibition, but not PI3K or CDK4/6 inhibition.Conclusions:Collectively, these findings suggest that while FGFR1 amplification confers broad resistance to ER, PI3K, and CDK4/6 inhibitors, mTOR inhibitors might have a unique therapeutic role in the treatment of patients with ER+/FGFR1+ MBC.

Supplementary figures

Purpose: This study aimed to identify biomarkers of resistance to endocrine therapy in estrogen receptor–positive (ER+) breast cancers treated with prolonged neoadjuvant letrozole.Experimental Design: We performed targeted DNA and RNA sequencing in 68 ER+ breast cancers from patients treated with preoperative letrozole (median, 7 months).Results: Twenty-four tumors (35%) exhibited a PEPI score ≥4 and/or recurred after a median of 58 months and were considered endocrine resistant. Integration of the 47 most upregulated genes (log FC > 1, FDR < 0.03) in letrozole-resistant tumors with transcription-binding data showed significant overlap with 20 E2F4-regulated genes (P = 2.56E−15). In patients treated with the CDK4/6 inhibitor palbociclib before surgery, treatment significantly decreased expression of 24 of the 47 most upregulated genes in letrozole-resistant tumors, including 18 of the 20 E2F4 target genes. In long-term estrogen-deprived ER+ breast cancer cells, palbociclib also downregulated all 20 E2F4 target genes and P-RB levels, whereas the ER downregulator fulvestrant or paclitaxel only partially suppressed expression of this set of genes and had no effect on P-RB. Finally, an E2F4 activation signature was strongly associated with resistance to aromatase inhibitors in the ACOSOG Z1031B neoadjuvant trial and with an increased risk of relapse in adjuvant-treated ER+ tumors in METABRIC.Conclusions: In tumors resistant to prolonged neoadjuvant letrozole, we identified a gene expression signature of E2F4 target activation. CDK4/6 inhibition suppressed E2F4 target gene expression in estrogen-deprived ER+ breast cancer cells and in patients' ER+ tumors, suggesting a potential benefit of adjuvant CDK4/6 inhibitors in patients with ER+ breast cancer who fail to respond to preoperative estrogen deprivation. Clin Cancer Res; 24(11); 2517–29. ©2018 AACR.

Adjuvant immunotherapy (IO) and targeted therapy (TT) have improved relapse-free survival (RFS) in patients with stage III melanoma, although about 25% of them relapse within a year. However, real-world data on treatment efficacy and safety as well as management of treatment recurrences are still limited. We retrospectively analyzed 113 patients with stage III melanoma who received at least one cycle of anti-PD-1 (nivolumab or pembrolizumab) or dabrafenib + trametinib as adjuvant therapy. Most of patients included into the analyses harbor BRAV600E mutation (66.4%) and had a stage IIIC melanoma (63.7%). Immunotherapy was administered in 48.7% of patients, whereas targeted therapy in 51.3% At data cut-off, median RFS was not reached with 12- and 24-months RFS of 81% and 64%, respectively. No new adverse events were registered. Thirty patients (26.5%) relapsed, mainly at distant sites. Patient treated with IO recurred mostly during adjuvant treatment (ON-treatment) while patients treated with TT relapsed at the end of treatment (OFF-treatment). At relapse, surgery, radiotherapy and systemic therapy were used alone or in combination. Among patients who started a first-line therapy, an excellent response switching to a different treatment was observed. Real-world outcomes and safety of adjuvant treatment for resected stage III melanoma appear comparable to clinical trials data. Moreover, management of recurrences depends on type of relapse (loco-regional vs distant) and timing (during vs OFF treatment). Furthermore, patients who relapse after adjuvant TT respond well to subsequent anti-PD1 based therapy.

Simple Summary Moving from the achievements of immune checkpoint inhibitors in metastatic lung cancer, the great promise of immunotherapy in resectable early-stage is to make a disease, often lethal until now for frequent post-surgery relapses, more curable. This review focuses on role of immunotherapy in early-stage non-small cell lung cancer. We summarize biological rationale, findings from clinical trials of adjuvant and neoadjuvant immune checkpoint inhibitors, questions about biomarkers and choice of endpoints, aiming to provide up-to-date information useful for clinical decision making. Lung cancer is the leading cause of cancer-related death worldwide. Since prognosis of early-stage non-small cell lung cancer (NSCLC) remains dismal for common relapses after curative surgery, considerable efforts are currently focused on bringing immunotherapy into neoadjuvant and adjuvant settings. Previously, perioperative chemotherapy showed only a modest but significative improvement in overall survival. The presence of broad tumor neoantigens load at primary tumor prior to surgery as well as the known immunosuppressive status following resection represent the main rationale for immunotherapy in early disease. Several trials have been conducted in recent years, leading to atezolizumab and nivolumab approval in the adjuvant and neoadjuvant setting, respectively, and perioperative immunotherapy in NSCLC remains a field of active clinical and preclinical investigation. Unanswered questions in perioperative therapy in NSCLC include the optimal sequence and timing of chemotherapy and immunotherapy, the potential of combination strategies, the role of predictive biomarkers for patient selection and the choice of useful endpoints in clinical investigation.

Major associations of medical oncologists remark that novel anticancer treatments should guarantee improvement of survival outcomes as well as of patients' quality of life (QoL). Herein, we investigated QoL assessment and reporting in phase III randomized controlled trials (RCTs) testing new drugs in metastatic non-small cell lung cancer (NSCLC), published between 2010 and 2021. We selected 172 RCTs for further analysis. Only 2/172 (1.2%) trial included QoL among primary study endpoints. Of note, 40/172 (23.3%) trials did not include QoL assessment among endpoints. The majority of RCTs (102/172, 59.3%) did not report QoL results in primary publications. Particularly, RCTs testing immunotherapy, target therapy and chemotherapy did not disclose QoL data in primary publications in 97.0%, 51.5% and 46.5% of cases, respectively. Next, we found that only 43/95 (45.3%) positive studies reported QoL results in primary articles. Of the 102 trials missing QoL data in primary manuscripts, only 21 (20.6%) disclosed QoL results in a secondary publication. Finally, we found a common fail in adherence to CONSORT-PROs items in publications reporting QoL results. In summary, our study reveals a relevant inadequate assessment and under-reporting of QoL in RCTs of novel systemic treatments for patients with metastatic NSCLC.

Background: FGFR1 amplification occurs in ~ 15% of ER+ breast cancers. In these tumors, nuclear FGFR1 has been shown to interact with DNA, but its role in transcriptional regulation is unclear. Thus, we investigated the genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer. Results: FGFR1 ChIP-Seq detected 4,412 DNA binding sites in CAMA1 ER+/FGFR1-amplified breast cancer cells cultured in estrogen-free conditions. Of these binding sites, 67% were enriched at promoter regions. ChIP-qPCR confirmed FGFR1 binding to several promoter regions in a second ER+/FGFR1-amplified cell line, MDA-MB-134, and a patient derived xenograft, HCI-011. To determine the nuclear FGFR1 interactome, we performed FLAG immunoprecipitation of mixed nuclear and chromatin fractions of CAMA1 cells transduced with a 3XFLAG-FGFR1 plasmid, followed by mass spectrometry (MS) of FLAG antibody pulldowns. MS revealed RNA Polymerase II subunits among the top nuclear FGFR1 interacting proteins. FGFR1 mainly bound Pol II phosphorylated on Ser5 (Pol II S5P), a marker of transcription initiation, in CAMA1, MDA-MB-134 and HCI-011 cell extracts. Pol II S5P ChIP-Seq revealed that 2,867/4,412 (65%) FGFR1 peaks were shared with Pol II S5P. ChIP-Seq also showed that 95% of FGFR1 peaks overlapped with both H3K4me3 and H3K27ac, markers of active transcription. Consistent with these results, RNA-Seq of CAMA1 cells showed that expression of FGFR1-bound genes was markedly higher than non FGFR1-bound genes (p Citation Format: Alberto Servetto, Rahul Kollipara, Luigi Formisano, Chang-Ching Lin, Kyung-min Lee, Dhivya R Sudhan, Ariella B Hanker, Sumanta Chatterjee, Angel Guerrero-Zotano, Paula Gonzalez-Ericsson, Saurabh Mendiratta, Hiroaki Akamatsu, Nicholas James, Ralf Kittler, Carlos L Arteaga. FGFR1 associates with gene promoters and regulates gene transcription: Implications for endocrine resistance in ER+/FGFR1-amplified breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS1-06.

The MYC oncogene is frequently amplified in triple-negative breast cancer (TNBC). Here, we show that MYC suppression induces immune-related hallmark gene set expression and tumor-infiltrating T cells in MYC-hyperactivated TNBCs. Mechanistically, MYC repressed stimulator of interferon genes (STING) expression via direct binding to the STING1 enhancer region, resulting in downregulation of the T-cell chemokines CCL5, CXCL10, and CXCL11. In primary and metastatic TNBC cohorts, tumors with high MYC expression or activity exhibited low STING expression. Using a CRISPR-mediated enhancer perturbation approach, we demonstrated that MYC-driven immune evasion is mediated by STING repression. STING repression induced resistance to PD-L1 blockade in mouse models of TNBC. Finally, a small-molecule inhibitor of MYC combined with PD-L1 blockade elicited a durable response in immune-cold TNBC with high MYC expression, suggesting a strategy to restore PD-L1 inhibitor sensitivity in MYC-overexpressing TNBC.

Aim: Comparison of first-line FOLFIRINOX (FFN) and nab-paclitaxel plus gemcitabine (NabGem) in patients with metastatic pancreatic ductal adenocarcinoma. Patients & methods: The authors analyzed data from 160 patients with metastatic pancreatic adenocarcinoma receiving first-line FFN (n=43) or NabGem (n=117). Results: FFN and NabGem were similar in median progression-free survival (24.43 vs 26.28weeks; hazard ratio [HR]: 0.88) and medial overall survival (47.43 vs 42.86weeks; HR: 0.90). Of the 43 patients receiving FFN, 26 (60.4%) were treated with second-line NabGem; 14/117 (12.0%) patients receiving NabGem received second-line FFN (p&nbsp

The 17q23 amplicon is associated with poor outcome in ER+ breast cancers, but the causal genes to endocrine resistance in this amplicon are unclear. Here, we interrogate transcriptome data from primary breast tumors and find that among genes in 17q23, PRR11 is a key gene associated with a poor response to therapeutic estrogen suppression. PRR11 promotes estrogen-independent proliferation and confers endocrine resistance in ER+ breast cancers. Mechanistically, the proline-rich motif-mediated interaction of PRR11 with the p85α regulatory subunit of PI3K suppresses p85 homodimerization, thus enhancing insulin-stimulated binding of p110-p85α heterodimers to IRS1 and activation of PI3K. PRR11-amplified breast cancer cells rely on PIK3CA and are highly sensitive to PI3K inhibitors, suggesting that PRR11 amplification confers PI3K dependence. Finally, genetic and pharmacological inhibition of PI3K suppresses PRR11-mediated, estrogen-independent growth. These data suggest ER+/PRR11-amplified breast cancers as a novel subgroup of tumors that may benefit from treatment with PI3K inhibitors and antiestrogens., The 17q23 amplicon is associated with poor outcome in ER+ breast cancers, but the causal genes responsible endocrine resistance in this region are unclear. In this study, the authors demonstrate that PRR11 located at 17q23, is critical for conferring endocrine resistance through activation of PI3K signalling and therefore propose PI3K inhibition as a treatment for PRR11-amplified breast cancers.

Background: CDK4/6 inhibitors have been approved in combination with endocrine therapy for the treatment of estrogen receptor positive (ER+) metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. Results: p21-activated kinase 1 (PAK1) is a serine/threonine kinase amplified in several human cancer types. Specifically, the survey of TCGA for copy number alterations and/or expression showed PAK1 amplification and/or overexpression in ~23% of ER+ breast cancer. To test the hypothesis that PAK1-amplification is related to CDK4/6 inhibitors resistance, we generated two ER+ breast cancer cell lines T47D (harbouring PAK1 amplification) and MCF7 (non PAK1-amplified) resistant to the combination of fulvestrant (ER downregulator) and abemaciclib (CDK4/6 inhibitor), namely T47DFAR and MCF7FAR. T47DFAR and MCF7FAR were obtained, exposing the parental cells to increasing doses of drugs. Interestingly, only the T47DFAR but not the MCF7FAR showed higher level of PAK1 expression and activation compared to the parental cell line. PAK1 pathway activation was evaluated measuring by immunoblot the levels of phosphorylation of PAK1, MEK, ERK and Beta-Catenin. Moreover, the genomic knockdown of PAK1 through RNAi causes a decrease in cell proliferation of T47DFAR, but not of MCF7FAR and parental cells, suggesting the role of PAK1 as a possible mediator of drug resistance. Conversely, overexpression of PAK1 in MCF7 and T47D parental cells was able to generate resistance to the drug combination. PAK-1 has shown to have a different role in the cytosol as pathways mediator and in the nucleus as transcriptional factor. Our data obtained by subcellular fractionation showed that after treatment with fulvestrant-abemaciclib, PAK1 protein accumulates into the cytosol in T47D-FAR compared to parental T47D, probably mediating an enhancement in cytoplasmatic cell signalling and pathway activation. To univocally demonstrate that PAK1 mediates tumor resistance to fulvestrant-abemaciclib combination, we are generating data from RNA-Seq and whole exome sequencing from T47DFAR and MCF7FAR and the relative parental cell lines. Also, we will assess the efficacy of PAK pharmacological inhibition in overcoming such resistance. Conclusions: In conclusion, our data suggest the role of PAK1 as a novel therapeutic target in ER+/PAK1-amplified breast cancer model resistant to endocrine therapy and CDK4/6 inhibitors. Citation Format: Stefania Belli, Alberto Servetto, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Fabiana Napolitano, Antonio Santaniello, Pietro De Placido, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Roberto Bianco, Luigi Formisano. Pak1 as a novel mediator of resistance to endocrine therapy and CDK4/6 inhibitors in ER+/PAK-1amplified 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 P3-06-08.

RB1 loss-of-function genomic alterations confer resistance to CDK4/6 inhibitors (CDK4/6i) and are enriched post treatment of CDK4/6i in estrogen receptor-positive (ER+) metastatic breast cancer. ER+/Rb-deficient breast cancer is a rising patient population in need of novel therapeutic strategies. Herein, we used a genome-wide CRISPR screen and identified protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in this refractory breast cancer subtype. sgRNA-induced depletion of PRMT5 arrested growth of MCF-7 and T47D RB1 knockout (RBKO) cells. PRMT5 catalyzes symmetric dimethylation of arginine (SDMA). In RBKO cells carrying doxycycline-inducible shRNA targeting the 3’UTR of PRMT5, rescue with wild-type but not an enzymatically dead mutant of PRMT5 restored cell growth, supporting that PRMT5 methyltrasferase activity is essential for growth of these cells. Gene set enrichment analysis (GSEA) of RNA-seq data revealed significant downregulation of cell cycle-related Hallmark gene signatures in RBKO cells treated with PRMT5 siRNA versus control siRNA. Both gene silencing and pharmacological blockade of PRMT5 with the small molecule inhibitor pemrametostat impeded G1-to-S cell cycle progression in MCF-7 and T47D RBKO cells and in lung, prostate, and triple-negative breast cancer cells with natural RB1 mutations or deletions, suggesting that PRMT5 inhibition can block the G1-to-S transition even in the absence of Rb. To identify the protein interactome of PRMT5 and the mechanism by which it promotes cell cycle progression in Rb-deficient cells, we performed proteomics analysis of Co-IP mass spectrometry and an SDMA post-translational modification scan and pinpointed FUS (fused in sarcoma) as a putative downstream effector of PRMT5. FUS is known to regulate RNA polymerase II (Pol II)-mediated transcription. Inhibition of PRMT5 with pemrametostat significantly reduced SDMA levels on FUS and dissociated FUS from Pol II as evidenced by FUS Co-IP and immunoblot analysis. ChIP-seq analysis revealed that treatment of RBKO cells with pemrametostat derepressed phosphorylation of Ser2 in the C-terminus of Pol II at transcription start sites (TSS) of genes involved in cell cycle progression. In accordance with the abnormal accumulation of pSer2 Pol II at TSS, pemrametostat treatment also resulted in an increased Pol II pausing index and an enrichment of intron retention splicing variants. Finally, therapeutic inhibition of PRMT5 with pemrametostat synergized with fulvestrant (a selective ER degrader) against growth of ER+/Rb-deficient breast cancer cell line- and patient-derived xenografts in mice, suggesting this combination as a novel therapeutic strategy for ER+/Rb-deficient metastatic breast cancers. Citation Format: Chang-Ching Lin, Tsung-Cheng Chang, Yunguan Wang, Yanfeng Zhang, Andrew Lemoff, Yisheng V. Fang, He Zhang, Dan Ye, Isabel Soria-Bretones, Alberto Servetto, Kyung-min Lee, Xuemei Luo, Joseph J. Otto, Hiroaki Akamatsu, David W. Cescon, Lin Xu, Yang Xie, Joshua T. Mendell, Ariella B. Hanker, Carlos L. Arteaga. PRMT5 is an actionable target in CDK4/6 inhibitor-resistant ER+/Rb-deficient breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3934.

We investigated the tumor microenvironment (TME) in estrogen receptor positive (ER+) primary breast cancers from stage I-III patients treated with estrogen deprivation (ED, induced with letrozole) for 10-21 days. We used primary breast cancer biopsies, collected from 198 patients, to assess tumor-infiltrating lymphocytes (TILs), transcriptomic profiles, and TME composition. Diagnostic biopsy and surgical on-treatment tumor specimens were collected; Ki67, ERα, PR, and HER2 expression were measured using automated quantitative analysis (AQUA). Tumors were categorized as estrogen deprivation-sensitive (ED-S) or -resistant (ED-R) based on the natural log (ln) of the Ki67 score (ln ≤1.0 or ≤2.7% Ki67+ cells vs. ln ≥2.0 or ≥7.4%, respectively) in the on-treatment (surgical) biopsy. Scoring of stromal (s)TILs, using hematoxylin and eosin staining of on-treatment specimens, revealed that sTILs were elevated in ED-R compared to ED-S tumors (p Citation Format: Fabiana Napolitano, Dhivya R. Sudhan, Yunguan Wang, Paula I. González-Ericsson, Luigi Formisano, Kyung-Min Lee, Chang-Ching Lin, María Rosario Chica-Parrado, Hongli Ma, Nathaniel Evans, Alberto Servetto, Saurabh Mendiratta, Spencer Barnes, Yisheng V. Fang, Justin M. Balko, Gordon B. Mills, Marilyne Labrie, Ariella B. Hanker, Carlos L. Arteaga. Immune cells infiltration and activation are higher in breast cancers resistant to antiestrogen therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3445.

Patients with locally advanced (LA) pancreatic ductal adenocarcinoma (PDAC) do not present distant metastases but are not eligible for surgery upfront. Chemotherapy regimens, such as FOLFIRINOX (FFN) or nab-paclitaxel plus gemcitabine (GemNab) in combination with loco-regional treatments are generally used in this setting. However, the best treatment choice is unknown. We retrospectively analyzed the information of 225 patients with stage II–III PDAC treated at our institution between October 2011 and December 2020. A total of 94 patients with LA PDAC who are non-eligible for surgery upfront received neoadjuvant FFN or GemNab. Of the 67 patients receiving FFN, 28 (41.8%) underwent surgery after neoadjuvant therapy. Of the 27 patients treated with GemNab, 6 (22.2%) became eligible for resection. The median overall survival (OS) was 85.1 weeks and 54.3 weeks in the FFN and GemNab groups, respectively (HR = 0.54, p = 0.0109). The median OS was 189.7 weeks and 76.4 weeks in the resected and unresected cohorts, respectively (HR = 0.25, p &lt, 0.0001). Neutropenia (37.3%), anemia (6.0%), and diarrhea (6.0%) in the FFN group and neutropenia (22.2%) and thrombocytopenia (18.5%) in the GemNab groups were the most frequent grade 3–4 side effects. Higher rates of thrombocytosis (p &lt, 0.0001) and peripheral edema (p &lt, 0.0001) were observed in the GemNab group. Our results suggest that the use of FFN is associated with more favorable clinical outcomes than GemNab for patients with LA PDAC. Future randomized and controlled clinical trials are needed to further elucidate the role of these regimens and loco-regional treatments in this setting.

Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists., Era+ breast cancer patients often develop resistance to endocrine therapy. Here, the authors show that FGFR1 amplification is a resistance mechanism to CDK4/6 inhibitor and endocrine therapy and that combined treatment with FGFR, CDK4/6, and anti-estrogens is a potential therapeutic strategy in Era+ breast cancer tumors.

Purpose: FGFR1 overexpression has been associated with endocrine resistance in ER+ breast cancer. We found FGFR1 localized in the nucleus of breast cancer cells in primary tumors resistant to estrogen suppression. We investigated a role of nuclear FGFR1 on gene transcription and antiestrogen resistance. Experimental Design: Tumors from patients treated with letrozole were subjected to Ki67 and FGFR1 IHC. MCF7 cells were transduced with FGFR1(SP-)(NLS) to promote nuclear FGFR1 overexpression. FGFR1 genomic activity in ER+/FGFR1-amplified breast cancer cells ± FOXA1 siRNA or ± the FGFR tyrosine kinase inhibitor (TKI) erdafitinib was examined by chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). The nuclear and chromatin-bound FGFR1 interactome was investigated by mass spectrometry (MS). Results: High nuclear FGFR1 expression in ER+ primary tumors positively correlated with post-letrozole Ki67 values. Nuclear FGFR1 overexpression influenced gene transcription and promoted resistance to estrogen suppression and to fulvestrant in vivo. A gene expression signature induced by nuclear FGFR1 correlated with shorter survival in the METABRIC cohort of patients treated with antiestrogens. ChIP-Seq revealed FGFR1 occupancy at transcription start sites, overlapping with active transcription histone marks. MS analysis of the nuclear FGFR1 interactome identified phosphorylated RNA-Polymerase II and FOXA1, with FOXA1 RNAi impairing FGFR1 recruitment to chromatin. Treatment with erdafitinib did not impair nuclear FGFR1 translocation and genomic activity. Conclusions: These data suggest nuclear FGFR1 contributes to endocrine resistance by modulating gene transcription in ER+ breast cancer. Nuclear FGFR1 activity was unaffected by FGFR TKIs, thus supporting the development of treatment strategies to inhibit nuclear FGFR1 in ER+/FGFR1 overexpressing breast cancer.

The development of cyclin-dependent kinases (CDK) 4 and 6 inhibitors represented a substantial breakthrough in the treatment of estrogen receptor positive (ER+), human epidermal growth factor receptor 2 (HER2) negative metastatic breast cancer. These drugs showed a significant clinical benefit in pivotal clinical trials. However, resistance eventually occurs, leading to disease progression. Next Generation Sequencing methodologies have been employed to investigate predictive biomarkers of response or resistance to CDK4/6 inhibitors. Whole exome and targeted sequencing of solid and liquid biopsies have revealed several possible genomic alterations associated with resistance. Notably, genomic alterations identified by DNA-sequencing did not fully recapitulate the entire landscape of resistance to CDK4/6 inhibitors. Gene expression analysis, such as RNA-Seq methodologies, have provided insights into transcriptional profiles and may need further application. Herein, we report the main findings derived from the use of NGS analysis in the context of resistance to CDK4/6 inhibitors in ER + breast cancer.

PURPOSE: FGFR1 overexpression has been associated with endocrine resistance in ER+ breast cancer. We found FGFR1 localized in the nucleus of breast cancer cells in primary tumors resistant to estrogen suppression. We investigated a role of nuclear FGFR1 on gene transcription and antiestrogen resistance. EXPERIMENTAL DESIGN: Tumors from patients treated with letrozole were subjected to Ki67 and FGFR1 IHC. MCF7 cells were transduced with FGFR1(SP-)(NLS) to promote nuclear FGFR1 overexpression. FGFR1 genomic activity in ER+/FGFR1-amplified breast cancer cells ± FOXA1 siRNA or ± the FGFR tyrosine kinase inhibitor (TKI) erdafitinib was examined by ChIP-Seq and RNA-Seq. The nuclear and chromatin-bound FGFR1 interactome was investigated by Mass Spectrometry (MS). RESULTS: High nuclear FGFR1 expression in ER+ primary tumors positively correlated with post-letrozole Ki67 values. Nuclear FGFR1 overexpression influenced gene transcription and promoted resistance to estrogen suppression and to fulvestrant in vivo. A gene expression signature induced by nuclear FGFR1 correlated with shorter survival in the METABRIC cohort of patients treated with antiestrogens. ChIP-Seq revealed FGFR1 occupancy at transcription start sites, overlapping with active transcription histone marks. MS analysis of the nuclear FGFR1 interactome identified phosphorylated RNA-Polymerase II and FOXA1, with FOXA1 RNAi impairing FGFR1 recruitment to chromatin. Treatment with erdafitinib did not impair nuclear FGFR1 translocation and genomic activity. CONCLUSIONS: These data suggest nuclear FGFR1 contributes to endocrine resistance by modulating gene transcription in ER+ breast cancer. Nuclear FGFR1 activity was unaffected by FGFR TKIs, thus supporting the development of treatment strategies to inhibit nuclear FGFR1 in ER+/FGFR1 overexpressing breast cancer.

The proto-oncogene c-Src is a non-receptor tyrosine kinase playing a key role in many cellular pathways, including cell survival, migration and proliferation. c-Src de-regulation has been observed in several cancer types, making it an appealing target for drug discovery efforts. Recent evidence emphasizes its crucial role not only in promoting oncogenic traits, but also in the acquisition and maintenance of cancer resistance to various chemotherapeutic or molecular target drugs. c-Src modulates epidermal growth factor receptor (EGFR) activation and amplifies its downstream oncogenic signals. In this review, we report several studies supporting c-Src kinase role in the intricate mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKIs). We further highlighted pre- and clinical progresses of combined treatment strategies made in recent years. Several pre-clinical data have encouraged the use of c-Src inhibitors in combination with EGFR inhibitors. However, clinical trials provided controversial outcomes in some cancer types. Despite c-Src inhibitors showed good tolerability in cancer patients, no incontrovertible and consistent clinical responses were recorded, supporting the idea that a better selection of patients is needed to improve clinical outcome. Currently, the identification of biological markers predictive of therapy response and the accurate molecular screening of cancer patients aimed to gain most clinical benefits become decisive and mandatory.

Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast and other cancers and tend to co-occur with HER3 (ERBB3) missense mutations. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against HER2-mutant tumors. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrate computational structural modeling with biochemical and cell biological analyses. Computational modeling predicts that the frequent HER3E928G kinase domain mutation enhances the affinity of HER2/HER3 and reduces binding of HER2 to its inhibitor neratinib. Co-expression of mutant HER2/HER3 enhances HER2/HER3 co-immunoprecipitation and ligand-independent activation of HER2/HER3 and PI3K/AKT, resulting in enhanced growth, invasiveness, and resistance to HER2-targeted therapies, which can be reversed by combined treatment with PI3Kα inhibitors. Our results provide a mechanistic rationale for the evolutionary selection of co-occurring HER2/HER3 mutations and the recent clinical observations that HER3 mutations are associated with a poor response to neratinib in HER2-mutant cancers.

We developed neratinib-resistant HER2-mutant cancer cells by gradual dose escalation. RNA sequencing identified TORC1 signaling as an actionable mechanism of drug resistance. Primary and acquired neratinib resistance in HER2-mutant breast cancer patient-derived xenografts (PDXs) was also associated with TORC1 hyperactivity. Genetic suppression of RAPTOR or RHEB ablated P-S6 and restored sensitivity to the tyrosine kinase inhibitor. The combination of the TORC1 inhibitor everolimus and neratinib potently arrested the growth of neratinib-resistant xenografts and organoids established from neratinib-resistant PDXs. RNA and whole-exome sequencing revealed RAS-mediated TORC1 activation in a subset of neratinib-resistant models. DNA sequencing of HER2-mutant tumors clinically refractory to neratinib, as well as circulating tumor DNA profiling of patients who progressed on neratinib, showed enrichment of genomic alterations that converge to activate the mTOR pathway.

Background: Pancreatic adenocarcinoma is the fourth leading cause of cancer-related death. In cases with metastasis, the combination of 5-fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) or gemcitabine-based chemotherapy regimens are considered the standard of care. However, the optimal sequence of these regimens is unclear. Methods: This retrospective study initially evaluated 186 patients with locally advanced/metastatic pancreatic cancer at three Italian institutions between February 2013 and October 2019. All patients had progressed after receiving gemcitabine-based first-line chemotherapy and were subsequently offered second-line FOLFIRINOX, FOLFOX-6, or FOLFIRI treatment. This study evaluated progression-free survival (PFS), overall survival from the start of second-line treatment (OS2), overall survival from the start of first-line treatment (OS1), and safety outcomes. Results: A total of 77 patients received ⩾4 cycles of second-line chemotherapy and were considered eligible: 15 patients received FOLFIRINOX, 32 patients received FOLFOX-6, and 30 patients received FOLFIRI. The FOLFIRINOX group had median PFS of 26.29 weeks and median OS2 of 47.86 weeks, while the FOLFIRI group had median PFS of 10.57 weeks and median OS2 of 25.00 weeks ( p = 0.038). No significant differences were observed between the FOLFIRINOX and FOLFOX-6 groups in terms of PFS (26.29 weeks versus 23.07 weeks) or OS2 (47.86 weeks versus 42.00 weeks). The most common grade 3–4 toxicities were anemia, neutropenia, and thrombocytopenia, which occurred more frequently in the FOLFIRINOX and FOLFOX-6 groups. Conclusion: Relative to the FOLFIRI regimen, the FOLFIRINOX regimen had a favorable toxicity profile and better survival outcomes. No significant differences were observed relative to the FOLFOX-6 regimen.

In the last few years, the treatment strategy in Non-Small Cell Lung Cancer (NSCLC) patients has been heavily modified by the introduction of the immune-checkpoint inhibitors. Anti-programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) therapy has improved both progression-free and the overall survival in almost all subgroups of patients, with or without PDL1 expression, with different degrees of responses. However, there are patients that are not benefitting from this treatment. A defined group of immune-checkpoint inhibitors non-responder tumours carry EGFR (epidermal growth factor receptor) mutations: nowadays, anti-PD-1/PD-L1 clinical trials often do not involve this type of patient and the use of immune-checkpoint inhibitors are under evaluation in this setting. Our review aims to elucidate the mechanisms underlying this resistance: we focused on evaluating the role of the tumour microenvironment, including infiltrating cells, cytokines, secreted factors, and angiogenesis, and its interaction with the tumour tissue. Finally, we analysed the possible role of immunotherapy in EGFR mutated tumours.

Purpose: While FGFR1 amplification has been described in breast cancer, the optimal treatment approach for FGFR1-amplified (FGFR1+) metastatic breast cancer (MBC) remains undefined. Experimental Design: We evaluated clinical response to endocrine and targeted therapies in a cohort of patients with hormone receptor–positive (HR+)/HER2− MBC and validated the functional role of FGFR1-amplification in mediating response/resistance to hormone therapy in vitro. Results: In the clinical cohort (N = 110), we identified that patients with FGFR1+ tumors were more likely to have progesterone receptor (PR)-negative disease (47% vs. 20%; P = 0.005), coexisting TP53 mutations (41% vs. 21%; P = 0.05), and exhibited shorter time to progression with endocrine therapy alone and in combination with CDK4/6 inhibitor, but not with a mTOR inhibitor (everolimus), adjusting for key prognostic variables in multivariate analysis. Furthermore, mTOR-based therapy resulted in a sustained radiological and molecular response in an index case of FGFR1+ HR+/HER2− MBC. In preclinical models, estrogen receptor–positive (ER+)/FGFR1-amplified CAMA1 human breast cancer cells were only partially sensitive to fulvestrant, palbociclib, and alpelisib, but highly sensitive to everolimus. In addition, transduction of an FGFR1 expression vector into ER+ T47D cells induced resistance to fulvestrant that could be overcome by added TORC1 inhibition, but not PI3K or CDK4/6 inhibition. Conclusions: Collectively, these findings suggest that while FGFR1 amplification confers broad resistance to ER, PI3K, and CDK4/6 inhibitors, mTOR inhibitors might have a unique therapeutic role in the treatment of patients with ER+/FGFR1+ MBC.

Background: Activating mutations in ERBB2 (HER2) are found in ~2% of solid tumors, including ~5% of metastatic breast cancers. The spectrum of HER2 mutations varies according to tumor type, with kinase domain (KD) missense mutations being most common in breast cancer, while exon 20 insertions are most common in non-small cell lung cancer (NSCLC). Breast cancer cells engineered with activating HER2 mutations show elevated HER2 phosphorylation and enhanced oncogenic growth. Activating mutations of other receptor tyrosine kinases such as EGFR affect receptor internalization, but whether HER2 mutations alter receptor internalization is not yet known. In addition, clinical responses to HER2 antibody-drug conjugates (ADCs) like T-DM1 have been noted in HER2-mutant NSCLC, raising the possibility that HER2 mutations alter receptor internalization, thus enhancing ADC sensitivity. Therefore, we aimed to 1) determine whether mutant HER2 receptor internalization is altered, and 2) determine whether altered internalization contributes to HER2 ADC sensitivity in HER2-mutant breast cancer. Methods: HER2 subcellular localization was investigated by immunofluorescence (IF) in MCF7 cells where HER2L755S, HER2V777L, or HER2L869R (or HER2WT) have been ‘knocked-in' using AAV-mediated recombination. We used ELISA-based endocytosis assays to quantify the internalization efficiency of trastuzumab-labeled HER2 in cell lines with WT and mutant HER2. Cell viability upon treatment with T-DM1 was tested using conventional 2D and 3D growth assays. Results: Partial colocalization of HER2 with clathrin, measured by IF, was observed in MCF7 cells, which was not affected by HER2 mutations. In the endocytosis assay, trastuzumab was inefficiently internalized over a period of 4 hours. In MCF7 or MCF10A breast epithelial cells, introduction of HER2L755S, HER2V777L, or HER2L869R did not significantly affect internalization following trastuzumab labeling, or after stimulation with EGF or neuregulin. In line with these results, MCF7 cells expressing HER2L755S, HER2V777L, or HER2L869R were not sensitive to T-DM1 in 2D cell viability assays or 3D Matrigel assays. In contrast, HER2 internalization was significantly more efficient in BT474 cells with wild-type HER2 amplification, which was accompanied by exquisite sensitivity of BT474 cells to T-DM1 both in vitro and in vivo. HER2 endocytic efficiency was significantly increased in MCF10A cells ectopically expressing HER2S310F, a mutation in the extracellular domain, or HER2A775>YVMA, a common insertion mutation in NSCLC. Conclusion: Breast cancer cells expressing HER2 KD missense mutations do not exhibit enhanced HER2 internalization and are not sensitive to T-DM1. However, cells with the HER2S310F mutation and the HER2A775>YVMA insertion showed enhanced HER2 endocytosis, consistent with clinical responses to T-DM1 in NSCLC patients with these mutations. Citation Format: Hiroaki Akamatsu, Rosa Mino, Marcel Mettlen, Dan Ye, Dhivya R. Sudhan, Albert Lin, Arnaldo Marin, Alberto Servetto, Kyung-min Lee, Sumanta Chatterjee, Sandra L. Schmid, Carlos L. Arteaga, Ariella B. Hanker. HER2 missense mutations in breast cancer cells do not alter HER2 internalization or sensitivity to T-DM1 [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 3675.

Background: FGFR1 amplification occurs in ~ 15% of estrogen receptor-positive (ER+) breast cancers. In these tumors, nuclear FGFR1 has been shown to interact with DNA, but its engagement in transcription regulation remains unclear. Thus, we investigated the mechanisms underpinning the genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer. Methods: FGFR1-ChIP-Seq was performed in CAMA1 ER+/FGFR1-amplified human breast cancer cells to identify genomic distribution of FGFR1. IP with FLAG antibody followed by Mass Spectrometry (MS) was carried out on nuclear plus chromatin fractions of CAMA1 cells overexpressing 3XFLAG-FGFR1 to uncover the nuclear FGFR1 interactome. Results: FGFR1-ChIP-Seq detected 4408 peaks in CAMA1 cells cultured in estrogen-free conditions, with marked enrichment of GC-rich consensus motifs. 67% of peaks were enriched at promoter regions. ChIP-PCR confirmed FGFR1 binding to several genomic loci in ER+/FGFR1-amplified cell lines (CAMA1 and MDA-MB-134) and PDX (HCI-011). Further, MS uncovered RNA Polymerase II subunits among the top nuclear FGFR1 interacting proteins. FGFR1 mainly bound Pol II phosphorylated on Ser5 (Pol II S5P), a marker of transcription initiation, in CAMA1, MDA-MB-134 and HCI-011 cell extracts. Pol II S5P-ChIP-Seq revealed that 65% (2867/4408) of FGFR1 peaks were shared with Pol II S5P in CAMA1 cells. Also, ChIP-Seq revealed that 95% of FGFR1 peaks overlapped with both H3K4me3 and H3K27ac, markers of active transcription. Consistent with these results, RNA-Seq of CAMA1 cells showed that expression of FGFR1-bound genes was markedly higher than non FGFR1-bound genes (p1), whose expression is likely regulated by nuclear FGFR1. A high signature score correlated with resistance to letrozole (p Conclusions: These findings support a prominent role for FGFR1 in the transcriptional machinery of breast cancer cells. Whether this transcriptional action is causal to antiestrogen resistance in ER+/FGFR1-amplified breast cancer is currently under investigation. Citation Format: Alberto Servetto, Rahul Kollipara, Luigi Formisano, Kyung-min Lee, Albert Lin, Dhivya R. Sudhan, Ariella B. Hanker, Sumanta Chatterjee, Hiroaki Akamatsu, Arnaldo Marin, Saurabh Mendiratta, Ralf Kittler, Carlos L. Arteaga. FGFR1 associates with gene promoters and regulates transcription in ER+/FGFR1-amplified breast cancer: Implications for endocrine resistance [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 1304.

The optimal therapeutic strategy for locally advanced pancreatic cancer patients (LAPC) has not yet been established. Our aim is to evaluate how surgery after neoadjuvant treatment with either FOLFIRINOX (FFN) or Gemcitabine-NabPaclitaxel (GemNab) affects the clinical outcome in these patients. LAPC patients treated at our institution were retrospectively analysed to reach this goal. The group characteristics were similar: 35 patients were treated with the FOLFIRINOX regimen and 21 patients with Gemcitabine Nab-Paclitaxel. The number of patients undergoing surgery was 14 in the FFN group (40%) and six in the GemNab group (28.6%). The median Disease-Free Survival (DFS) was 77.10 weeks in the FFN group and 58.65 weeks in the Gem Nab group (p = 0.625), while the median PFS in the unresected group was 49.4 weeks in the FFN group and 30.9 in the GemNab group (p = 0.0029, 95% CI 0.138&ndash, 0.862, HR 0.345). The overall survival (OS) in the resected population needs a longer follow up to be completely assessed, while the median overall survival (mOS) in the FFN group was 72.10 weeks and 53.30 weeks for the GemNab group (p = 0.06) in the unresected population. Surgery is a valuable option for LAPC patients and it is able to induce a relevant survival advantage. FOLFIRINOX and Gem-NabPaclitaxel should be offered as first options to pancreatic cancer patients in the locally advanced setting.

The HER2 tyrosine kinase inhibitor (TKI) neratinib is clinically active in patients with HER2-mutant cancers. However, responses are heterogeneous across tumor types and generally short lived, suggesting mechanisms of de novo and acquired resistance. We developed neratinib-resistant HER2-mutant 5637 bladder and OVCAR8 ovarian cancer cells by gradual dose escalation. RNA sequencing identified TORC1 signaling as an actionable mechanism of drug resistance. Treatment with the TORC1 inhibitor everolimus or genetic inactivation of RAPTOR or RHEB ablated p-S6 levels and restored sensitivity to the TKI. The combination of everolimus and neratinib potently arrested growth of neratinib-resistant xenografts. RNA-seq and RAS activity assays revealed significant enrichment of the RAS pathway in neratinib-resistant cells. siRNA mediated knockdown of RAS isoforms also abrogated p-S6 and neratinib resistance. Finally, DNA sequencing of HER2-mutant tumors from patients clinically refractory to neratinib showed enrichment of somatic alterations associated with aberrant activation of the mTOR pathway.

In several tumors the PI3K/AKT/mTOR pathway is frequently disrupted, an event that results in uncontrolled cell proliferation and tumor growth. Through the years, several compounds have been developed to inhibit the pathway at different steps: the mammalian target of rapamycin (mTOR) seemed to be the most qualified target. However, this kinase has such a key role in cell survival that mechanisms of resistance are rapidly developed. Nevertheless, clinical results obtained with mTOR inhibitors in breast cancer, renal cell carcinoma, neuroendocrine tumors and mantle cell lymphoma push oncologists to actively further develop these drugs, maybe by better selecting the population to which they are offered, through the research of predictive factors of responsiveness. In this review, we aim to describe mechanisms of resistance to mTOR inhibitors, from preclinical and clinical perspectives.

Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast cancers. The HER2 tyrosine kinase inhibitor (TKI) neratinib has shown clinical activity against cancers harboring HER2 activating mutations. Co-occurring HER2 and HER3 (ERBB3) mutations have been reported to co-occur in patients with breast cancer, suggesting the possibility of cooperativity of both oncogenes. Interrogation of the Project GENIE database revealed that gain-of-function missense mutations in HER2 and HER3 exhibit statistically significant co-occurence [q value=0.006 (breast cancer); q=1.01x10−26 (all cancer types)]. On the other hand, HER3 mutations were nearly absent in cancers harboring HER2 in-frame insertions and cancers with HER2 amplification. In breast cancer, co-occurring HER3 mutations were mutually exclusive with PIK3CA mutations, suggestive of potential functional redundancy. Sixty-eight unique breast cancers from cBioPortal, GENIE, and Foundation Medicine were found to harbor co-occurring mutations in HER2 and HER3, the most common of which were L755S, V777L, L869R/Q, and S310F, each with HER3E928G. Computational structural modeling suggested that the E928G substitution in HER3 enhances the interface energy between HER2 and HER3 kinase domains. This was confirmed by co-immunoprecipitation assays in HEK293 cells transfected with wild-type (WT) or mutant HER2 and HER3. This binding was further enhanced between HER3E928G and HER2 missense mutants. Further, structural modeling of L755S, V777L, and L869R/Q in HER2 revealed that each of these mutations increase HER2 kinase activation. Accordingly, each of these HER2 mutants, as well has HER2S310F in the extracellular domain, increased ligand-independent P-HER2 and P-HER3 in HEK293 cells. P-HER3 levels were the highest HEK293 in cells co-expressing HER2 and HER3 mutations. Similar results were observed in MCF10A breast epithelial cells stably expressing mutant HER2 and mutant HER3. Enhanced activation of the PI3K pathway (P-AKT and P-S6) was observed in cells expressing both mutations compared to cells expressing mutant HER2 with HER3WT or vice-versa. MCF10A HER2WT/HER3E982G cells did not form colonies in ligand-free 3D Matrigel assays, whereas cells expressing HER2L755S/HER3WT formed organized acini. In contrast, HER2L755S/HER3E928G acini were highly invasive. Addition of the HER3 ligand neuregulin (NRG) to HER2L755S/HER3WT cells phenocopied the effect of HER3E928G. Interestingly, MCF10A cells expressing the HER2Y772_A775 (YVMA) insertion together with HER3WT also formed invasive acini in the absence of NRG, suggesting that this HER2 insertion mutation is more transforming than HER2 missense mutations. Similar results were obtained with invasion assays using Matrigel-coated chambers. Finally, we examined the effects of HER2/HER3 co-mutations on sensitivity to HER2 inhibitors. Structural analysis was performed of the HER3/HER2 kinase domain complex docked with neratinib. HER3E928G, when bound to HER2, is located close to the neratinib-binding pocket of HER2 and was predicted to reduce HER2/neratinib binding affinity. Similarly, co-expression of HER3E928G reduced the ability of neratinib to inhibit P-HER3, P-AKT, P-S6, and growth of MCF10A cells expressing HER2/HER3 co-mutations. The PI3Kα inhibitor alpelisib restored neratinib sensitivity. Conclusions: Co-expression of mutant HER2 and mutant HER3 promotes ligand-independent HER2/HER association, HER3 phosphorylation, and cancer cell invasion via enhanced activation of the PI3K pathway; this enhanced signaling output is incompletely blocked by neratinib. Therefore, breast cancers expressing co-occurring HER2 and HER3 mutations may require the addition of a PI3Kα inhibitor to a HER2 TKI. Citation Format: Ariella B. Hanker, Harikrishna Sekar Jayanthan, Dan Ye, Chang-Ching Lin, Hiroaki Akamatsu, Jonathan H. Sheehan, James P. Koch, Dhivya R. Sudhan, Monica Red Brewer, Alberto Servetto, Jie He, Vincent A. Miller, Alshad S. Lalani, Jens Meiler, Carlos L. Arteaga. Co-occurring gain-of-function mutations in HER2 and HER3 modulate HER2/HER3 activation, breast cancer progression, and HER2 inhibitor sensitivity [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 GS6-04.