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Mechanisms driving resistance to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in hormone receptor–positive (HR+) breast cancer have not been clearly defined. Whole-exome sequencing of 59 tumors with CDK4/6i exposure revealed multiple candidate resistance mechanisms including RB1 loss, activating alterations in AKT1, RAS, AURKA, CCNE2, ERBB2, and FGFR2, and loss of estrogen receptor expression. In vitro experiments confirmed that these alterations conferred CDK4/6i resistance. Cancer cells cultured to resistance with CDK4/6i also acquired RB1, KRAS, AURKA, or CCNE2 alterations, which conferred sensitivity to AURKA, ERK, or CHEK1 inhibition. Three of these activating alterations—in AKT1, RAS, and AURKA—have not, to our knowledge, been previously demonstrated as mechanisms of resistance to CDK4/6i in breast cancer preclinically or in patient samples. Together, these eight mechanisms were present in 66% of resistant tumors profiled and may define therapeutic opportunities in patients.Significance:We identified eight distinct mechanisms of resistance to CDK4/6i present in 66% of resistant tumors profiled. Most of these have a therapeutic strategy to overcome or prevent resistance in these tumors. Taken together, these findings have critical implications related to the potential utility of precision-based approaches to overcome resistance in many patients with HR+ metastatic breast cancer.This article is highlighted in the In This Issue feature, p. 1079

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Differentially expressed genes

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FGFR alterations in different cohorts

Supplementary Data from Acquired FGFR and FGF Alterations Confer Resistance to Estrogen Receptor (ER) Targeted Therapy in ER+ Metastatic Breast Cancer

clinicopathologic info

Complete exome data

Gene sets defined and used in this study and Signature strength per sample

Biopsy purity and ploidy

Durable control of invasive solid tumors necessitates identifying therapeutic resistance mechanisms and effective drug combinations. In this work, we used a network-based mathematical model to identify sensitivity regulators and drug combinations for the PI3Kα inhibitor alpelisib in estrogen receptor positive (ER+) PIK3CA-mutant breast cancer. The model-predicted efficacious combination of alpelisib and BH3 mimetics, for example, MCL1 inhibitors, was experimentally validated in ER+ breast cancer cell lines. Consistent with the model, FOXO3 downregulation reduced sensitivity to alpelisib, revealing a novel potential resistance mechanism. Cell line–specific sensitivity to combinations of alpelisib and BH3 mimetics depended on which BCL2 family members were highly expressed. On the basis of these results, newly developed cell line–specific network models were able to recapitulate the observed differential response to alpelisib and BH3 mimetics. This approach illustrates how network-based mathematical models can contribute to overcoming the challenge of cancer drug resistance. Significance: Network-based mathematical models of oncogenic signaling and experimental validation of its predictions can identify resistance mechanisms for targeted therapies, as this study demonstrates for PI3Kα-specific inhibitors in breast cancer.

Background: CDK4/6 inhibitors have emerged as the standard of care for HR+ MBC. However, there is limited insight into the potential benefit of abemaciclib following prior progression on palbociclib or ribociclib. Based on a multi-center cohort of patients with HR+ MBC who had received abemaciclib after prior palbociclib progression (Wander SA et al ASCO 2019), we have previously reported that abemaciclib after prior CDK4/6i progression was well tolerated and that a subset of patients derived durable clinical benefit. Identifying molecular predictors of sensitivity to abemaciclib after prior CDK4/6i progression constitutes an important area of research. Given the high frequency of ESR1 mutations in HR+ MBC with antiestrogen resistance, we evaluated the translational impact of ESR1 mutations in mediating response to abemaciclib in this setting. Methods: To evaluate abemaciclib sensitivity in ESR1 mutant cell lines, T47D HR+ breast cancer cells were modified to over-express multiple mutant ESR1 isoforms via lentiviral infection and antibiotic selection. These isoforms included ESR1 Y537S, Y537N, and D538G. In an additional T47D cell line, RB1 expression was knocked down via CRISPR. The resulting derivative cell lines were grown in the absence of estrogen (via charcoal-stripped serum, CSS) or in escalating doses of abemaciclib. Cell viability was measured via cell-titer-glo assay. For clinical validation, we identified patients with MBC who had ESR1 mutations detected by targeted sequencing of cell-free DNA (cfDNA), via CLIA certified Guardant assay, and had abemaciclib exposure following prior progression on palbociclib or ribociclib in the existing multi-center cohort from six US institutions. Results: All ESR1 mutant derivative cells demonstrated enhanced growth in estrogen deprivation compared to GFP controls, as expected, and were similarly sensitive to escalating doses of abemaciclib monotherapy in vitro, suggesting that ESR1 mutations do not confer resistance to abemaciclib. Interestingly, two patients with ESR1 mutations (in the absence of concurrent driver alterations in RB1, FGFR, CCNE2, and ERBB2) demonstrated progression on palbociclib and sensitivity to abemaciclib. In one patient, cfDNA obtained prior to palbociclib and fulvestrant exposure failed to reveal any ESR1 alteration. Following progression on palbociclib, and prior to sequential exposure to abemaciclib, an ESR1 Y537N alteration was identified. The patient went on to receive 16 months of abemaciclib monotherapy. In a second patient, an ESR1 D538G alteration was identified following progression on palbociclib and fulvestrant. The patient had several intervening regimens, and subsequently went on to receive abemaciclib and fulvestrant for 16 months. RB1-null T47D cells were resistant to abemaciclib monotherapy in vitro, as expected and, in the clinical dataset, the presence of alterations in previously identified genomic mediators of CDK4/6i resistance, such as RB1, were associated with progression on both palbociclib and abemaciclib. Conclusions: HR+ breast cancer cells expressing mutant ESR1 isoforms were resistant to estrogen deprivation but retained sensitivity to abemaciclib in vitro. Furthermore, patients harboring ESR1 mutations via targeted sequencing of cfDNA, in the absence of other known mediators of CDK4/6i resistance, were shown to derive clinical benefit from abemaciclib following prior progression on palbociclib. These results suggest that patients with HR+ MBC, ESR1 mutation, and clinical resistance to anti-estrogen treatment and palbociclib may be candidates for abemaciclib treatment. Further research is warranted to confirm these novel translational observations. Citation Format: Seth A. Wander, Hyo S. Han, Gabriela N. Johnson, Maxwell R. Lloyd, Pingping Mao, Utthara Nayar, Kailey Kowalski, Casey R. Stein, Veronica Mariotti, Leslie SL Kim, Maren Levin, Jing Xi, Apurva Pandey, Siobhan Dunne, Azadeh Nasrazadani, Adam Brufsky, Kevin Kalinsky, Cynthia X Ma, Joyce O’Shaughnessy, Nikhil Wagle, Aditya Bardia. Esr1 mutation as a potential predictor of abemaciclib benefit following prior cdk4/6 inhibitor (cdk4/6i) progression in hormone receptor-positive (hr+) metastatic breast cancer (mbc): A translational investigation [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 PS5-10.

Purpose:To identify clinically relevant mechanisms of resistance to ER-directed therapies in ER+ breast cancer.Experimental Design:We conducted a genome-scale functional screen spanning 10,135 genes to investigate genes whose overexpression confer resistance to selective estrogen receptor degraders. In parallel, we performed whole-exome sequencing in paired pretreatment and postresistance biopsies from 60 patients with ER+ metastatic breast cancer who had developed resistance to ER-targeted therapy. Furthermore, we performed experiments to validate resistance genes/pathways and to identify drug combinations to overcome resistance.Results:Pathway analysis of candidate resistance genes demonstrated that the FGFR, ERBB, insulin receptor, and MAPK pathways represented key modalities of resistance. The FGFR pathway was altered via FGFR1, FGFR2, or FGF3 amplifications or FGFR2 mutations in 24 (40%) of the postresistance biopsies. In 12 of the 24 postresistance tumors exhibiting FGFR/FGF alterations, these alterations were acquired or enriched under the selective pressure of ER-directed therapy. In vitro experiments in ER+ breast cancer cells confirmed that FGFR/FGF alterations led to fulvestrant resistance as well as cross-resistance to the CDK4/6 inhibitor palbociclib. RNA sequencing of resistant cell lines demonstrated that FGFR/FGF induced resistance through ER reprogramming and activation of the MAPK pathway. The resistance phenotypes were reversed by FGFR inhibitors, a MEK inhibitor, and/or a SHP2 inhibitor.Conclusions:Our results suggest that FGFR pathway is a distinct mechanism of acquired resistance to ER-directed therapy that can be overcome by FGFR and/or MAPK pathway inhibitors.

Mechanisms driving resistance to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in hormone receptor–positive (HR+) breast cancer have not been clearly defined. Whole-exome sequencing of 59 tumors with CDK4/6i exposure revealed multiple candidate resistance mechanisms including RB1 loss, activating alterations in AKT1, RAS, AURKA, CCNE2, ERBB2, and FGFR2, and loss of estrogen receptor expression. In vitro experiments confirmed that these alterations conferred CDK4/6i resistance. Cancer cells cultured to resistance with CDK4/6i also acquired RB1, KRAS, AURKA, or CCNE2 alterations, which conferred sensitivity to AURKA, ERK, or CHEK1 inhibition. Three of these activating alterations—in AKT1, RAS, and AURKA—have not, to our knowledge, been previously demonstrated as mechanisms of resistance to CDK4/6i in breast cancer preclinically or in patient samples. Together, these eight mechanisms were present in 66% of resistant tumors profiled and may define therapeutic opportunities in patients.Significance:We identified eight distinct mechanisms of resistance to CDK4/6i present in 66% of resistant tumors profiled. Most of these have a therapeutic strategy to overcome or prevent resistance in these tumors. Taken together, these findings have critical implications related to the potential utility of precision-based approaches to overcome resistance in many patients with HR+ metastatic breast cancer.This article is highlighted in the In This Issue feature, p. 1079

Background: The AKT1 oncogene represents a central node in the well-characterized PI3K/AKT/mTOR signal transduction pathway involved in the regulation of cellular proliferation as well as therapeutic resistance. With the emergence of multiple targeted therapies for HR+ MBC, we sought to explore the prevalence of AKT1 alterations, the spectrum of co-occurring mutations, and any potential impact on response to endocrine-based therapy, including CDK 4/6 and PIK3CA inhibitors. Methods: Presence or absence of AKT1 mutation was interrogated via cell-free DNA (cfDNA) analysis (Guardant assay) among patients with HR+/HER2- MBC receiving treatment at the Massachusetts General Hospital who underwent routine clinical cfDNA testing. Genomic sequencing results were then compared to detailed clinical annotation based upon retrospective chart review. In the laboratory, HR+/HER2- T47D breast cancer cells were transfected with AKT1, under the control of a doxycycline-inducible promoter, to induce AKT1 overexpression. Sensitivity to escalating doses of fulvestrant and palbociclib were interrogated via cell-titer-glo viability assay in vitro. Results: Among 272 patients with HR+/HER2- MBC, we identified 13 patients with AKT1 mutation (4.7%). 12/13 (92%) patients had a canonical AKT1E17K alteration, one patient (8%) had an AKT1 E49K alteration. AKT1 mutations occurred concurrently with alterations in ESR1 (31%), PIK3CA (23%), TP53 (23%), ERBB2 (15%), and RB1 (8%). All patients had been exposed to some form of anti-estrogen prior to sequencing, either via (neo)adjuvant therapy and/or in the metastatic setting; 5/13 (38%) had exposure to a CDK4/6 inhibitor prior to cfDNA sequencing. Following the identification of an AKT1 alteration, examples of rapid clinical progression on fulvestrant, CDK4/6i, and/or everolimus were identified, however the presence of an AKT1 alteration was not sufficient to predict pan-resistance to these agents, as counter-examples of clinical benefit were also identified. In an index patient with an AKT1 mutation and prior progression on letrozole and palbociclib, subsequent treatment with fulvestrant and abemaciclib did not yield clinical benefit, highlighting the potential role of AKT1 in mediating clinical resistance to endocrine therapy and CDK4/6i. In the pre-clinical model, HR+/HER2- breast cancer cells demonstrated resistance to both fulvestrant and palbociclib following upregulation of exogenous AKT1 expression. Updated clinical data with additional patients and response to regimens incorporating an anti-estrogen, a CDK4/6 inhibitor, an mTOR inhibitor, and/or a PIK3CA inhibitor will be presented at the meeting. Conclusions: Activating AKT1 mutations can be identified via targeted sequencing of cfDNA in patients with HR+/HER2- MBC, and may play an important role in mediating resistance to anti-estrogens, CDK4/6 inhibitors, and other emerging targeted therapies. Heterogeneity in response to endocrine-based therapy following the identification of an AKT1 alteration may be related to tumor-extrinsic factors, the AKT1 allelic fraction, or cooperativity between multiple resistance drivers. Further research is needed to confirm these findings and guide optimal therapeutic sequencing strategies for patients with HR+/HER2- AKT1 mutant MBC. Citation Format: Seth Wander, Pingping Mao, Maxwell Lloyd, Kailey Kowalski, Gabriela N. Johnson, Giuliana Malvarosa, Beverly Moy, Leif W. Ellisen, John Iafrate, Nikhil Wagle, Aditya Bardia. AKT1 alterations following exposure to endocrine-based therapy in patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC): Clinical and functional implications [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 P6-10-08.

Background: The CDK4/6 inhibitors have emerged as standard first- or second-line regimens in combination with an antiestrogen for patients with HR+/HER2- MBC. While these agents convey significant clinical benefit in many patients, intrinsic resistance can occur and, in patients who respond, acquired resistance is unfortunately inevitable. Despite their widespread use, we have limited insight into the molecular mechanisms governing response and resistance to these agents. Methods: Whole exome sequencing (WES) was performed on metastatic tumor biopsies from 58 patients (pts) with HR+/HER2- MBC who received a CDK4/6 inhibitor with or without an antiestrogen at the Dana-Farber Cancer Institute, including 7 pts with pre/post-exposure biopsy pairs. Among these biopsies, 69.5% were characterized as resistant (intrinsic or acquired) and 30.5% were characterized as sensitive. To validate putative resistance mediators identified in patient samples, HR+/HER2- breast cancer cells were modified via CRISPR knockout or lentiviral overexpression. Sensitivity of these cells to antiestrogens and CDK4/6i was interrogated via cell-titer-glo assay. In parallel, HR+/HER2- breast cancer cells were cultured to resistance in the presence of an escalating dose of CDK4/6i. Derivative cell lines were subjected to western blotting in an effort to interrogate the putative resistance mediators identified in pts. Novel dependencies were identified in these derivative cell lines via treatment with targeted therapeutic agents in vitro. Results: WES of tumors with CDK4/6i exposure revealed candidate mechanisms of resistance including biallelic RB1 disruption (n=4, 10%) and activating events in AKT1 (n=5, 12.5%), RAS (n=4, 10%), aurora kinase A (AURKA, n=11, 27.5%), and cyclin E2 (CCNE2, n=6, 15%). Convergent evolution toward biallelic RB1 disruption was identified in a single patient with one pre- and two post-exposure biopsies, while acquisition of AKT1 mutation and amplification was identified in two separate instances. Knockout of RB1 and overexpression of AKT1, KRAS G12D, AURKA, and CCNE2 provoked CDK4/6i and antiestrogen resistance in vitro. Breast cancer cells cultured to resistance in CDK4/6i demonstrated concordant acquisition of RB1 downregulation, RAS/ERK activation, AURKA overexpression, and CCNE2 overexpression. Derivative resistant cell lines with RB1 loss or AURKA gain demonstrated enhanced sensitivity to a novel AURKA inhibitor (LY3295668), while cells with RAS activation were highly sensitive to ERK inhibition (via LY3214996). CCNE2-overexpressing cells were highly sensitive to prexasertib, a CHEK1 inhibitor. Conclusions: The genomic landscape of resistance to CDK4/6i is heterogeneous with multiple potential mediators that play well-established roles in cell division and oncogenic signal transduction. We present novel mechanisms of clinical resistance including activation of AKT1 and RAS family oncogenes as well as amplification of AURKA and CCNE2. These drivers were able to provoke resistance to CDK4/6i in vitro. Finally, in each case, a novel dependency was identified which is readily translatable into the clinic. These results underscore the potential of next-generation sequencing as a critical tool to enable identification of resistance mediators, while also suggesting that the presence of specific genomic alterations may define new therapeutic opportunities in CDK4/6i-resistant HR+ MBC. Citation Format: Seth A. Wander, Ofir Cohen, Xueqian Gong, Gabriela N. Johnson, Jorge Buendia-Buendia, Maxwell Lloyd, Dewey Kim, Flora Luo, Pingping Mao, Karla Helvie, Kailey Kowalski, Utthara Nayar, Stephen Parsons, Ricardo Martinez, Lacey Litchfield, Xiang Ye, Chun Ping Yu, Valerie Jansen, Levi A. Garraway, Eric P. Winer, Sara M. Tolaney, Nancy U. Lin, Sean Buchanan, Nikhil Wagle. The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in patients with hormone receptor-positive (HR+)/HER2- metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-09.

Durable control of invasive solid tumors necessitates identifying therapeutic resistance mechanisms and effective drug combinations. A promising approach to tackle the cancer drug resistance problem is to build mechanistic mathematical models of the signaling network of cancer cells, and explicitly model the dynamics of information flow through this network under distinct genetic conditions and in response to perturbations.In this work, we used a network-based mathematical model to identify sensitivity factors and drug combinations for the PI3Kα inhibitor alpelisib, which was recently approved for ER+PIK3CAmutant breast cancer. We experimentally validated the model-predicted efficacious combination of alpelisib and BH3 mimetics (e.g. MCL1 inhibitors) in ER+ breast cancer cell lines. We also experimentally validated the reduced sensitivity to alpelisib caused by FOXO3 knockdown, which is a novel potential resistance mechanism. Our experimental results showed cell line-specific sensitivity to the combination of alpelisib and BH3 mimetics, which was driven by the choice of BH3 mimetics. We find that cell lines were sensitive to the addition of either MCL1 inhibitor s63845 alone or in combination with BCL-XL/BCL-2 inhibitor navitoclax, and that the need for the combination of both BH3 mimetics was predicted by the expression of BCL-XL. Based on these results, we developed cell line-specific network models that are able to recapitulate the observed differential response to alpelisib and BH3 mimetics, and also incorporate the most recent knowledge on resistance and response to PI3Kα inhibitors.Overall, we present an approach for the development, experimental testing, and refining of mathematical models, which we apply to the context of PI3Kα inhibitor drug resistance in breast cancer. Our approach predicted and validated PI3Kα inhibitor sensitivity factors (FOXO3 knockdown) and drug combinations (BH3 mimetics), and illustrates that network-based mathematical models can contribute to overcoming the challenge of cancer drug resistance.

Background: Deciphering the molecular landscape of resistance to the CDK4/6 inhibitors represents a critically important question for patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC). Emerging insights from sequencing efforts suggest that inactivating alterations in the RB1 tumor suppressor occur in a small minority of patients and that a variety of heterogeneous mediators provoke resistance in patient samples. Proteins implicated in CDK4/6i resistance include cell cycle regulators such as cyclin E1/2, CDK6, and aurora kinase as well as known oncogenic signal transduction mediators involved in activation of the RAS-MEK and AKT-mTOR pathways. The insulin-like growth factor 1 receptor (IGF1R) has been implicated in modulating anti-estrogen resistance, and IGF1R inhibitors are currently in various stages of pre-clinical and clinical development. Methods: We identified patients with amplification events in IGF1R from a database containing targeted sequencing of solid tumor samples obtained from patients with HR+ MBC enrolled on a research biopsy protocol. Tumor biopsies may have been obtained at various points during each patient’s clinical treatment course. HR+ T47D cells were modified to over-express IGF1R via lentiviral infection and selection. Derivative cell lines were treated with IGF-1 ligand and downstream activation of the PI3K/AKT and RAS/MEK pathways were assessed via western blotting. Control cells (expressing GFP) were mixed with IGF1R-expressing cells 1:1 and cultured in the presence of IGF-1 ligand and palbociclib or other drugs, for 1-3 weeks. At the timepoint of interest, cells were harvested and the relative proportion of GFP or IGF1R-expressing cells were interrogated via flow cytometry. Results: We identified seven patients with HR+ MBC and IGF1R amplifications via targeted sequencing of tumor biopsies. Five of these patients had exposure to CDK4/6i-based therapy in the metastatic setting. Three patients demonstrated intrinsic resistance to CDK4/6i treatment (with duration Conclusions: IGF1R amplification events were identified in tumor biopsy samples that reflect either intrinsic or acquired resistance to CDK4/6i-based therapy. HR+ breast cancer cells which over-express IGF1R demonstrate enrichment under palbociclib drug selection in a flow cytometry-based competition assay, which was abrogated by concurrent use of a MEK or IGF1R inhibitor. These results suggest that IGF1R may join the increasingly heterogeneous landscape of CDK4/6i resistance mediators. Further exploration of this possibility is warranted. A subset of patients with IGF1R-mediated CDK4/6i resistance could benefit from therapeutic strategies designed to downregulate MEK or IGF1R activity. Citation Format: Seth A. Wander, Pingping Mao, Maxwell R. Lloyd, Gabriela N. Johnson, Kailey Kowalski, Utthara Nayar, Lillian M. Guenther, Kimberly Stegmaier, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Igf1r mediates cdk4/6 inhibitor (cdk4/6i) resistance in tumor samples and in cellular models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-08.

To identify clinically relevant mechanisms of resistance to ER-directed therapies in ERWe conducted a genome-scale functional screen spanning 10,135 genes to investigate genes whose overexpression confer resistance to selective estrogen receptor degraders. In parallel, we performed whole-exome sequencing in paired pretreatment and postresistance biopsies from 60 patients with ERPathway analysis of candidate resistance genes demonstrated that the FGFR, ERBB, insulin receptor, and MAPK pathways represented key modalities of resistance. The FGFR pathway was altered viaOur results suggest that FGFR pathway is a distinct mechanism of acquired resistance to ER-directed therapy that can be overcome by FGFR and/or MAPK pathway inhibitors.

Clinical resistance mechanisms to CDK4/6 inhibitors in HR+ breast cancer have not been clearly defined. Whole exome sequencing of 59 tumors with CDK4/6i exposure revealed multiple candidate resistance mechanisms including RB1 loss, activating alterations in AKT1, RAS, AURKA, CCNE2, ERBB2, and FGFR2, and loss of ER expression. In vitro experiments confirmed that these alterations conferred CDK4/6i resistance. Cancer cells cultured to resistance with CDK4/6i also acquired RB1, KRAS, AURKA, or CCNE2 alterations, which conferred sensitivity to AURKA, ERK, or CHEK1 inhibition. Besides inactivation of RB1, which accounts for ∼5% of resistance, seven of these mechanisms have not been previously identified as clinical mediators of resistance to CDK4/6 inhibitors in patients. Three of these—RAS activation, AKT activation, and AURKA activation—have not to our knowledge been previously demonstrated preclinically. Together, these eight mechanisms were present in 80% of resistant tumors profiled and may define therapeutic opportunities in patients.SignificanceWe identified eight distinct mechanisms of resistance to CDK4/6 inhibitors present in 80% of resistant tumors profiled. Most of these have a therapeutic strategy to overcome or prevent resistance in these tumors. Taken together, these findings have critical implications related to the potential utility of precision-based approaches to overcome resistance in many patients with HR+ MBC.

Beyond acquired mutations in the estrogen receptor (ER), mechanisms of resistance to ER-directed therapies in ER+ breast cancer have not been clearly defined. We conducted a genome-scale functional screen spanning 10,135 genes to investigate genes whose overexpression confer resistance to selective estrogen receptor degraders. Pathway analysis of candidate resistance genes demonstrated that the FGFR, ERBB, insulin receptor, and MAPK pathways represented key modalities of resistance. In parallel, we performed whole exome sequencing in paired pre-treatment and post-resistance biopsies from 60 patients with ER+ metastatic breast cancer who had developed resistance to ER-targeted therapy. The FGFR pathway was altered via FGFR1, FGFR2, or FGF3/FGF4 amplifications or FGFR2 mutations in 24 (40%) of the post-resistance biopsies. In 12 of the 24 post-resistance tumors exhibiting FGFR/FGF alterations, these alterations were not detected in the corresponding pre-treatment tumors, suggesting that they were acquired or enriched under the selective pressure of ER-directed therapy. In vitro experiments in ER+ breast cancer cells confirmed that FGFR/FGF alterations led to fulvestrant resistance as well as cross-resistance to the CDK4/6 inhibitor palbociclib, through activation of the MAPK pathway. The resistance phenotypes were reversed by FGFR inhibitors and, to a lesser extent, MEK inhibitors, suggesting potential treatment strategies. Significance A genome-scale overexpression screen revealed a broad spectrum of resistance mechanisms against SERDs, which can provide a resource for researchers studying resistance to ER-directed therapies as well as the biology of estrogen receptor dependencies in ER+ breast cancer. We demonstrate that activating FGFR/FGF alterations are a mechanism of acquired resistance to ER-directed therapies and CDK4/6 inhibitors in ER+ metastatic breast cancer and can be overcome by combination therapy targeting both the ER and the FGFR pathway. The detection of targetable, clonally acquired genetic alterations in metastatic tumor biopsies highlights the value of serial tumor testing to dissect mechanisms of resistance in human breast cancer and its potential application in directing clinical management.

Resistance to therapy in metastatic breast cancer patients results from acquired mutations and selection of resistant clones. Understanding the mechanisms of resistance in the clinical setting is complicated by the lack of genomic characterization of tumor specimens. This case report describes a patient with ER+ and dynamic HER2+ status with exposure to a range of different therapies, suggesting that inference of tumor subclonal composition and evolution can be leveraged to study treatment resistance. A female patient was diagnosed with estrogen receptor positive (ER+) and human epidermal growth factor receptor positive (HER2+) stage I (T1N0) breast cancer. After receiving standard of care adjuvant chemotherapy (AC-TH) and Tamoxifen for two years, the patient developed metastatic disease to the liver. Upon metastatic disease, she received first line treatment with the trastuzumab emtansine antibody-drug conjugate (T-DM1). After fifteen months, enlargement of supraclavicular and retroperitoneal lymph nodes was observed, Letrozole was added to the treatment regimen until the patient progressed after eight months. Subsequent therapy with Fulvestrant and the CDK4/6 inhibitor Palbocicilb was administered for three months until the patient developed brain metastases. We analyzed biopsies taken at different time points of metastatic disease and, using whole exome sequencing (WES), reconstructed the subclonal architecture of each of the tumor specimens and inferred the evolutionary history of these clones. These results suggest potential mechanisms of resistance to the different therapies administered during disease progression. Biopsies obtained originated from a retroperitoneal lymph node at 3 years after diagnosis, a liver metastasis at 4 years after diagnosis and a brain metastasis after 4 years and 3 months after diagnosis. WES was performed on these tumor specimens and characterization of mutational events and copy number alterations was made using standard analytical pipelines. Inference of subclonal populations in the three tumor specimens analyzed revealed presence of a founding clone with mutations common to the three metastatic biopsies with alterations in TP53 p.K305* and PIK3CA p.E545K. A clone unique to the ER+/HER2- retroperitoneal lymph node metastasis shows no known driver mutations. In the liver metastasis (ER+/HER2-), we observed the presence of a dominant clone (cellular prevalence 93.5%) with mutation in ESR1 p.G442R and another subclone detected at low cellular prevalence (~5%) with an amplification in the ERBB2 gene. In the brain metastasis (ER+/HER2-), the latest tumor specimen obtained, we observed clonal selection of the ERBB2-amplified subpopulation (clonal prevalence 60%). The subclonal population dynamics and the different alterations in the clones present at different time points of tumor evolution provide a rationale to understand the therapeutic sensitivity and resistance observed in this patient. The ER+/HER2- retroperitoneal lymph node metastasis which appeared under T-DM1 treatment, suggests that this subclone is resistant to T-DM1, explained by the HER2 negative status, and was selected while the patient was on this regime. At the time of appearance of this clone, Letrozole was added to the treatment and a clone with an ESR1 p.G442R mutation appeared and dominated the tumor population, suggesting that this mutation provides resistance to the aromatase inhibitor. When treatment was switched to Fulvestrant and Palbociclib, a potentially dormant clone with ER+ and HER2+ features re-appeared in the tumor. Evolutionary analysis of this patient's tumor specimens underscores the importance of multiple therapies directed at all tumor vulnerabilities present, since targeting a single driver feature of the tumor will result in selection of other resistant subclones. Citation Format: Jorge E. Buendia-Buendia, Sonia Pernas, Ofir Cohen, Ping Ping Mao, Kailey Kowalski, Nancy Lin, Eric Winer, Nikhil Wagle. Clonal heterogeneity and tumor evolution inform treatment resistance in a patient with HER2+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-09.

While recent studies have begun characterizing the metastatic breast cancer (MBC) genomics, our understanding of mechanisms of acquired endocrine-resistance, their induced cell-state, and their altered drug-response profile, remains lacking. We collected biopsies from patients with MBC with detailed clinicopathologic features. To date we profiled 520 exomes, and 291 transcriptomes, with 126 patients having multiple biopsy exomes. Curated endocrine-relapse set include 60 patients with pre-treatment and post-relapse exomes. Characterization of candidate mechanisms of resistance (MOR) included 909 RNA-seq profiles of T47D cells with introduced MOR under various drugs. In the acquired endocrine resistance cohort, as expected, we found frequent ESR1 acquired mutations (13 pt, 22%). Additionally, we identified acquired activating SNVs and amplifications in oncogenic receptor tyrosine kinases (RTKs) in 18/60 (30%), including EGF family - HER2 (n=7), ERRB3 (R525Q), and EGFR (S116F), and FGF family - FGFR1 (n=5), FGFR2 (n=4), and FGF3 amplicon (n=4). RNA-seq of T47D cells overexpressing HER2 activating mutations revealed a distinct cell-state (HER2-ACT). Similarly, FGFR activation revealed a district FGFR-ACT state. The transcriptional signatures of these HER2 and FGFR states were remarkably similar (odd-ratio= 91, p= 2.64E-94). To characterize the cell-state common to HER and FGFR, we defined RTK-ACT with 358 overlapping marker genes (see table). Canonical (estradiol) ER signaling is slightly elevated in RTK-ACT, however this state is strongly associated with growth-factor driven ER signaling, suggesting reprogramming of ER from AF2, to AF1 signaling. Consistent with this, RTK-ACT had significantly higher MAPK activation. Additionally, RTK-ACT Induced stronger similarity to Basal-state, enrichment in motility/migration, mesenchymal, and stem-like features, with top genes including CDH3, MBP7, and S100, ETV, DUSP, SPRY families (see table). To study RTK-driven state in-vivo, we analyzed RNA-seq from our MBC biopsies, and compared tumors with activating RTK mutations (n=38) with WT (n=118), and inferred activated RTK in-vivo (RTK.ACT.iv). Our in-vitro and in-vivo states show significant overlap (OR=4.71, p=9.42E-20). Furthermore, characterization of RTK.ACT.iv, recapitulated all the cell-state features observed in RTK.ACT - including higher growth-factors ER signaling, MAPK, and basal-like state (see table). We further studied the viability and transcription of these RTKs under various drugs (12 treatments), including fulvestrant, palbociclib, and specific tyrosine kinase inhibitors (TKIs) - Neratinib (pan-HER inhibitor) and FIIN-3 (FGFR-i). We found that HER2-ACT and FGFR-ACT signatures remain robust when treated with fulvestrant, palbociclib, and their combinations, as compared to TKIs suppression (see table). in-line with our viability results - suggesting intrinsic resistance to CDK4/6i and sensitivity to TKIs. This study demonstrated that activating RTKs constitute of prevalent modality of acquired resistance to endocrine therapies, inducing a distinct state with clinical implications - suggesting the potential benefit of combination therapies with specific TKIs over CDK4/6i. The common MAPK activity and our preliminary results - suggests the potential of convergence-node targeting strategy with added MEK or SHP2 inhibition. TableGene set AGene set BOdds-ratioP-value (two-sided)Fisher''s Exact test matHER2-ACT -FGFR-ACT -912.64E-9469, 131, 131, 22704HER2 S653C, L755S, V777L, L869R vs. GFP, under DMSO, rank genes based on the logFC (top 200)FGFR1, FGFR2 (WT, N550K, M538I, K660N) vs. GFP, Parental, Under DMSO, rank genes based on the logFC (top 200)HER2-ACT_1000 -FGFR-ACT_1000 -18.98.28E-248358, 642, 642, 21758HER2 S653C, L755S, V777L, L869R vs. GFP, under DMSO, rank genes based on the logFC- top 1000FGFR1, FGFR2 (WT, N550K, M538I, K660N) vs. GFP, Parental, Under DMSO, rank genes based on the logFC - top 1000RTK-ACTHALLMARK_ESTROGEN_RESPONSE_EARLY, MSigDB3.030.004229, 191, 349, 22474RTK-ACTER driven by Growth Factors, PMID: 208897186.771.74E-059, 86, 349, 22585RTK-ACTMEK_UP.V1_UP (MAPK), MSigDB10.95.72E-1827, 169, 331, 22496RTK-ACTRAS ONCOGENE (MAPK), PMID: 162730928.445.77E-1220, 158, 338, 22553RTK-ACTWU_CELL_MIGRATION, PMID 187243907.648.96E-1119, 165, 339, 22502RTK-ACTHUPER_BREAST_BASAL_VS_LUMINAL_UP, PMID 17409405131.38E-079, 45, 349, 22621RTK-ACTHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, MSigDB3.770.00031411, 189, 347, 22477RTK-ACTLIM_MAMMARY_STEM_CELL_UP, PMID 203461513.119.56E-0622, 467, 336, 22205RTK-ACTRTK-ACT.iv -4.719.42E-2060, 940, 298, 21992In MBC biopsies, compare RTK mutations with WT, rank genes based on the logFC - top 1000RTK-ACT.iv (in-vivo)HALLMARK_ESTROGEN_RESPONSE_EARLY, MSigDB1.950.020316, 184, 984, 22088RTK-ACT.ivER driven by Growth Factors, PMID: 208897182.640.0076310, 85, 990, 22193RTK-ACT.ivMEK_UP.V1_UP (MAPK), MSigDB2.863.91E-0522, 174, 978, 22098RTK-ACT.ivRAS ONCOGENE (MAPK), PMID: 162730921.620.13212, 166, 988, 22152RTK-ACT.ivWU_CELL_MIGRATION, PMID 187243903.573.60E-0725, 159, 975, 22115RTK-ACT.ivHUPER_BREAST_BASAL_VS_LUMINAL_UP, PMID 174094059.515.43E-1016, 38, 984, 22235RTK-ACT.ivHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, MSigDB2.090.0073817, 183, 983, 22090RTK-ACT.ivLIM_MAMMARY_STEM_CELL_UP, PMID 203461511.420.089229, 460, 971, 21819HER2-ACTHER2-ACT - Fulv3006.53E-183109, 91, 91, 22750HER2-ACTHER2-ACT - Palbo2331.46E-163101, 99, 99, 22749HER2-ACTHER2.ACT - Fulv + Palbo2197.64E-15999, 101, 101, 22742HER2-ACTHER2.ACT - Neratinib63.19.78E-7257, 143, 143, 22707HER2-ACTHER2.ACT - Fulv + Neratinib59.33.53E-6855, 145, 145, 22724FGFR-ACTFGFR.ACT - Palbo19003.2e-319157, 43, 43, 22778FGFR-ACTFGFR.ACT - Fulv + Palbo12501.38E-290148, 52, 52, 22767FGFR-ACTFGFR.ACT - Fulv8661.72E-266140, 60, 60, 22764FGFR-ACTFGFR.ACT - Palbo + FIIN.31062.88E-10474, 126, 126, 22710FGFR-ACTFGFR.ACT - Fulv + Palbo + FIIN.378.41.14E-8464, 136, 136, 22705FGFR-ACTFGFR.ACT - FIIN.367.42.16E-7559, 141, 141, 22730FGFR-ACTFGFR.ACT - Fulv + FIIN.336.89.47E-4541, 159, 159, 22733 Citation Format: Ofir Cohen, Pingping Mao, Utthara Nayar, Jorge E Buendia-Bue, Dewey Kim, Esha Jain, Karla Helvie, Daniel Abravanel, Kailey J Kowalski, Christian Kapstad, Samuel Freeman, Victor Adalsteinsson, Seth A Wander, Adrienne G Waks, Gad Getz, Aviv Regev, Eric Winer P Winer, Nancy U Nancy U. Lin, Nikhil Wagle. Acquired activating mutations in RTKs confer endocrine resistance in ER+ metastatic breast cancer through ER-reprogramming, MAPK signaling, and an induced stem-like cell state [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-02.

Durable control of invasive solid tumors is thwarted by the lack of knowledge of effective drug combinations and of the acquired and intrinsic resistance mechanisms of drugs. In an effort to tackle this problem, the SU2C-NSF-TVF Drug Combination Convergence Team is using mechanistic models of cancer cell signaling based on therapeutic and cell line data in order to identify elements within cancer cells that might eventually be exploited through therapeutic combinations. Here we present a comprehensive mechanistic network model of signal transduction in ER+ PIK3CA-mutant breast cancer. Focusing on PI3K inhibitors, the model recapitulates known resistance mechanisms and predicts other possibilities for resistance: loss of RB1, FOXO3, P27, or PRAS40. To test these predictions, we analyzed genome-wide CRISPR screens of two breast cell lines in the presence of PI3K inhibitors (BYL719 and GDC0032) and found that the predicted genes (RB1, FOXO3, P27, and PRAS40) were significantly enriched in the screens. Some of these resistance genes (e.g. loss of RB1) were found to be cell-line specific and follow-up experiments in RB1-KO cells confirmed the cell-line-specific nature of PI3K-inhibitor resistance. The model also reveals known and novel combinatorial interventions that are more effective than PI3K inhibition alone. For example, the model predicts that the combination of PI3K inhibitors with inhibitors of anti-apoptotic protein MCL1 would be effective. Follow up experiments in cell lines using cell viability assays, cell death analyses, and dynamic BH3 profiling experiments to determine increases in apoptotic priming upon treatment confirmed that MCL1 inhibitors (S63845) enhance the effect of PI3K inhibitors (BYL719) and that this combinatorial effect is cell-line-specific, similarly to what was found in the resistance genes case. In conclusion, the model predicted drug resistance mechanisms and effective drug combinations, some of which were verified experimentally and found to be cell-line-specific. Next iterations of the model will incorporate the identified discrepancies and newly identified resistance mechanisms to drugs of clinical interest. Citation Format: Jorge Gómez Tejeda Zañudo, Pingping Mao, Joan Montero, Guotai Xu, Kailey J. Kowalski, Gabriela N. Johnson, José Baselga, Maurizio Scaltriti, Anthony G. Letai, Nikhil Wagle, Reka Albert. Network modeling of drug resistance mechanisms and drug combinations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 675.