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Clinical Trial
. 2020 Aug;10(8):1174-1193.
doi: 10.1158/2159-8290.CD-19-1390. Epub 2020 May 13.

The Genomic Landscape of Intrinsic and Acquired Resistance to Cyclin-Dependent Kinase 4/6 Inhibitors in Patients with Hormone Receptor-Positive Metastatic Breast Cancer

Seth A Wander #  1   2   3   4 , Ofir Cohen #  1   2   4 , Xueqian Gong #  5 , Gabriela N Johnson  1   2   4 , Jorge E Buendia-Buendia  1   2   4 , Maxwell R Lloyd  1   2 , Dewey Kim  1   2   4 , Flora Luo  1   2   3   4 , Pingping Mao  1   2   3   4 , Karla Helvie  1   2 , Kailey J Kowalski  1   2   4 , Utthara Nayar  1   2   3   4 , Adrienne G Waks  1   2   3   4 , Stephen H Parsons  5 , Ricardo Martinez  5 , Lacey M Litchfield  5 , Xiang S Ye  5 , Chunping Yu  5 , Valerie M Jansen  5 , John R Stille  5 , Patricia S Smith  5 , Gerard J Oakley  5 , Quincy S Chu  6 , Gerald Batist  7 , Melissa E Hughes  1   2 , Jill D Kremer  5 , Levi A Garraway  1   2   3   4   5 , Eric P Winer  2   3 , Sara M Tolaney  2   3 , Nancy U Lin  2   3 , Sean G Buchanan  5 , Nikhil Wagle  8   2   3   4
Affiliations
Clinical Trial

The Genomic Landscape of Intrinsic and Acquired Resistance to Cyclin-Dependent Kinase 4/6 Inhibitors in Patients with Hormone Receptor-Positive Metastatic Breast Cancer

Seth A Wander et al. Cancer Discov. 2020 Aug.

Abstract

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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Figures

Figure 1.
Figure 1.. The genomic landscape of CDK4/6i resistance is heterogeneous, with multiple potential driver events.
(a) Biopsy phenotypes were assigned as sensitive, acquired resistance, or intrinsic resistance based upon timing of the biopsy relative to CDK4/6i exposure (d - days), best radiographic response (BRR), and duration of treatment. Patients were categorized as experiencing clinical benefit on CDK4/6i if interval restaging demonstrated a response or disease stability for at least six months. (b) Mutational matrix (CoMut) depicting the genomic landscape of the CDK4/6i cohort (n = 59 biopsies, 58 patients). Copy number alterations and mutational events in select genes of interest are shown. Clinical parameters (shown at the top) include receptor status, anti-estrogen agent, CDK4/6 inhibitor, best radiographic response (P – progression, R – response, S – stable), biopsy phenotype (S – sensitive, IR – intrinsic resistance, AR – acquired resistance), treatment duration (days), biopsy timing relative to treatment initiation (days), time since metastatic diagnosis (days), and number of lines of prior treatment. (c) Phenotype distribution plot demonstrating a higher frequency of copy number amplifications in Aurora Kinase A (AURKA) among resistant biopsies (AR + IR, left) compared to sensitive biopsies (right, 0.0081, Welch test). (d) Bar plot visualization of mutational (M) and/or copy number alterations (A – amplification, LA – low amplification) in select genes. The proportional enrichment (fraction of samples demonstrating alteration) in sensitive biopsies (left, blue) and resistant biopsies (AR + IR, right, red) is included.
Figure 2.
Figure 2.. Acquired resistance to CDK4/6i in patients with pre-treatment and post-progression biopsies demonstrates convergent evolution of biallelic RB1 disruption and evolved AKT1 activation.
Phylogenetic analysis depicting the evolutionary history for seven patients with acquired alterations, with clonal evolutionary dynamics demonstrating: (a) acquired polyclonal ESR1 mutations after aromatase inhibition, followed by convergent evolution of RB1 inactivation, with different RB1-inactivating mutations acquired in two parallel sibling clones; (b) Acquired AKT1 amplification; (c) No notable candidate for acquired mechanism of resistance (MOR); (d) Acquired AKT1 (W80R) mutation; (e) No notable candidate for acquired MOR; (f) Acquired inactivation of DNA Mismatch Repair Protein (MLH3); and (g) Acquired activating ESR1 mutation (Y537S) and amplification in AKT3. Branch colors in the phylogenetic trees match colors in the cell clouds for each patient, such that each color represents a subclone within the tumor cell population. Branch lengths are proportional to the number of mutations, private to that subclone, and the prevalence of each subclone is represented by relative proportion of colored cells in the cell clouds.
Figure 3.
Figure 3.. Clinical vignettes for candidate resistance drivers in representative patients (RB1, AKT1, RAS, and CCNE2).
Clinical vignettes including treatment sequence, timing of metastatic progression, and available biopsies with key genomic findings are provided for the following - (a) four patients with biallelic alterations in RB1, including a patient with multiple biopsies and convergent evolution toward RB1 disruption (top, phylogenetic analysis for this patient is provided in Figure 2A). (b) Three patients with acquired alterations in AKT1 following progression on CDK4/6i. In the first (top), a new mutation in AKT1 W80R was identified. In the second (middle), a baseline alteration (AKT1 L52H) was identified at the time of diagnosis; at the time of progression on CDK4/6i, two biopsies were obtained – both demonstrating the baseline AKT1 L52H mutation, one also demonstrating an acquired amplification of the wild-type AKT1 protein (phylogenetic analyses for these patients are provided in Figure 2B and D). (c) Three patients with resistance to CDK4/6i and RAS-family alterations (including two instances of KRAS G12D and one instance of HRAS mutation). (d) Three patients with intrinsic resistance to CDK4/6i and amplification events in CCNE2.
Figure 4.
Figure 4.. Candidate genomic alterations provoke CDK4/6i resistance in vitro.
(a) T47D cells were modified via CRISPR-mediated downregulation (RB1) or lentiviral overexpression (AKT1, KRAS G12D, AURKA, CCNE2) to interrogate potential resistance mediators identified in patient biopsy samples. Western blotting with the indicated antibodies is included. (b-f) Modified T47D cells were exposed to escalating doses of CDK4/6i (palbociclib – left, abemaciclib – right) and viability was estimated via cell-titer-glo (CTG) assay. Control (CRISPR non-targeting guide or GFP) cells are plotted along with the resistance driver of interest (RB1 – b, AKT1 – c, KRAS G12D – d, AURKA – e, CCNE2 – f). Parental and variant cell lines are normalized to vehicle control and viability is plotted as a function of increasing CDK4/6i (graphed as triplicate average +/− standard deviation). All variants provoke CDK4/6i resistance (to both palbociclib and abemaciclib) in vitro in T47D cells. Corresponding IC50 values are included in Supplemental Table 7.
Figure 5.
Figure 5.. Candidate mutations emerge in cell lines cultured under CDK4/6i selective pressure and define new therapeutic dependencies in vitro.
(a) Breast cancer cell lines (T47D, MCF7, MDA-MB-361) were cultured long-term to resistance in the presence of CDK4/6i (palbociclib, abemaciclib). The resulting cell lines which emerged were subjected to western blotting for putative mediators of drug resistance (RB1, AKT1, KRAS/ERK, AURKA, and CCNE2). (b-c) T47D cells cultured to resistance in the presence of abemaciclib demonstrated low levels of RB1 expression (T47D-AR1) and increased sensitivity to the AURKA inhibitor LY3295668. MDA-MB-361 cells cultured to resistance in the presence of abemaciclib demonstrated high levels of ERK activation (361-AR1) and increased sensitivity to the ERK inhibitor LY3214996. MDA-MB-361 cells cultured to resistance in the presence of palbociclib demonstrated high levels of AURKA (361-PR1) and increased sensitivity to the AURKA inhibitor LY3295668. MCF7 cells cultures to resistance in the presence of palbociclib demonstrated increased levels of CCNE2 (MCF7-PR1) and increased sensitivity to the CHEK1 inhibitor prexasertib.
Figure 6.
Figure 6.. A novel aurora kinase A inhibitor demonstrates therapeutic efficacy in a patient with metastatic HR+ breast cancer after progression on CDK4/6i.
(a) A patient with locally advanced HR+/HER2- breast cancer developed metastatic recurrence on adjuvant tamoxifen. She received CDK4/6i and letrozole in the first line setting with prolonged clinical benefit (>3 years). At progression, she was placed on trial with the AURKA inhibitor LY3295668; she subsequently experienced prolonged disease control ~11 months. Baseline staging studies following progression on CDK4/6i in the patient described are included (top); she had osseous metastatic disease and visceral disease limited to the foci noted in the liver. Two interval restaging studies (top) demonstrate disease stability/mild response. Liver biopsy obtained at the time of progression on CDK4/6i and prior to LY3295668 demonstrated high Ki67 and high RB1 protein expression via immunohistochemistry (IHC, 10x) (bottom). (b) Schematic diagram demonstrating the potential utility of next-generation sequencing following progression on CDK4/6i; actionable alterations in RB1, ERBB2, FGFR2, AKT1, RAS, AURKA, and CCNE2 could dictate informed selection of targeted therapies as indicated.
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