PKMYT1 is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER+ Breast Cancer.

Endocrine therapies (ET) with CDK4/6 inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of 22 ER+ breast cancer patient-derived xenografts (PDXs) demonstrated that PKMYT1, a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX organoids and xenografts, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.

Selective CDK7 Inhibition Suppresses Cell Cycle Progression and MYC Signaling While Enhancing Apoptosis in Therapy-resistant Estrogen Receptor-positive Breast Cancer.

PURPOSE: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms. EXPERIMENTAL DESIGN: We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models. RESULTS: We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models. CONCLUSIONS: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments.

High FOXA1 levels induce ER transcriptional reprogramming, a pro-metastatic secretome, and metastasis in endocrine-resistant breast cancer.

Aberrant activation of the forkhead protein FOXA1 is observed in advanced hormone-related cancers. However, the key mediators of high FOXA1 signaling remain elusive. We demonstrate that ectopic high FOXA1 (H-FOXA1) expression promotes estrogen receptor-positive (ER+) breast cancer (BC) metastasis in a xenograft mouse model. Mechanistically, H-FOXA1 reprograms ER-chromatin binding to elicit a core gene signature (CGS) enriched in ER+ endocrine-resistant (EndoR) cells. We identify Secretome14, a CGS subset encoding ER-dependent cancer secretory proteins, as a strong predictor for poor outcomes of ER+ BC. It is elevated in ER+ metastases vs. primary tumors, irrespective of ESR1 mutations. Genomic ER binding near Secretome14 genes is also increased in mutant ER-expressing or mitogen-treated ER+ BC cells and in ER+ metastatic vs. primary tumors, suggesting a convergent pathway including high growth factor receptor signaling in activating pro-metastatic secretome genes. Our findings uncover H-FOXA1-induced ER reprogramming that drives EndoR and metastasis partly via an H-FOXA1/ER-dependent secretome.

A Multiparameter Molecular Classifier to Predict Response to Neoadjuvant Lapatinib plus Trastuzumab without Chemotherapy in HER2+ Breast Cancer.

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

Can adults with developmental dyslexia apply statistical knowledge to a new context?

We investigated transfer of artificial grammar learning in adults with and without dyslexia in 3 experiments. In Experiment 1, participants implicitly learned an artificial grammar system and were tested on new items that included the same symbols. In Experiment 2, participants were given practice with letter strings and then tested on strings created with a different letter set. In Experiment 3, participants were given practice with shapes and then tested on strings created with different shapes. Results show that in Experiment 1, both groups demonstrated utilization of pre-trained instances in the subsequent grammaticality judgement task, while in Experiments 2 (orthographic) and 3 (nonorthographic), only typically developed participants demonstrated application of knowledge from training to test. A post hoc analysis comparing between the experiments suggests that being trained and tested on an orthographic task leads to better performance than a nonorthographic task among typically developed adults but not among adults with dyslexia. Taken together, it appears that following extensive training, individuals with dyslexia are able to form stable representations from sequential stimuli and use them in a subsequent task that utilizes strings of similar symbols. However, the manipulation of the symbols challenges this ability.

Self-perception and perceived parental perception in adolescent girls with anorexia nervosa.

Negative self-perception is associated with poor outcomes in adults with anorexia nervosa (AN). Our study aimed to assess the association between the self-perception of female adolescents with AN and how these adolescents perceive the attitudes of their parents toward them on the severity and short-term outcome of their illness. For this purpose, we assessed 30 adolescent girls hospitalized with AN and 30 female controls. Self-perception and perceived parental attitudes were assessed using the Structural Analysis of Social Behavior (SASB), according to which self-perception is formed via close relations with significant others in early life. Patients with AN responded to the SASB and to questionnaires assessing eating disorder (ED) symptomatology and emotional distress at both admission and discharge. Controls were similarly assessed once. We found that patients with AN showed a more negative self-perception than controls. Negative self-perception was associated with negative perceptions of the mothers' attitudes toward the girls. There was no between-group difference in the perceived perception of the fathers' attitude to the girls. Self-perception and perceived parental attitudes were associated with the severity of ED symptoms and emotional distress. Finally, an improvement was found in self-perception and perceived maternal attitudes toward the girl from admission to discharge, alongside a decrease in the severity of ED symptoms and emotional distress. Self-perception at admission was associated with ED pathology and emotional distress at discharge. These findings suggest that self-perception and perceived parental attitudes toward the adolescent with AN may be associated with the severity of the illness and its short-term outcome.

A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer.

Most invasive lobular breast cancers (ILC) are of the luminal A subtype and are strongly hormone receptor-positive. Yet, ILC is relatively resistant to tamoxifen and associated with inferior long-term outcomes compared with invasive ductal cancers (IDC). In this study, we sought to gain mechanistic insights into these clinical findings that are not explained by the genetic landscape of ILC and to identify strategies to improve patient outcomes. A comprehensive analysis of the epigenome of ILC in preclinical models and clinical samples showed that, compared with IDC, ILC harbored a distinct chromatin state linked to gained recruitment of FOXA1, a lineage-defining pioneer transcription factor. This resulted in an ILC-unique FOXA1-estrogen receptor (ER) axis that promoted the transcription of genes associated with tumor progression and poor outcomes. The ILC-unique FOXA1-ER axis led to retained ER chromatin binding after tamoxifen treatment, which facilitated tamoxifen resistance while remaining strongly dependent on ER signaling. Mechanistically, gained FOXA1 binding was associated with the autoinduction of FOXA1 in ILC through an ILC-unique FOXA1 binding site. Targeted silencing of this regulatory site resulted in the disruption of the feed-forward loop and growth inhibition in ILC. In summary, ILC is characterized by a unique chromatin state and FOXA1-ER axis that is associated with tumor progression, offering a novel mechanism of tamoxifen resistance. These results underscore the importance of conducting clinical trials dedicated to patients with ILC in order to optimize treatments in this breast cancer subtype. SIGNIFICANCE: A unique FOXA1-ER axis in invasive lobular breast cancer promotes disease progression and tamoxifen resistance, highlighting a potential therapeutic avenue for clinical investigations dedicated to this disease. See related commentary by Blawski and Toska, p. 3668.

Endocrine Therapy-Resistant Breast Cancer Cells Are More Sensitive to Ceramide Kinase Inhibition and Elevated Ceramide Levels Than Therapy-Sensitive Breast Cancer Cells.

ET resistance is a critical problem for estrogen receptor-positive (ER+) breast cancer. In this study, we have investigated how alterations in sphingolipids promote cell survival in ET-resistant breast cancer. We have performed LC-MS-based targeted sphingolipidomics of tamoxifen-sensitive and -resistant MCF-7 breast cancer cell lines. Follow-up studies included treatments of cell lines and patient-derived xenograft organoids (PDxO) with small molecule inhibitors; cytometric analyses to measure cell death, proliferation, and apoptosis; siRNA-mediated knockdown; RT-qPCR and Western blot for gene and protein expression; targeted lipid analysis; and lipid addback experiments. We found that tamoxifen-resistant cells have lower levels of ceramides and hexosylceramides compared to their tamoxifen-sensitive counterpart. Upon perturbing the sphingolipid pathway with small molecule inhibitors of key enzymes, we identified that CERK is essential for tamoxifen-resistant breast cancer cell survival, as well as a fulvestrant-resistant PDxO. CERK inhibition induces ceramide-mediated cell death in tamoxifen-resistant cells. Ceramide-1-phosphate (C1P) partially reverses CERK inhibition-induced cell death in tamoxifen-resistant cells, likely through lowering endogenous ceramide levels. Our findings suggest that ET-resistant breast cancer cells maintain lower ceramide levels as an essential pro-survival mechanism. Consequently, ET-resistant breast cancer models have a unique dependence on CERK as its activity can inhibit de novo ceramide production.

NPY1R exerts inhibitory action on estradiol-stimulated growth and predicts endocrine sensitivity and better survival in ER-positive breast cancer.

G Protein-Coupled Receptors (GPCRs) represent the largest superfamily of cell-surface proteins. However, the expression and function of majority of GPCRs remain unexplored in breast cancer (BC). We interrogated the expression and phosphorylation status of 398 non-sensory GPCRs using the landmark BC proteogenomics and phosphoproteomic dataset from The Cancer Genome Atlas. Neuropeptide Y Receptor Y1 (NPY1R) gene and protein expression were significantly higher in Luminal A tumors versus other BC subtypes. The trend of NPY1R gene, protein, and phosphosite (NPY1R-S368s) expression was decreasing in the order of Luminal A, Luminal B, Basal, and human epidermal growth factor receptor 2 (HER2) subtypes. NPY1R gene expression increased in response to estrogen and reduced with endocrine therapy in estrogen receptor-positive (ER+) BC cells and xenograft models. Conversely, NPY1R expression decreased in ER+ BC cells resistant to endocrine therapies (estrogen deprivation, tamoxifen, and fulvestrant) in vitro and in vivo. NPY treatment reduced estradiol-stimulated cell growth, which was reversed by NPY1R antagonist (BIBP-3226) in ER+ BC cells. Higher NPY1R gene expression predicted better relapse-free survival and overall survival in ER+ BC. Our study demonstrates that NPY1R mediates the inhibitory action of NPY on estradiol-stimulated growth of ER+ BC cells, and its expression serves as a biomarker to predict endocrine sensitivity and survival in ER+ BC patients.

Interferon Signaling in Estrogen Receptor-positive Breast Cancer: A Revitalized Topic.

Cancer immunology is the most rapidly expanding field in cancer research, with the importance of immunity in cancer pathogenesis now well accepted including in the endocrine-related cancers. The immune system plays an essential role in the development of ductal and luminal epithelial differentiation in the mammary gland. Originally identified as evolutionarily conserved antipathogen cytokines, interferons (IFNs) have shown important immune-modulatory and antineoplastic properties when administered to patients with various types of cancer, including breast cancer. Recent studies have drawn attention to the role of tumor- and stromal-infiltrating lymphocytes in dictating therapy response and outcome of breast cancer patients, which, however, is highly dependent on the breast cancer subtype. The emerging role of tumor cell-inherent IFN signaling in the subtype-defined tumor microenvironment could influence therapy response with protumor activities in breast cancer. Here we review evidence with new insights into tumor cell-intrinsic and tumor microenvironment-derived IFN signaling, and the crosstalk of IFN signaling with key signaling pathways in estrogen receptor-positive (ER+) breast cancer. We also discuss clinical implications and opportunities exploiting IFN signaling to treat advanced ER+ breast cancer.

Artificial grammar learning is facilitated by distributed practice: Evidence from a letter reordering task.

Previous studies have shown that distributed practice-a training strategy that is known to facilitate memory-is likely to result in greater learning than massed practice. This effect has been demonstrated largely in explicit tasks. The purpose of this study was to test whether statistical learning of artificial grammar is affected by the lag between learning sessions overall, and by high and low complexity stimuli (as measure by chunk strength). Two groups (spaced-short and spaced-long) learned strings of letters created according to a set of rules and were required to produce new strings using given letter sets. For the spaced-short group, the two learning sessions, each including training and a test phase, took place sequentially with a 10-min break, whereas for the spaced-long group, learning sessions were distributed across two days (1-day lag). Overall results showed improved performance following spaced-long practice compared to spaced-short practice. The results also indicated that in the low chunk strength strings (indicating high complexity), both groups demonstrated similar improvement from first to second testing, while in the high chunk strength strings (indicating low complexity), improvement in letter reordering performance was significantly higher when the learning sessions were distributed across two days. This pattern of findings suggests that stimuli complexity affects the extent to which distributed practice enhance artificial grammar learning.

Restoring order at the cell cycle border: Co-targeting CDK4/6 and CDK2.

Overcoming resistance to CDK4/6 inhibitors is a major clinical challenge. In this issue of Cancer Cell, Freeman-Cook et al. study mechanisms of resistance to CDK4/6 inhibitors by employing a CRISPRa screen. They identify the cyclin E-CDK2 axis and Myc signaling as key pathways of resistance and develop PF-06873600, a selective CDK2/4/6 inhibitor.

Management of hormone receptor-positive, human epidermal growth factor 2-negative metastatic breast cancer.

Estrogen receptor (ER) is the major driver of most metastatic breast cancers (mBCs). Endocrine therapy (ET) is the most effective treatment for ER + mBC, but its effectiveness is limited by high rates of de novo and acquired resistance. A growing understanding of the biological characteristics and complexity of the ER pathway and the mechanisms of ET resistance has led to the development of a new generation of targeted therapies. One such mechanism is the cell cycle signaling pathways, which lead to the development of cyclin-dependent kinase 4/6 inhibitors (CDK4/6is) that have, in turn, transformed the management of such tumors. Another important mechanism is the alteration of the phosphatidylinositol 3'-kinase/AKT/mammalian target of rapamycin pathway. Drugs targeting each component of these pathways are currently used in clinical practice, and several more are in development. As a result, a myriad of new targeted therapies are consistently being added to the clinical oncologist armamentarium. Navigating the evolving and highly complex treatment landscape of HR + /HER2- mBC remains both an art and a challenge. In this review, we discuss the biological features of HR + /HER2- mBC and the different mechanisms of resistance to ET. We also discuss the management of mBC as the disease changes from endocrine-sensitive to endocrine-resistant.

A novel role of ADGRF1 (GPR110) in promoting cellular quiescence and chemoresistance in human epidermal growth factor receptor 2-positive breast cancer.

While G protein-coupled receptors (GPCRs) are known to be excellent drug targets, the second largest family of adhesion-GPCRs is less explored for their role in health and disease. ADGRF1 (GPR110) is an adhesion-GPCR and has an important function in neurodevelopment and cancer. Despite serving as a poor predictor of survival, ADGRF1's coupling to G proteins and downstream pathways remain unknown in cancer. We evaluated the effects of ADGRF1 overexpression on tumorigenesis and signaling pathways using two human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC) cell-line models. We also interrogated publicly available clinical datasets to determine the expression of ADGRF1 in various BC subtypes and its impact on BC-specific survival (BCSS) and overall survival (OS) in patients. ADGRF1 overexpression in HER2+ BC cells increased secondary mammosphere formation, soft agar colony formation, and % of Aldefluor-positive tumorigenic population in vitro and promoted tumor growth in vivo. ADGRF1 co-immunoprecipitated with both Gαs and Gαq proteins and increased cAMP and IP1 when overexpressed. However, inhibition of only the Gαs pathway by SQ22536 reversed the pro-tumorigenic effects of ADGRF1 overexpression. RNA-sequencing and RPPA analysis revealed inhibition of cell cycle pathways with ADGRF1 overexpression, suggesting cellular quiescence, as also evidenced by cell cycle arrest at the G0/1 phase and resistance to chemotherapy in HER2+ BC. ADGRF1 was significantly overexpressed in the HER2-enriched BC compared to luminal A and B subtypes and predicted worse BCSS and OS in these patients. Therefore, ADGRF1 represents a novel drug target in HER2+ BC, warranting discovery of novel ADGRF1 antagonists.

Neratinib plus trastuzumab is superior to pertuzumab plus trastuzumab in HER2-positive breast cancer xenograft models.

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

The bone microenvironment increases phenotypic plasticity of ER+ breast cancer cells.

Estrogen receptor-positive (ER+) breast cancer exhibits a strong bone tropism in metastasis. How the bone microenvironment (BME) impacts ER signaling and endocrine therapy remains poorly understood. Here, we discover that the osteogenic niche transiently and reversibly reduces ER expression and activities specifically in bone micrometastases (BMMs), leading to endocrine resistance. As BMMs progress, the ER reduction and endocrine resistance may partially recover in cancer cells away from the osteogenic niche, creating phenotypic heterogeneity in macrometastases. Using multiple approaches, including an evolving barcoding strategy, we demonstrated that this process is independent of clonal selection, and represents an EZH2-mediated epigenomic reprogramming. EZH2 drives ER+ BMMs toward a basal and stem-like state. EZH2 inhibition reverses endocrine resistance. These data exemplify how epigenomic adaptation to BME promotes phenotypic plasticity of metastatic seeds, fosters intra-metastatic heterogeneity, and alters therapeutic responses. Our study provides insights into the clinical enigma of ER+ metastatic recurrences despite endocrine therapies.

Endocrine-Based Treatments in Clinically-Relevant Subgroups of Hormone Receptor-Positive/HER2-Negative Metastatic Breast Cancer: Systematic Review and Meta-Analysis.

A precise assessment of the efficacy of first-/second-line endocrine therapies (ET) ± target therapies (TT) in clinically-relevant subgroups of hormone receptor-positive (HR+)/HER2-negative metastatic breast cancer (MBC) has not yet been conducted. To improve our current knowledge and support clinical decision-making, we thus conducted a systematic literature search to identify all first-/second-line phase II/III randomized clinical trials (RCT) of currently approved or most promising ET ± TT. Then, we performed a meta-analysis to assess progression-free (PFS) and/or overall survival (OS) benefit in several clinically-relevant prespecified subgroups. Thirty-five RCT were included (17,595 patients). Pooled results show significant reductions in the risk of relapse or death of 26-41% and 12-27%, respectively, depending on the clinical subgroup. Combination strategies proved to be more effective than single-agent ET (PFS hazard ratio (HR) range for combinations: 0.60-0.65 vs. HR range for single agent ET: 0.59-1.37; OS HR range for combinations: 0.74-0.87 vs. HR range for single agent ET: 0.68-0.98), with CDK4/6-inhibitors(i) + ET being the most effective regimen. Single agent ET showed comparable efficacy with ET+TT combinations in non-visceral (p = 0.63) and endocrine sensitive disease (p = 0.79), while mTORi-based combinations proved to be a valid therapeutic option in endocrine-resistant tumors, as well as PI3Ki + ET in PIK3CA-mutant tumors. These results strengthen international treatment guidelines and can aid therapeutic decision-making.

Stimulus variation-based training enhances artificial grammar learning.

The current study was designed to explore whether statistical learning ability is affected by the diversity of the stimulus set used in the training phase. The effect of stimulus diversity was assessed by controlling and manipulating the number of exposures to a given set and the number of unique strings presented to the learner during the training phase. 147 students participated in two studies. In the unvaried stimulus study, 71 participants learned the same basic set of 15 exemplars, once(15 × 1 exposure), twice (15 × 2 exposures = 30 total strings) and 3 times (15 × 3 exposures = 45 total strings). In the varied stimulus study, 75 participants learned 15, 30 and 45, all of which were unique, unrepeated exemplars. All groups were asked to classify test strings for their grammaticality following training. Results of the d' measures in the unvaried stimulus study indicate similar performance across the groups. Conversely, the results of the varied stimulus study show that the group presented with 45 unique strings performed significantly better than the baseline group (15 strings). Analysis of the differences across the equivalent groups in the two studies (15 × 2 exposures vs. 30 unique strings and 15 × 3 exposures vs. 45 unique strings) indicates differences in performance only between the group who was presented with the same 15 strings three times and the group presented with 45 unrepeated strings. Taken together, our results shed additional light on the central role of stimulus variation in Artificial Grammar Learning.