Stacy Moulder’s research while affiliated with Eli Lilly and Company and other places

Triple-negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer. The pillar of treatment is chemotherapy, but only half of the patients have a complete response and good survival. To resolve inter- and intra-tumoral heterogeneity and determine their clinical associations, we performed single-cell RNA-sequencing and spatial transcriptomics methods including Visium and Xenium on treatment-naïve samples of TNBC patients in the ARTEMIS clinical trial. We find that TNBC was classified into 4 major archetypes at patient level: luminal secretory-associated, basal-associated, immunoreactive, and luminal androgen receptor. At cell level, cancer cells exhibited intratumoral heterogeneity in 13 gene expression metaprograms. The TNBC tumor microenvironment (TME) consisted of 49 distinct immune and stromal cell states, many of which were reprogrammed relative to normal breast tissues from disease-free women. We further identified 8 ecotypes of cancer cells and TME cell states that co-occurred among patients and were associated with specific archetypes and chemotherapy response groups. In contrast to previous work on T-cells, our data showed the importance of macrophage cell states and cancer cell metaprograms for interferon signaling, HLA expression and cell cycle activity that were associated with chemotherapy response. To facilitate a clinical application, we developed a 13-gene-based model to predict response. Collectively, this study provides new insights into the natural biology of untreated TNBC tumors and their association with chemotherapy response. Citation Format Yun Yan, Yiyun Lin, Tapsi Kumar, Shanshan Bai, Aatish Thennavan, Jianzhuo Li, Tuan Tran, Min Hu, Mitchell Rao, Anna Casasent, Elizabeth Ravenberg, Gaiane Margishvili Rauch, Alyson Clayborn, Debu Tripathy, Alastair Thompson, Bora Lim, Lei Huo, Stacy Moulder, Clinton Yam, Nicholas Navin. Decoding the archetypes and ecotypes of triple-negative breast cancer in responses to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6438.

Metaplastic breast cancer (MpBC) is a highly chemoresistant subtype of breast cancer with no standardized therapy options. A clinical study in anthracycline-refractory MpBC patients suggested that nitric oxide synthase (NOS) inhibitor NG-monomethyl-l-arginine (L-NMMA) may augment anti-tumor efficacy of taxane. We report that NOS blockade potentiated response of human MpBC cell lines and tumors to phosphoinositide 3-kinase (PI3K) inhibitor alpelisib and taxane. Mechanistically, NOS blockade leads to a decrease in the S-nitrosylation of c-Jun NH2-terminal kinase (JNK)/c-Jun complex to repress its transcriptional output, leading to enhanced tumor differentiation and associated chemosensitivity. As a result, combined NOS and PI3K inhibition with taxane targets MpBC stem cells and improves survival in patient-derived xenograft models relative to single-/dual-agent therapy. Similarly, biopsies from MpBC tumors that responded to L-NMMA+taxane therapy showed a post-treatment reversal of epithelial-to-mesenchymal transition and decreased stemness. Our findings suggest that combined inhibition of iNOS and PI3K is a unique strategy to decrease chemoresistance and improve clinical outcomes in MpBC.

Triple negative breast cancer (TNBC) accounts for 15–20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.

Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer commonly treated with neoadjuvant chemotherapy (NAC). While about 50% of patients achieve a pathological complete response (pCR), the remainder often develop resistance and metastasis, leading poor survivals. Currently, the factors linking pre-treatment cancer cells and the tumor microenvironment (TME) to NAC response are unclear. To investigate this question, we conducted single-cell RNA sequencing on fresh pre-treatment core biopsy samples collected from patients in the ARTEMIS clinical trial. We identified 13 gene expression meta-programs of cancer cells and about 50 cell states of immune and stromal cell types. Based on the cancer cells alone, we identified four TNBC archetypes at patient-level: luminal secretory-associated (LSA), basal-associated (BA), immunoreactive (IR), and luminal androgen receptor (LAR). Notably, the archetype BA and IR were associated with non-pCR and pCR, respectively. Additionally, we found the TNBC ecosystem was composed of eight eco-hubs, reflecting the co-occurrence patterns of cancer and TME cell states. These eco-hubs revealed different cell community across archetypes and NAC outcomes. For example, an eco-hub with a co-occurrence of interferon response-related cancer cells and immune cells was prevalent in the archetype IR and pCR patients. To facilitate a clinical application, we further developed a 13-gene-based machine learning model to predict NAC response. In summary, these results provide new insights into the cellular determinants of TNBC biology and chemotherapeutic response. Citation Format: Yun Yan, Yiyun Lin, Tapsi Kumar, Shanshan Bai, Jianzhuo Li, Tuan Tran, Min Hu, Elizabeth Ravenberg, Maia Rauch, Alyson Clayborn, Alastair Thompson, Bora Lim, Lei Huo, Stacy Moulder, Clinton Yam, Nicholas Navin. Decoding the archetypes and eco-hubs of triple-negative breast cancer in responses to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6933.

Chemotherapy, along with the PD1 inhibitor pembrolizumab, is the recommended standard of care for patients with primary triple negative breast cancer (TNBC). Nearly half of TNBC patients treated with standard neoadjuvant chemotherapy (NACT) have excellent responses. However, those patients with significant residual cancer burden (RCB) after NACT are at high risk of recurrence or metastatic relapse within two years. Due to the challenges posed by the inter- and intratumoral heterogeneity of TNBC, it is critical to develop appropriate models for predicting and overcoming therapy resistance. We established a collection of 92 orthotopic patient-derived xenograft (PDX) models of TNBC from 84 patients before, during, and after NACT while they were enrolled in the ARTEMIS trial (NCT02276443) at MD Anderson Cancer Center. Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), after a 3-month course of additional chemotherapy and/or different experimental therapies in cases of AC resistance (post-NACT), and from the metastatic lesions of two patients. The collection includes 12 longitudinal sets. Models were established from chemo-sensitive and -resistant tumors, but engraftment success was higher from those cancers that proved to be treatment resistant (RCB II/III). The PDX collection includes models encompassing a broad heterogeneity of chemotherapy responses, histologic features, and molecular TNBC subtypes. In addition, the majority of models develop spontaneous lung metastases. Whole exome sequencing demonstrated conservation of mutations between patient tumors and corresponding PDX models, with TP53 being the most commonly mutated gene. A similar comparison of patient and PDX transcriptomes revealed conservation of signaling pathway signatures, with those related to immune and stromal interactions being the most variable, as anticipated when adapting human tumors to growth in mice. The subclonal architecture of the tumors exhibited little overall change throughout NACT, suggesting that selection for resistant subclones is not likely to be a significant contributor to resistance. Tumor cells from the longitudinal PDX collections were subjected to high throughput drug susceptibility profiling to identify targetable vulnerabilities associated with resistance to NACT. We found that post-NACT tumors exhibited enhanced sensitivity to drugs targeting protein homeostasis pathways. Preclinical studies with pevonedistat validated the neddylation pathway as an effective target in chemoresistant TNBC. Citation Format: Amanda L. Rinkenbaugh, Yuan Qi, Reid T. Powell, Shirong Cai, Jiansu Shao, Faiza Baameur Hancock, Lei Guo, Xiaomei Zhang, Sabrina Jeter-Jones, Chunxiao Fu, Rebekah Gould, Jason B. White, Clifford Stephan, Gloria V. Echeverria, Peter J. Davies, Stacy Moulder, W. Fraser Symmans, Jeffrey T. Chang, Helen Piwnica-Worms. PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6620.

Background Recent advances have been made in targeting the phosphoinositide 3-kinase pathway in breast cancer. Phosphatase and tensin homolog (PTEN) is a key component of that pathway. Objective To understand the changes in PTEN expression over the course of the disease in patients with triple-negative breast cancer (TNBC) and whether PTEN copy number variation (CNV) by next-generation sequencing (NGS) can serve as an alternative to immunohistochemistry (IHC) to identify PTEN loss. Methods We compared PTEN expression by IHC between pretreatment tumors and residual tumors in the breast and lymph nodes after neoadjuvant chemotherapy in 96 patients enrolled in a TNBC clinical trial. A correlative analysis between PTEN protein expression and PTEN CNV by NGS was also performed. Results With a stringent cutoff for PTEN IHC scoring, PTEN expression was discordant between pretreatment and posttreatment primary tumors in 5% of patients (n = 96) and between posttreatment primary tumors and lymph node metastases in 9% (n = 33). A less stringent cutoff yielded similar discordance rates. Intratumoral heterogeneity for PTEN loss was observed in 7% of the patients. Among pretreatment tumors, PTEN copy numbers by whole exome sequencing (n = 72) were significantly higher in the PTEN-positive tumors by IHC compared with the IHC PTEN-loss tumors (p < 0.0001). However, PTEN-positive and PTEN-loss tumors by IHC overlapped in copy numbers: 14 of 60 PTEN-positive samples showed decreased copy numbers in the range of those of the PTEN-loss tumors. Conclusion Testing various specimens by IHC may generate different PTEN results in a small proportion of patients with TNBC; therefore, the decision of testing one versus multiple specimens in a clinical trial should be defined in the patient inclusion criteria. Although a distinct cutoff by which CNV differentiated PTEN-positive tumors from those with PTEN loss was not identified, higher copy number of PTEN may confer positive PTEN, whereas lower copy number of PTEN would necessitate additional testing by IHC to assess PTEN loss. Trial registration NCT02276443.

Simple Summary High stromal tumor-infiltrating lymphocytes (sTILs) are associated with improved pathologic complete response (pCR) in triple-negative breast cancer (TNBC). In this study of 408 patients enrolled in a prospective early-stage TNBC neoadjuvant chemotherapy trial, we aimed to identify clinicopathologic features that could be combined with sTILs to better predict pCR. Applying a training set and a testing set, we found that integrating high Ki-67 (cutoff > 35%) and high sTIL (cutoff ≥ 20%) in a model of computed response scores could predict a pCR rate of 65%. This model may refine the selection of early-stage TNBC patients for neoadjuvant clinical trials evaluating de-escalation strategies. Abstract High stromal tumor-infiltrating lymphocytes (sTILs) are associated with improved pathologic complete response (pCR) in triple-negative breast cancer (TNBC). We hypothesize that integrating high sTILs and additional clinicopathologic features associated with pCR could enhance our ability to predict the group of patients on whom treatment de-escalation strategies could be tested. In this prospective early-stage TNBC neoadjuvant chemotherapy study, pretreatment biopsies from 408 patients were evaluated for their clinical and demographic features, as well as biomarkers including sTILs, Ki-67, PD-L1 and androgen receptor. Multivariate logistic regression models were developed to generate a computed response score to predict pCR. The pCR rate for the entire cohort was 41%. Recursive partitioning analysis identified ≥20% as the optimal cutoff for sTILs to denote 35% (143/408) of patients as having high sTILs, with a pCR rate of 59%, and 65% (265/408) of patients as having low sTILs, with a pCR rate of 31%. High Ki-67 (cutoff > 35%) was identified as the only predictor of pCR in addition to sTILs in the training set. This finding was verified in the testing set, where the highest computed response score encompassing both high sTILa and high Ki-67 predicted a pCR rate of 65%. Integrating Ki67 and sTIL may refine the selection of early stage TNBC patients for neoadjuvant clinical trials evaluating de-escalation strategies.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks the expression of estrogen receptor (ER), progesterone receptor (PR) and HER2 and therefore have limited hormonal treatment options. Neoadjuvant chemotherapy (NAC) is backbone of treatment for TNBC, and about 50% of patients respond well leading to pathological complete response (pCR). However, the remaining patients develop resistance to NAC and progress to metastatic disease and poor survival in 1-2 years after the initial treatment. Previous studies have performed bulk RNA expression profiling of TNBC patients and identified 5-6 subgroups of patients, however these studies could not resolve expression programs at single cell resolution to distinguish between the tumor cells and different components of the tumor microenvironment (TME). Here we performed scRNA-seq of pre-treatment fresh core biopsy tissue samples from TNBC patients in the ARTEMIS clinical trial and compared these data between pCR and non-pCR patients to identify programs associated with response to NAC. We also compared these data to scRNA-seq data from patients with disease-free breast tissue to understand the basic biology of TNBC and identify cell types that are reprogrammed in malignant disease. Using the single cell tumor cell data, we identified 4 archetypes of TNBC which represent patient-level intertumor expression programs: luminal secretory-like (LS), basal/luminal-like (BL), immunoregulatory (IM), and luminal androgen receptor (LAR). Notably, the archetype BL was associated with non-pCR, while IM was associated with pCR. We further identified 13 metatraits, which are unique intratumoral expression programs that are shared across patients. Across the cancer cells, we identified 13 metatraits such as cell cycling, stress, hypoxia, interferon response, HLA, partial epithelial-mesenchymal transition, and endoplasmic reticulum stress, many of which corresponded to NAC response. In the immune compartment, we found 15 myeloid cell states, 14 T/NK cell states, and 6 B cell states, several of which corresponded to pCR/non-pCR. Similarly, in the stromal compartment, there were 4 fibroblast cell states, 4 pericyte cell states, and 7 endothelial cell subtypes, of which several cell states were associated with NAC response. Overall, these data report the natural biology of TNBC patients and malignant cell states that are reprogrammed in malignant disease, as well as their correspondence to NAC response, providing new data to predict which TNBC patients are likely to respond to chemotherapy. Citation Format: Tapsi Kumar, Yiyun Lin, Yun Yan, Shanshan Bai, Jianzhuo Li, Tuan Tran, Min Hu, Elizabeth Ravenberg, Maia Rauch, Alyson Clayborn, Alastair Thompson, Lei Huo, Stacy Moulder, Clinton Yam, Nicholas Navin. Decoding the natural biology of triple-negative breast cancer and response to chemotherapy by single-cell transcriptomics [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 2147.

Metaplastic breast cancer (MpBC) is a rare and highly chemoresistant breast cancer subtype, with a median survival of 8 months after metastatic disease, and no standard therapeutic options. MpBCs are enriched for epithelial-to-mesenchymal transition (EMT)/cancer stem cell (CSC) markers, produce high levels of nitric oxide (NO), and have a hyperactive phosphoinositide 3-kinase (PI3K) signaling pathway. NO activates multiple oncogenic pathways, such as PI3K and transforming growth factor beta (TGFβ), a regulator of EMT. Therefore, we hypothesized that pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) could augment the efficacy of α-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models. Immunostaining analysis revealed that MpBC PDX tumors had elevated co-expression of iNOS and pAkt (60% vs 23%, p=0.04) relative to triple-negative breast cancer (TNBC) PDX tumors. MpBC PDX tumors had higher RPL39 A14V (66% vs 4.7%, p< 0.00) and PIK3CA hotspot mutation rates (50% vs 19.1%, p=0.31) than TNBC PDX tumors. L-NMMA was synergistic with alpelisib in MpBC cell lines with PIK3CA/PIK3R1 mutations and antagonistic in PIK3CA-wild type and PTEN-deleted models. In vivo evaluation using MpBC PDX tumors found that L-NMMA augmented the efficacy of alpelisib in reducing tumor volume in PIK3CA-mutated MpBC PDX models. Transcriptomic analysis found gene sets associated with EMT reversal, such as the formation of cornified envelope (NES = 2.04 Nom p<0.00) and keratinization pathway (NES = 2.06, Nom p<0.00), were enriched pathways in MpBC PDX tumors that responded to combination therapy. Pharmacological/genomic inhibition of iNOS reversed EMT in MpBC cells, by decreased expression of Zeb1, TGFβ, Snail, Vimentin, and increased expression of E-cadherin and ZO-1 in immunoblotting analysis. MpBC cells with NOS2 knockout acquired an epithelial-like cellular morphology, and this reversal of EMT rendered MpBC cells more sensitive to alpelisib and taxane-chemotherapy. MpBC PDX tumors that responded to combination therapy also exhibited a reversal in EMT, with a decrease in aldehyde dehydrogenase (ALDH1), a CSC marker. Combination therapy also reduced tumor-initiating ability, enhanced chemosensitivity, and improved overall survival in MpBC PDX models. These studies paralleled a phase 1b/2 clinical trial with L-NMMA+taxane chemotherapy in a cohort of anthracycline-refractory MpBC patients (NCT02834403). The clinical benefit rate was 40% (6/15), overall response rate was 20% (3/15), and one patient achieved a pathologic complete response. Relative to baseline tumors, the responder end-of-treatment tumors had undergone reversal of EMT, with decreased expression of iNOS and ALDH1. We find that combining L-NMMA and alpelisib is a novel therapeutic strategy to treat MpBC, and combination therapy is being tested in a first multicenter phase 2 study for patients with MpBC. Citation Format: Tejaswini P. Reddy, Akshjot Puri, Liliana Guzman-Rojas, Christoforos Thomas, Wei Qian, Jianying Zhou, Hong Zhao, Xiaoxian Li, Bijan Mahboubi, Adrian Oo, Cho Young-Jae, Baek Kim, Jose Thaiparambil, Maria Florencia Chervo, Roberto Rosato, Camila Ayerbe, Noah Giese, Stacy Moulder, Helen Piwnica-Worms, Funda Meric-Bernstam, Jenny C. Chang. NOS inhibition reverses epithelial-to-mesenchymal transition and synergizes with alpelisib in metaplastic 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 3447.